subject:( Tandem affinity purification)

Northeastern University

1. Doherty, Michael Francis. Proteomic analysis reveals CACN-1 as an integral component of the spliceosome in C. elegans.

Degree: MS, Department of Biology, 2013, Northeastern University

URL: http://hdl.handle.net/2047/d20003320

► Cell migration is an essential physiological activity that controls processes such as embryonic development and tissue formation in all animals. cacn-1 is a conserved, novel… (more)

Subjects/Keywords: C. elegans; cell migration; mass spectrometry; proteomics; RNAi; tandem affinity purification; Biology; Molecular Biology

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APA (6^th Edition):

Doherty, M. F. (2013). Proteomic analysis reveals CACN-1 as an integral component of the spliceosome in C. elegans. (Masters Thesis). Northeastern University. Retrieved from http://hdl.handle.net/2047/d20003320

Chicago Manual of Style (16^th Edition):

Doherty, Michael Francis. “Proteomic analysis reveals CACN-1 as an integral component of the spliceosome in C. elegans.” 2013. Masters Thesis, Northeastern University. Accessed January 18, 2021. http://hdl.handle.net/2047/d20003320.

MLA Handbook (7^th Edition):

Doherty, Michael Francis. “Proteomic analysis reveals CACN-1 as an integral component of the spliceosome in C. elegans.” 2013. Web. 18 Jan 2021.

Vancouver:

Doherty MF. Proteomic analysis reveals CACN-1 as an integral component of the spliceosome in C. elegans. [Internet] [Masters thesis]. Northeastern University; 2013. [cited 2021 Jan 18]. Available from: http://hdl.handle.net/2047/d20003320.

Council of Science Editors:

Doherty MF. Proteomic analysis reveals CACN-1 as an integral component of the spliceosome in C. elegans. [Masters Thesis]. Northeastern University; 2013. Available from: http://hdl.handle.net/2047/d20003320

2. SUNG YING YING. INVESTIGATING THE ROLE OF NORA IN ASYMMETRIC DIVISION OF DROSOPHILA NEURAL STEM CELLS.

Degree: 2011, National University of Singapore

URL: http://scholarbank.nus.edu.sg/handle/10635/30963

Subjects/Keywords: Asymmetric division; Drosophila; neuroblast; aPKC; Nora; tandem affinity purification

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APA (6^th Edition):

YING, S. Y. (2011). INVESTIGATING THE ROLE OF NORA IN ASYMMETRIC DIVISION OF DROSOPHILA NEURAL STEM CELLS. (Thesis). National University of Singapore. Retrieved from http://scholarbank.nus.edu.sg/handle/10635/30963

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

YING, SUNG YING. “INVESTIGATING THE ROLE OF NORA IN ASYMMETRIC DIVISION OF DROSOPHILA NEURAL STEM CELLS.” 2011. Thesis, National University of Singapore. Accessed January 18, 2021. http://scholarbank.nus.edu.sg/handle/10635/30963.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

YING, SUNG YING. “INVESTIGATING THE ROLE OF NORA IN ASYMMETRIC DIVISION OF DROSOPHILA NEURAL STEM CELLS.” 2011. Web. 18 Jan 2021.

Vancouver:

YING SY. INVESTIGATING THE ROLE OF NORA IN ASYMMETRIC DIVISION OF DROSOPHILA NEURAL STEM CELLS. [Internet] [Thesis]. National University of Singapore; 2011. [cited 2021 Jan 18]. Available from: http://scholarbank.nus.edu.sg/handle/10635/30963.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

YING SY. INVESTIGATING THE ROLE OF NORA IN ASYMMETRIC DIVISION OF DROSOPHILA NEURAL STEM CELLS. [Thesis]. National University of Singapore; 2011. Available from: http://scholarbank.nus.edu.sg/handle/10635/30963

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

3. 김, 민재. Identification of novel Fc-interacting proteins involved in the inflammation induced by internalizing anti-DNA antibody.

Degree: 2020, Ajou University

URL: http://repository.ajou.ac.kr/handle/201003/19243 ; http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000029530

►

비공개

ABSTRACT i TABLE OF CONTENTS ii LIST OF FIGURES iii LIST OF TABLES iv I. INTRODUCTION 1 II. MATERIALS AND METHODS 8 A. Cell… (more)

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비공개

ABSTRACT i TABLE OF CONTENTS ii LIST OF FIGURES iii LIST OF TABLES iv I. INTRODUCTION 1 II. MATERIALS AND METHODS 8 A. Cell culture 8 B. Construction of expression vectors and peptide 8 C. Protein preparation using HEK293F cell 9 D. Antibodies 9 E. Lentiviral vector construction 10 F. Establishment of stable cell lines 11 G. Tandem affinity purification (TAP) 11 H. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and in-gel tryptic digestion 12 I. Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis 13 J. Protein Microarray 13 K. Enzyme-Linked Immunosorbent assay (ELISA) 14 L. Confocal microscopy 14 M. Western blotting 15 N. Immunoprecipitation (IP) 15 O. Quantitative RT-PCR (qRT-PCR) 16 P. In vivo ubiquitination assay 17 Q. Statistical analysis 17 III. RESULTS 18 A. Production of IL-8 and TNF-α induced by cell-internalizable anti-DNA IgG is not mediated by TRIM21-signaling pathway. 18 B. Tandem affinity purification (TAP) is used as a method for the identification of novel Fc-interacting proteins in cell cytosol. 23 C. Extracellular tandem affinity purification (ExTAP) is designed to preserve the structural integrity of IgG-Fc used for identification of novel Fc-receptors in cell cytosol. 28 D. Human IgG Fc-interacting proteins are identified by three independent TAP assays coupled with mass-spectrometry analysis. 33 E. The binding between Fc-interacting proteins and purified IgCw-γκ is confirmed in HEK293T cells. 37 F. The binding between Fc-interacting proteins and internalizing 3D8 IgG is confirmed in HEK293T cells. 41 G. Production of IL-8 and TNF-α response to treatment of 3D8 IgG is reduced by knockdown of Fc-interacting proteins in THP-1 cells. 44 H. Ubiquitination of cell-internalizable 3D8 IgG is not mediated by TRIM21 in THP-1 cells. 48 I. Ubiquitination of 3D8 IgG is increased by TRIM21 knockdown in THP-1 cells. 53 J. β-TrCP2 ubiquitinates 3D8 IgG. 57 IV. DISCUSSION 60 V. CONCLUSION 64 REFERENCES 65 국문요약 72

Doctor

Advisors/Committee Members: 대학원 의생명과학과, 201325003, 김, 민재.

Subjects/Keywords: Anti-DNA antibody; TRIM21; β-TrCP2; Tandem affinity purification(TAP); 항-DNA 항체; 일렬 친화도 정제

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

김, �. (2020). Identification of novel Fc-interacting proteins involved in the inflammation induced by internalizing anti-DNA antibody. (Thesis). Ajou University. Retrieved from http://repository.ajou.ac.kr/handle/201003/19243 ; http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000029530

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

김, 민재. “Identification of novel Fc-interacting proteins involved in the inflammation induced by internalizing anti-DNA antibody.” 2020. Thesis, Ajou University. Accessed January 18, 2021. http://repository.ajou.ac.kr/handle/201003/19243 ; http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000029530.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

김, 민재. “Identification of novel Fc-interacting proteins involved in the inflammation induced by internalizing anti-DNA antibody.” 2020. Web. 18 Jan 2021.

Vancouver:

김 �. Identification of novel Fc-interacting proteins involved in the inflammation induced by internalizing anti-DNA antibody. [Internet] [Thesis]. Ajou University; 2020. [cited 2021 Jan 18]. Available from: http://repository.ajou.ac.kr/handle/201003/19243 ; http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000029530.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

김 �. Identification of novel Fc-interacting proteins involved in the inflammation induced by internalizing anti-DNA antibody. [Thesis]. Ajou University; 2020. Available from: http://repository.ajou.ac.kr/handle/201003/19243 ; http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000029530

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of Vienna

4. Aspalter, Irene. Hunting for co-factors of endosomal toll-like receptors.

Degree: 2009, University of Vienna

URL: http://othes.univie.ac.at/4358/

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Toll-like-Rezeptoren (TLRs) sind transmembran Proteine, welche für die Erkennung von Pathogen assoziierten molekularer Strukturen (PAMPs) in der angeborenen Immunität wichtig sind. Zehn strukturell sehr ähnliche… (more)

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Toll-like-Rezeptoren (TLRs) sind transmembran Proteine, welche für die Erkennung von Pathogen assoziierten molekularer Strukturen (PAMPs) in der angeborenen Immunität wichtig sind. Zehn strukturell sehr ähnliche TLRs wurden bis jetzt entdeckt, welche entweder in der Plasmamembran (TLR2, -4, -5, -6 und -11) oder in der Membran von Endosomen (TLR3, -7, -8 und -9) lokalisiert sind. Durch Ligandenbindung können TLRs eine intrazelluläre Signalkaskade induzieren, wodurch die Expression von Zytokine, Interferone und Proteine zur Verteidigung der Zelle und des Organismus angeschaltet wird. Obwohl die zehn TLRs strukturell stark konserviert sind, kann ein breites Spektrum an strukturell sehr unterschiedlichen Liganden gebunden werden. Die Komplexität der Bindung zwischen TLRs und PAMPs ist am besten bekannt für die Bindung von Lipopolysaccharid (LPS) durch TLR4, welche drei Cofaktoren benötigt. Die zentrale Hypothese meiner Diplomarbeit geht davon aus, dass TLRs nicht alleine an ihre Liganden binden, sondern verschiede Co-Faktoren benötigen und in Komplexe eingebunden sind, wie es für TLR4 der Fall ist. Um Rezeptor assoziierte Co-Faktoren zu identifizieren wurde systematisch nach endosomalen TLR Komplexen, die für eine angeborene Immunantwort nach viraler Infektion notwendig sind, gesucht. Eine interdisziplinäre Strategie welche Biochemie, Zellbiologie, Bioinformatik und Proteomik umfasst, wurde angewendet. Die murinen, endosomalen TLRs (3, 7, 8 und 9) wurden am C-Terminus mit einer GS-Tandem Affinity-Purification (TAP) Kassette versehen. Diese Konstrukte wurden verwendet, um stabile RAW264.7 Makrophagen Zelllinien, mittels retroviralem Gen-Transfers, zu erzeugen. Um die richtige Prozessierung und Lokalisierung der rekombinanten Proteine zu überprüfen, wurde ein Glycosylierungszustand der vier Rezeptoren überprüft. Die Lysate der stabilen RAW264.7 Zell-Linien wurden für Tandem-Affinity-Purification verwendet, wobei spezielle Bedingungen für endosomale Transmembranproteine angewendet wurden. Anschließend wurden die aufgereinigten Eluate im Massenspektrometer analysiert. Nach bioinformatischer Analyse, basierend auf der CeMM-pulldown Datenbank und Peptidanreicherung, wurde eine Liste von sieben Kandidaten erstellt. Um zu überprüfen, ob diese sieben Kandidaten tatsächlich mit den endosomalen TLRs interagieren wurde Co-Immunprezipitation angewendet, wobei die Interaktion von zwei Kandidaten mit TLRs gezeigt werden konnte. Des Weiteren wurden funktionelle Analysen für diese beiden Kandidaten durchgeführt, um Aufschluss über deren Funktion in der angeborenen Immunität zu erhalten. Diese Arbeit soll dazu dienen das Verständnis von Toll-like Rezeptor vermittelter Pathogen Erkennung und der darauffolgenden Immunantwort zu erhöhen.

Toll-like receptors (TLRs) are trans-membrane proteins that mediate the recognition of pathogen associated molecular patterns (PAMPs) in innate immunity. Ten structurally highly related TLRs have been identified in mammals, which are either located at the plasma membrane (TLR2, -4, -5, -6…

Subjects/Keywords: 42.13 Molekularbiologie; 42.32 Virologie; 44.45 Immunologie; Makrophagen / Toll-like Rezeptoren / Endosom / Tandem-Affinity-Purification / Proteomik / Angeborene Immunität / Pathogen; macrophages / toll-like receptors / endosome / tandem-affinity-purification / proteomics / innate immunity / pathogen / pattern recognition receptor

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Aspalter, I. (2009). Hunting for co-factors of endosomal toll-like receptors. (Thesis). University of Vienna. Retrieved from http://othes.univie.ac.at/4358/

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Aspalter, Irene. “Hunting for co-factors of endosomal toll-like receptors.” 2009. Thesis, University of Vienna. Accessed January 18, 2021. http://othes.univie.ac.at/4358/.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Aspalter, Irene. “Hunting for co-factors of endosomal toll-like receptors.” 2009. Web. 18 Jan 2021.

Vancouver:

Aspalter I. Hunting for co-factors of endosomal toll-like receptors. [Internet] [Thesis]. University of Vienna; 2009. [cited 2021 Jan 18]. Available from: http://othes.univie.ac.at/4358/.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Aspalter I. Hunting for co-factors of endosomal toll-like receptors. [Thesis]. University of Vienna; 2009. Available from: http://othes.univie.ac.at/4358/

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Universiteit Utrecht

5. Mulder, K.W. Functional and genetic analysis of the Ccr4-Not complex in Yeast.

Degree: 2006, Universiteit Utrecht

URL: http://dspace.library.uu.nl:8080/handle/1874/19930

► Transcription is a complex process that is regulated at multiple levels. This involves a wide variety of multi-subunit protein-complexes including the basal (or general) transcription… (more)

▼ Transcription is a complex process that is regulated at multiple levels. This involves a wide variety of multi-subunit protein-complexes including the basal (or general) transcription factors and RNA polymerase II. One of the major obstacles for the transcription machinery is chromatin, which consists of DNA wrapped around an octamer composed of the histone core particle (two molecules of H2A, H2B, H3 and H4) and the linker histone H1. One of the complexes involved in regulation of transcription is the nine-subunit Ccr4-Not complex. Initially, components of this complex were identified as negative regulators of the non-consensus TATA box driven transcription of the HIS3 gene. In addition, some data also suggest an additional positive role in transcription. Recently, Ccr4p and Caf1p were identified as the major cytoplasmic deadenylases in yeast, indicating that his complex functions in both transcription and mRNA turnover. In addition, human CNOT4 displays RING-finger mediated UbcH5B-dependent ubiquitylation activity. To address the physiological cones-quences of the ubiquitylation activity of Not4p, we used Saccharomyces cerevisiae (bakers' yeast) as a model organism. First, site-directed mutagenesis of the RING-finger of yeast Not4p identified residues required for interaction with Ubc4p and Ubc5p, the yeast orthologs of UbcH5B. Subsequent in vitro assays with purified Ccr4-Not complexes showed Not4p mediated E3 ligase activity, which was dependent on the interaction with Ubc4p. Next, we performed synthetic genetic array (SGA) analyses using not4Δ and not4L35A alleles. This indicates involvement of the RING-finger of Not4p in transcription, ubiquitylation and DNA damage responses. In addition, we found overlapping phenotypes for deletions of UBC4 and mutants encoding single amino acid substitutions of the RING-finger of Not4p. Together, the results presented in chapter 2 show that Not4p functions as an E3 ligase by modulating Ubc4p/Ubc5p mediated stress responses in vivo. Furthermore, genetic experiments have indicated a role for the Ccr4-Not complex in the response to hydroxyurea (HU)-induced replication stress and ionizing radiation. This response includes transcriptional induction of the four genes constituting the ribonucleotide reductase (RNR) enzymatic complex, RNR1-4, and degradation of its inhibitor, Sml1p. In chapter 3, we investigated the mechanism of the HU sensitivity conferred by mutation of CCR4-NOT genes. We found that the ubiquitin protein ligase activity of Not4p does not play a role in HU-induced Sml1p degradation. We showed, however, that the HU sensitivity of ccr4-not mutant strains correlated very well with a defect in accumulation of RNR2, RNR3 and RNR4 mRNA after HU or MMS treatment. Chromatin immuno-precipitation experiments showed that TBP, RNA polymerase II and Set1p recruitment to the activated RNR3 locus was defective in cells lacking NOT4. Moreover, RNR3 promoter activity was not induced by HU treatment of these cells. Together, these experiments show that induction of RNR gene…

Subjects/Keywords: Geneeskunde; Ccr4-Not complex; transcription; deadenylation; histone modification; ubiquitylation; Saccharomyces Cerevisiae; synthetic genetic array analysis; chromatin immunoprecipitation; tandem affinity purification; phosphorylation

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Mulder, K. W. (2006). Functional and genetic analysis of the Ccr4-Not complex in Yeast. (Doctoral Dissertation). Universiteit Utrecht. Retrieved from http://dspace.library.uu.nl:8080/handle/1874/19930

Chicago Manual of Style (16^th Edition):

Mulder, K W. “Functional and genetic analysis of the Ccr4-Not complex in Yeast.” 2006. Doctoral Dissertation, Universiteit Utrecht. Accessed January 18, 2021. http://dspace.library.uu.nl:8080/handle/1874/19930.

MLA Handbook (7^th Edition):

Mulder, K W. “Functional and genetic analysis of the Ccr4-Not complex in Yeast.” 2006. Web. 18 Jan 2021.

Vancouver:

Mulder KW. Functional and genetic analysis of the Ccr4-Not complex in Yeast. [Internet] [Doctoral dissertation]. Universiteit Utrecht; 2006. [cited 2021 Jan 18]. Available from: http://dspace.library.uu.nl:8080/handle/1874/19930.

Council of Science Editors:

Mulder KW. Functional and genetic analysis of the Ccr4-Not complex in Yeast. [Doctoral Dissertation]. Universiteit Utrecht; 2006. Available from: http://dspace.library.uu.nl:8080/handle/1874/19930

6. Niu, Xiaofan. Gene Regulatory Machinery and Proteomics of Sexual Reproduction in Phytophthora infestans.

Degree: Plant Pathology, 2010, University of California – Riverside

URL: http://www.escholarship.org/uc/item/0qw9h17b

► Mating is important for the survival of Phytophthora infestans, the causal agent of potato and tomato late blight. The mating process increases population fitness and… (more)

▼ Mating is important for the survival of Phytophthora infestans, the causal agent of potato and tomato late blight. The mating process increases population fitness and produces thick-walled oospores that can survive harsh environmental conditions. To better understand the mechanisms that regulate of sexual reproduction in P. infestans, the promoters of two mating-induced genes, PITG_002525 and PITG_00483, were studied in order to identify their cis-regulatory elements. PITG_02525 encodes an elicitin-like protein, and PITG_00483 encodes a PUMILIO (Pum) protein. The promoter of PITG_02525 was sequentially deleted and fused with a beta-glucuronidase reporter gene to identify its regulatory motifs. The full length promoter was found to be active in young and mature, and male and female, structures. The deletion analysis identified a potential repressor element and an activator. Similar studies of the pum promoter showed expression both during oosporogenesis and sporulation. One promoter element was found to be essential for the expression of the gene in both stages. Fusing this element with a minimal promoter indicated that it was sufficient for gene expression during sporulation, but not in mating. The RNA binding domain (RBD) of Pum then was used as a model to develop a system for P. infestans protein interaction studies. Expression of the RBD with a tandem affinity purification tag designed with P. infestans codon bias, allowed the successful purification of the Pum RBD with a potential binding partner. This system is adaptable for future studies of other protein interactions. A MudPIT global proteomics comparison of oospores with nonsporulating hyphae (NSH) was then conducted to profile the protein composition of oospores and identify important proteins for germination. It was proposed that the proteins detected in dormant oospores were synthesized during early oosporogenesis and stored for germination. Our study identified proteins that are more abundant in oospores belonging to several functional groups. These included enzymes involved in carbohydrate and fatty acid oxidation to produce energy, and anti-oxidant and anti-stress proteins that may protect the germ tubes, and secreted proteins that could be involved in host infection. This revealed that P. infestans has a very well designed life cycle.

Subjects/Keywords: Plant Pathology; MudPIT; Phytophthora infestans; Protein tandem affinity purification; Proteomics; Sexual reproduction; Transcription regulation

…71 CHAPTER II Developing the tandem affinity purification system for protein interaction… …61-64 CHAPTER II Developing the tandem affinity purification system for protein… …fusing the RBD with a tandem affinity purification (TAP) and transformating the…

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Niu, X. (2010). Gene Regulatory Machinery and Proteomics of Sexual Reproduction in Phytophthora infestans. (Thesis). University of California – Riverside. Retrieved from http://www.escholarship.org/uc/item/0qw9h17b

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Niu, Xiaofan. “Gene Regulatory Machinery and Proteomics of Sexual Reproduction in Phytophthora infestans.” 2010. Thesis, University of California – Riverside. Accessed January 18, 2021. http://www.escholarship.org/uc/item/0qw9h17b.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Niu, Xiaofan. “Gene Regulatory Machinery and Proteomics of Sexual Reproduction in Phytophthora infestans.” 2010. Web. 18 Jan 2021.

Vancouver:

Niu X. Gene Regulatory Machinery and Proteomics of Sexual Reproduction in Phytophthora infestans. [Internet] [Thesis]. University of California – Riverside; 2010. [cited 2021 Jan 18]. Available from: http://www.escholarship.org/uc/item/0qw9h17b.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Niu X. Gene Regulatory Machinery and Proteomics of Sexual Reproduction in Phytophthora infestans. [Thesis]. University of California – Riverside; 2010. Available from: http://www.escholarship.org/uc/item/0qw9h17b

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of South Florida

7. Desai, Shruti. Regulation of Positive Regulatory Domain I- Binding Factor 1 and Its Role in Mantle Cell Lymphoma.

Degree: 2010, University of South Florida

URL: https://scholarcommons.usf.edu/etd/1613

► The human positive regulatory domain I binding factor 1 (PRDI-BF1/PRDM1) promotes differentiation of mature B cells into antibody secreting plasma cells. In contrast ectopic expression… (more)

▼ The human positive regulatory domain I binding factor 1 (PRDI-BF1/PRDM1) promotes differentiation of mature B cells into antibody secreting plasma cells. In contrast ectopic expression of PRDM1 in lymphoma cells can lead to inhibition of proliferation or apoptosis. However, little is currently known about the regulation of PRDM1. The first study presented demonstrates that in lymphoma cells stimulation through the B cell receptor rapidly induces endogenous PRDM1 at the level of transcription. This study provides evidence that the PRDM1 promoter is preloaded and poised for activation in the B cell lines. The transcription factor PU.1 is shown to be required for B cell receptor induced expression of PRDM1 in lymphoma cells and in PU.1 positive myeloma cells. Furthermore, activation is associated with loss of the co-repressor TLE4 from the PU.1 complex. The second study establishes the requirement for PRDM1 in Mantle cell lymphoma (MCL) response to Bortezomib. MCL, an aggressive form of B cell lymphoma, has poor disease- free survival rate. The proteasome inhibitor, Bortezomib, is approved for treatment of relapsed and refractory MCL. However, the precise mechanism of action of Bortezomib is not well understood. Bortezomib rapidly induces transcription of PRDM1 along with apoptosis in MCL cell lines and primary MCL tumor samples. Knockdown of PRDM1 inhibits Bortezomib-induced apoptosis, while ectopic expression of PRDM1 alone leads to apoptosis in MCL. MKI67 and PCNA, which are required for proliferation and survival, were identified as novel direct targets of PRDM1 in MCL. Chromatin immunoprecipitation and knockdown studies reveal specific repression of MKI67 and PCNA is mediated by PRDM1 in response to Bortezomib. Furthermore promoter studies demonstrate that PRDM1 functions through a specific site in the proximal promoter region of PCNA and through a distal upstream repression domain on the MKI67 promoter. Together these findings establish PRDM1 as a key mediator of Bortezomib activity in MCL through suppression of proliferation and survival genes. The third study presented demonstrates use of Tandem affinity purification technique followed by mass spectrometry to identify PRDM1 and Reptin52 protein interactions. The observations in this study provide preliminary evidence of novel mechanism of regulation of PRDM1 protein function.

Subjects/Keywords: prdm1; gene regulation; b cell lymphoma; protease inhibitor; tandem affinity purification; American Studies; Arts and Humanities

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Desai, S. (2010). Regulation of Positive Regulatory Domain I- Binding Factor 1 and Its Role in Mantle Cell Lymphoma. (Thesis). University of South Florida. Retrieved from https://scholarcommons.usf.edu/etd/1613

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Desai, Shruti. “Regulation of Positive Regulatory Domain I- Binding Factor 1 and Its Role in Mantle Cell Lymphoma.” 2010. Thesis, University of South Florida. Accessed January 18, 2021. https://scholarcommons.usf.edu/etd/1613.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Desai, Shruti. “Regulation of Positive Regulatory Domain I- Binding Factor 1 and Its Role in Mantle Cell Lymphoma.” 2010. Web. 18 Jan 2021.

Vancouver:

Desai S. Regulation of Positive Regulatory Domain I- Binding Factor 1 and Its Role in Mantle Cell Lymphoma. [Internet] [Thesis]. University of South Florida; 2010. [cited 2021 Jan 18]. Available from: https://scholarcommons.usf.edu/etd/1613.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Desai S. Regulation of Positive Regulatory Domain I- Binding Factor 1 and Its Role in Mantle Cell Lymphoma. [Thesis]. University of South Florida; 2010. Available from: https://scholarcommons.usf.edu/etd/1613

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

8. Mulder, K.W. Functional and genetic analysis of the Ccr4-Not complex in Yeast.

Degree: 2006, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/19930 ; URN:NBN:NL:UI:10-1874-19930 ; urn:isbn:978-90-393-4421-7 ; URN:NBN:NL:UI:10-1874-19930 ; https://dspace.library.uu.nl/handle/1874/19930

► Transcription is a complex process that is regulated at multiple levels. This involves a wide variety of multi-subunit protein-complexes including the basal (or general) transcription… (more)

▼ Transcription is a complex process that is regulated at multiple levels. This involves a wide variety of multi-subunit protein-complexes including the basal (or general) transcription factors and RNA polymerase II. One of the major obstacles for the transcription machinery is chromatin, which consists of DNA wrapped around an octamer composed of the histone core particle (two molecules of H2A, H2B, H3 and H4) and the linker histone H1. One of the complexes involved in regulation of transcription is the nine-subunit Ccr4-Not complex. Initially, components of this complex were identified as negative regulators of the non-consensus TATA box driven transcription of the HIS3 gene. In addition, some data also suggest an additional positive role in transcription. Recently, Ccr4p and Caf1p were identified as the major cytoplasmic deadenylases in yeast, indicating that his complex functions in both transcription and mRNA turnover. In addition, human CNOT4 displays RING-finger mediated UbcH5B-dependent ubiquitylation activity. To address the physiological cones-quences of the ubiquitylation activity of Not4p, we used Saccharomyces cerevisiae (bakers' yeast) as a model organism. First, site-directed mutagenesis of the RING-finger of yeast Not4p identified residues required for interaction with Ubc4p and Ubc5p, the yeast orthologs of UbcH5B. Subsequent in vitro assays with purified Ccr4-Not complexes showed Not4p mediated E3 ligase activity, which was dependent on the interaction with Ubc4p. Next, we performed synthetic genetic array (SGA) analyses using not4Δ and not4L35A alleles. This indicates involvement of the RING-finger of Not4p in transcription, ubiquitylation and DNA damage responses. In addition, we found overlapping phenotypes for deletions of UBC4 and mutants encoding single amino acid substitutions of the RING-finger of Not4p. Together, the results presented in chapter 2 show that Not4p functions as an E3 ligase by modulating Ubc4p/Ubc5p mediated stress responses in vivo. Furthermore, genetic experiments have indicated a role for the Ccr4-Not complex in the response to hydroxyurea (HU)-induced replication stress and ionizing radiation. This response includes transcriptional induction of the four genes constituting the ribonucleotide reductase (RNR) enzymatic complex, RNR1-4, and degradation of its inhibitor, Sml1p. In chapter 3, we investigated the mechanism of the HU sensitivity conferred by mutation of CCR4-NOT genes. We found that the ubiquitin protein ligase activity of Not4p does not play a role in HU-induced Sml1p degradation. We showed, however, that the HU sensitivity of ccr4-not mutant strains correlated very well with a defect in accumulation of RNR2, RNR3 and RNR4 mRNA after HU or MMS treatment. Chromatin immuno-precipitation experiments showed that TBP, RNA polymerase II and Set1p recruitment to the activated RNR3 locus was defective in cells lacking NOT4. Moreover, RNR3 promoter activity was not induced by HU treatment of these cells. Together, these experiments show that induction of RNR gene…

Subjects/Keywords: Ccr4-Not complex; transcription; deadenylation; histone modification; ubiquitylation; Saccharomyces Cerevisiae; synthetic genetic array analysis; chromatin immunoprecipitation; tandem affinity purification; phosphorylation

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Mulder, K. W. (2006). Functional and genetic analysis of the Ccr4-Not complex in Yeast. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/19930 ; URN:NBN:NL:UI:10-1874-19930 ; urn:isbn:978-90-393-4421-7 ; URN:NBN:NL:UI:10-1874-19930 ; https://dspace.library.uu.nl/handle/1874/19930

Chicago Manual of Style (16^th Edition):

Mulder, K W. “Functional and genetic analysis of the Ccr4-Not complex in Yeast.” 2006. Doctoral Dissertation, University Utrecht. Accessed January 18, 2021. https://dspace.library.uu.nl/handle/1874/19930 ; URN:NBN:NL:UI:10-1874-19930 ; urn:isbn:978-90-393-4421-7 ; URN:NBN:NL:UI:10-1874-19930 ; https://dspace.library.uu.nl/handle/1874/19930.

MLA Handbook (7^th Edition):

Mulder, K W. “Functional and genetic analysis of the Ccr4-Not complex in Yeast.” 2006. Web. 18 Jan 2021.

Vancouver:

Mulder KW. Functional and genetic analysis of the Ccr4-Not complex in Yeast. [Internet] [Doctoral dissertation]. University Utrecht; 2006. [cited 2021 Jan 18]. Available from: https://dspace.library.uu.nl/handle/1874/19930 ; URN:NBN:NL:UI:10-1874-19930 ; urn:isbn:978-90-393-4421-7 ; URN:NBN:NL:UI:10-1874-19930 ; https://dspace.library.uu.nl/handle/1874/19930.

Council of Science Editors:

Mulder KW. Functional and genetic analysis of the Ccr4-Not complex in Yeast. [Doctoral Dissertation]. University Utrecht; 2006. Available from: https://dspace.library.uu.nl/handle/1874/19930 ; URN:NBN:NL:UI:10-1874-19930 ; urn:isbn:978-90-393-4421-7 ; URN:NBN:NL:UI:10-1874-19930 ; https://dspace.library.uu.nl/handle/1874/19930

University of St. Andrews

9. Carr, Sharon. Adenovirus and its interaction with host cell proteins .

Degree: 2007, University of St. Andrews

URL: http://hdl.handle.net/10023/219

► The E1B55k gene in adenovirus type 5 was studied by generating recombinant viruses in which the E1B55k gene was N-terminally tagged with either a cyan… (more)

Subjects/Keywords: Adenovirus; E1B55k; SUMO; Recombinant; Tandem affinity purification; TAP; CFP; Cyan fluorescent protein; Lysine to arginine mutation; SUMO consensus sequence

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Carr, S. (2007). Adenovirus and its interaction with host cell proteins . (Thesis). University of St. Andrews. Retrieved from http://hdl.handle.net/10023/219

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Carr, Sharon. “Adenovirus and its interaction with host cell proteins .” 2007. Thesis, University of St. Andrews. Accessed January 18, 2021. http://hdl.handle.net/10023/219.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Carr, Sharon. “Adenovirus and its interaction with host cell proteins .” 2007. Web. 18 Jan 2021.

Vancouver:

Carr S. Adenovirus and its interaction with host cell proteins . [Internet] [Thesis]. University of St. Andrews; 2007. [cited 2021 Jan 18]. Available from: http://hdl.handle.net/10023/219.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Carr S. Adenovirus and its interaction with host cell proteins . [Thesis]. University of St. Andrews; 2007. Available from: http://hdl.handle.net/10023/219

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

10. Mulder, K.W. Functional and genetic analysis of the Ccr4-Not complex in Yeast.

Degree: 2006, University Utrecht

URL: https://dspace.library.uu.nl/handle/1874/19930 ; URN:NBN:NL:UI:10-1874-19930 ; 1874/19930 ; urn:isbn:9789039344217 ; URN:NBN:NL:UI:10-1874-19930 ; https://dspace.library.uu.nl/handle/1874/19930

► Transcription is a complex process that is regulated at multiple levels. This involves a wide variety of multi-subunit protein-complexes including the basal (or general) transcription… (more)

▼ Transcription is a complex process that is regulated at multiple levels. This involves a wide variety of multi-subunit protein-complexes including the basal (or general) transcription factors and RNA polymerase II. One of the major obstacles for the transcription machinery is chromatin, which consists of DNA wrapped around an octamer composed of the histone core particle (two molecules of H2A, H2B, H3 and H4) and the linker histone H1. One of the complexes involved in regulation of transcription is the nine-subunit Ccr4-Not complex. Initially, components of this complex were identified as negative regulators of the non-consensus TATA box driven transcription of the HIS3 gene. In addition, some data also suggest an additional positive role in transcription. Recently, Ccr4p and Caf1p were identified as the major cytoplasmic deadenylases in yeast, indicating that his complex functions in both transcription and mRNA turnover. In addition, human CNOT4 displays RING-finger mediated UbcH5B-dependent ubiquitylation activity. To address the physiological cones-quences of the ubiquitylation activity of Not4p, we used Saccharomyces cerevisiae (bakers' yeast) as a model organism. First, site-directed mutagenesis of the RING-finger of yeast Not4p identified residues required for interaction with Ubc4p and Ubc5p, the yeast orthologs of UbcH5B. Subsequent in vitro assays with purified Ccr4-Not complexes showed Not4p mediated E3 ligase activity, which was dependent on the interaction with Ubc4p. Next, we performed synthetic genetic array (SGA) analyses using not4Δ and not4L35A alleles. This indicates involvement of the RING-finger of Not4p in transcription, ubiquitylation and DNA damage responses. In addition, we found overlapping phenotypes for deletions of UBC4 and mutants encoding single amino acid substitutions of the RING-finger of Not4p. Together, the results presented in chapter 2 show that Not4p functions as an E3 ligase by modulating Ubc4p/Ubc5p mediated stress responses in vivo. Furthermore, genetic experiments have indicated a role for the Ccr4-Not complex in the response to hydroxyurea (HU)-induced replication stress and ionizing radiation. This response includes transcriptional induction of the four genes constituting the ribonucleotide reductase (RNR) enzymatic complex, RNR1-4, and degradation of its inhibitor, Sml1p. In chapter 3, we investigated the mechanism of the HU sensitivity conferred by mutation of CCR4-NOT genes. We found that the ubiquitin protein ligase activity of Not4p does not play a role in HU-induced Sml1p degradation. We showed, however, that the HU sensitivity of ccr4-not mutant strains correlated very well with a defect in accumulation of RNR2, RNR3 and RNR4 mRNA after HU or MMS treatment. Chromatin immuno-precipitation experiments showed that TBP, RNA polymerase II and Set1p recruitment to the activated RNR3 locus was defective in cells lacking NOT4. Moreover, RNR3 promoter activity was not induced by HU treatment of these cells. Together, these experiments show that induction of RNR gene…

Subjects/Keywords: Ccr4-Not complex; transcription; deadenylation; histone modification; ubiquitylation; Saccharomyces Cerevisiae; synthetic genetic array analysis; chromatin immunoprecipitation; tandem affinity purification; phosphorylation

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Mulder, K. W. (2006). Functional and genetic analysis of the Ccr4-Not complex in Yeast. (Doctoral Dissertation). University Utrecht. Retrieved from https://dspace.library.uu.nl/handle/1874/19930 ; URN:NBN:NL:UI:10-1874-19930 ; 1874/19930 ; urn:isbn:9789039344217 ; URN:NBN:NL:UI:10-1874-19930 ; https://dspace.library.uu.nl/handle/1874/19930

Chicago Manual of Style (16^th Edition):

Mulder, K W. “Functional and genetic analysis of the Ccr4-Not complex in Yeast.” 2006. Doctoral Dissertation, University Utrecht. Accessed January 18, 2021. https://dspace.library.uu.nl/handle/1874/19930 ; URN:NBN:NL:UI:10-1874-19930 ; 1874/19930 ; urn:isbn:9789039344217 ; URN:NBN:NL:UI:10-1874-19930 ; https://dspace.library.uu.nl/handle/1874/19930.

MLA Handbook (7^th Edition):

Mulder, K W. “Functional and genetic analysis of the Ccr4-Not complex in Yeast.” 2006. Web. 18 Jan 2021.

Vancouver:

Mulder KW. Functional and genetic analysis of the Ccr4-Not complex in Yeast. [Internet] [Doctoral dissertation]. University Utrecht; 2006. [cited 2021 Jan 18]. Available from: https://dspace.library.uu.nl/handle/1874/19930 ; URN:NBN:NL:UI:10-1874-19930 ; 1874/19930 ; urn:isbn:9789039344217 ; URN:NBN:NL:UI:10-1874-19930 ; https://dspace.library.uu.nl/handle/1874/19930.

Council of Science Editors:

Mulder KW. Functional and genetic analysis of the Ccr4-Not complex in Yeast. [Doctoral Dissertation]. University Utrecht; 2006. Available from: https://dspace.library.uu.nl/handle/1874/19930 ; URN:NBN:NL:UI:10-1874-19930 ; 1874/19930 ; urn:isbn:9789039344217 ; URN:NBN:NL:UI:10-1874-19930 ; https://dspace.library.uu.nl/handle/1874/19930

11. Zhao, Hongwei. A Proteomic Study of Plant Messenger RNA Cleavage and Polyadenylation Specificity Factors and the Establishment of an In Vitro Cleavage Assay System.

Degree: PhD, Botany, 2008, Miami University

URL: http://rave.ohiolink.edu/etdc/view?acc_num=miami1218547019

► Cleavage and polyadenylation of precursor messenger RNA (pre-mRNA) is an essential processing event during mRNA maturation. Most of the understanding of the protein factors… (more)

▼ Cleavage and polyadenylation of precursor messenger RNA (pre-mRNA) is an essential processing event during mRNA maturation. Most of the understanding of the protein factors required for this process comes from yeast and mammals in which successful in vitro cleavage and polyadenylation systems have been extensively employed. In contrast, limited information about plant polyadenylation factors and their functions has been largely from bioinformatics and genetics studies more recently, due to the lack of a plant in vitro system. Using tandem affinity purification (TAP) methods, plant cleavage and polyadenylation specificity factor (CPSF) was isolated and identified from Arabidopsis thaliana. By compiling the in vivo interaction data from TAP purification, as well as available yeast two-hybrid assay data from literature, a plant CPSF model is proposed. According to this model, the Arabidopsis CPSF possesses AtCPSF30, AtCPSF73-I, AtCPSF73-II, AtCPSF100, AtCPSF160, AtFY, and AtFIPS5. Among them, AtCPSF100 serves as a core on which other factors are associated, except for AtFIPS5. AtFIPS5 associates to the CPSF complex indirectly by interacting with AtCPSF30. Protein factors from other complexes, such as AtSYM5, AtCLPS3 and AtPCFS4, may associate with CPSF through AtCPSF30 directly or indirectly. These results show that plant CPSF shares many similarities with its yeast and mammalian counterparts, despite some obvious distinct features. To reconstitute an in vitro assay system for plant mRNA 3'-end processing, protein extracts from Arabidopsis suspension cell cultures over-expressing some polyadenylation-related protein factors, such as AtCPSF30, AtCPSF73-I, AtCPSF73-II, and AtCLPS3, were successfully used in regenerating the cleavage reaction. The characteristics of the plant in vitro cleavage system were further studied by using one of these over-expressed factors, AtCPSF73-I. This in vitro cleavage system has the expected features: cleaves the substrate RNA at the same locations as when processed in vivo; the cleavage activity is dependent on the polyadenylation signals; and the cleavage leaves a 3'-hydroxyl group at the 3'-end for polyadenylation. Analysis of the system also showed the potential involvement of both endonuclease and 3'-5'exonuclease activities. An endo-exonuclease dualism model was proposed for pre-mRNA cleavage in plants. This pioneering system will serve as a platform to further explore plant mRNA 3'-end formation mechanisms and other RNA processing events. Advisors/Committee Members: Li, Qingshun (Advisor).

Subjects/Keywords: Biogeochemistry; Botany; Molecular Biology; Cleavage and polyadenylation specificity factor; CPSF; tandem affinity purification; TAP; in vitro cleavage

…of a plant in vitro system. Using tandem affinity purification (TAP) methods… …22 Tandem affinity purification method and its application in the proteomic study of plant… …questions, I performed tandem affinity purification to isolate the Arabidopsis CPSF complex, and… …purification, as well as available yeast two-hybrid assay data from literature, a plant CPSF model is… …31 TAP purification…

8 more images…

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Zhao, H. (2008). A Proteomic Study of Plant Messenger RNA Cleavage and Polyadenylation Specificity Factors and the Establishment of an In Vitro Cleavage Assay System. (Doctoral Dissertation). Miami University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=miami1218547019

Chicago Manual of Style (16^th Edition):

Zhao, Hongwei. “A Proteomic Study of Plant Messenger RNA Cleavage and Polyadenylation Specificity Factors and the Establishment of an In Vitro Cleavage Assay System.” 2008. Doctoral Dissertation, Miami University. Accessed January 18, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=miami1218547019.

MLA Handbook (7^th Edition):

Zhao, Hongwei. “A Proteomic Study of Plant Messenger RNA Cleavage and Polyadenylation Specificity Factors and the Establishment of an In Vitro Cleavage Assay System.” 2008. Web. 18 Jan 2021.

Vancouver:

Zhao H. A Proteomic Study of Plant Messenger RNA Cleavage and Polyadenylation Specificity Factors and the Establishment of an In Vitro Cleavage Assay System. [Internet] [Doctoral dissertation]. Miami University; 2008. [cited 2021 Jan 18]. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1218547019.

Council of Science Editors:

Zhao H. A Proteomic Study of Plant Messenger RNA Cleavage and Polyadenylation Specificity Factors and the Establishment of an In Vitro Cleavage Assay System. [Doctoral Dissertation]. Miami University; 2008. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1218547019

University of Helsinki

12. Lång, Mika. Purification of alphavirus replication complexes.

Degree: Department of Food and Environmental Sciences; Helsingfors universitet, Agrikultur- och forstvetenskapliga fakulteten, Institutionen för livsmedels- och miljövetenskaper, 2017, University of Helsinki

URL: http://hdl.handle.net/10138/229812

►

Alfavirukset ovat yksijuosteisia positiivisäkeisiä RNA-viruksia, jotka ovat aiheuttaneet lukuisia epidemioita viimeisten vuosikymmenten aikana. Virusten genomi koodaa neljää ei-rakenteellista proteiinia, jotka osallistuvat virusperäisen RNA:n monistamiseen. Translaatiotuote… (more)

▼

Alfavirukset ovat yksijuosteisia positiivisäkeisiä RNA-viruksia, jotka ovat aiheuttaneet lukuisia epidemioita viimeisten vuosikymmenten aikana. Virusten genomi koodaa neljää ei-rakenteellista proteiinia, jotka osallistuvat virusperäisen RNA:n monistamiseen. Translaatiotuote on polyproteiini, josta yksittäiset ei-rakenteelliset proteiinit pilkotaan. Eirakenteelliset proteiinit tuottavat genomista negatiivisäikeen, joka toimii templaattina posiitivisäikeiden tuotannossa. Negatiivisesta templaatti-RNA:sta monistetaan subgenominen RNA, joka koodaa rakenteellisia proteiineja. Genomisen ja subgenomisen RNA:n tuotanto tapahtuu ei-rakenteellisista proteiineista koostuvissa replikaatiokomplekseissa, jotka sijaitsevat kuroumissa solukalvolla ja endolysosomisilla vesikkeleillä. Infektion lisäksi viruksen proteiineja ja templaattia koodaavien plasmidien transfektoimisen avulla kyetään muodostamaan replikaatiokomplekseja soluissa. Replikaatiokompleksien toiminnan ja rakenteen tutkimiseksi niitä on kyettävä puhdistamaan soluista. Tässä työssä replikaatiokompleksien puhdistamiseksi käytettiin kahta eri menetelmää. Ensimmäisessä menetelmässä nisäkässolut infektoitiin elävillä viruksilla ja replikaatiokompleksien siirtyminen endolysosomiin vesikkeleihin solukalvolta estettiin solujen lääkekäsittelyllä. Solut rikottiin ja tumat poistettiin sentrifugoimalla. Supernatantti sedimentoitiin ultrasentrifugaatiolla kerrosgradientissa. Replikaatiokompleksit sisältävää fraktiota puhdistettiin edelleen flotaatiolla, jossa fraktion osien annettiin asettua tiheyden mukaan. Replikaatiokompleksit sisältävää fraktiota tutkittiin elektronimikroskopian sekä proteiini- ja lipidianalyysien avulla. Toisessa puhdistusmenetelmässä nisäkässoluihin transfektoitiin templaatti-RNA:ta ja replikaasipolyproteiinia koodaavat plasmidit, jotka saivat replikaatiokompleksit muodostumaan soluissa. Polyproteiinisekvenssissä oli myös jakso, joka koodasi hemagglutiniinipeptidin osaksi replikaatiokompleksin polymeraasina toimivaa ei-rakenteellista proteiinia. Soluista poistettiin tumat sentrifugaatiolla ja replikaatiokompleksit sidottiin supernatantista hemagglutiniinivasta-aineella. Sitoutuneet replikaatiokompleksit irroitettiin Laemmlin puskuriliuoksella ja puhdistumista tutkittiin Western blotting -menetelmällä. Elektronimikroskoopilla havaittiin replikaatiokompleksin sisältäviä kuroumia ja vasta-aineilla voitiin todentaa eirakenteellisten proteiinien läsnäolo. Lukumäärällisesti kuroumia havaittiin vähän ja näytteissä oli myös soluperäisiä epäpuhtauksia. Replikaatiokompleksien puhdistuminen vasta-aineaffiniteetilla oli hyvin vähäistä, mutta puhdistuminen oli suurempaa kuin näytteissä, joissa ei ollut hemagglutiniinimerkkiä. Vaikka merkittävää replikaatiokompleksien puhdistumista ei saavutettu, työn aikana kehitettiin molempia menetelmiä ja saavutettiin replikaatiokompleksien osittainen puhdistuminen infektoiduista soluista.

Alphaviruses are single-stranded positive-sense RNA viruses that have been the cause of numerous epidemics in the past decades. The viral…

Subjects/Keywords: alphavirus; replication complex; purification; Semliki Forest virus; affinity purification; ultracentrifugation; Mikrobiologia; Microbiology; Mikrobiologi; alphavirus; replication complex; purification; Semliki Forest virus; affinity purification; ultracentrifugation

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Lång, M. (2017). Purification of alphavirus replication complexes. (Masters Thesis). University of Helsinki. Retrieved from http://hdl.handle.net/10138/229812

Chicago Manual of Style (16^th Edition):

Lång, Mika. “Purification of alphavirus replication complexes.” 2017. Masters Thesis, University of Helsinki. Accessed January 18, 2021. http://hdl.handle.net/10138/229812.

MLA Handbook (7^th Edition):

Lång, Mika. “Purification of alphavirus replication complexes.” 2017. Web. 18 Jan 2021.

Vancouver:

Lång M. Purification of alphavirus replication complexes. [Internet] [Masters thesis]. University of Helsinki; 2017. [cited 2021 Jan 18]. Available from: http://hdl.handle.net/10138/229812.

Council of Science Editors:

Lång M. Purification of alphavirus replication complexes. [Masters Thesis]. University of Helsinki; 2017. Available from: http://hdl.handle.net/10138/229812

University of Toronto

13. Gulersen, Moti. Characterization of NP22 and its Potential Role in NMDA Receptor-mediated Transmission.

Degree: 2011, University of Toronto

URL: http://hdl.handle.net/1807/30617

►

N-methyl D-aspartate (NMDA) receptors represent integral signal transducers for excitatory glutamate neurotransmission. While NMDA receptors are critical for synaptic plasticity, the molecular events underlying this… (more)

Subjects/Keywords: NMDA; neuroscience; protein interactions; NP22; synaptic plasticity; affinity purification; 0419; 0383

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Gulersen, M. (2011). Characterization of NP22 and its Potential Role in NMDA Receptor-mediated Transmission. (Masters Thesis). University of Toronto. Retrieved from http://hdl.handle.net/1807/30617

Chicago Manual of Style (16^th Edition):

Gulersen, Moti. “Characterization of NP22 and its Potential Role in NMDA Receptor-mediated Transmission.” 2011. Masters Thesis, University of Toronto. Accessed January 18, 2021. http://hdl.handle.net/1807/30617.

MLA Handbook (7^th Edition):

Gulersen, Moti. “Characterization of NP22 and its Potential Role in NMDA Receptor-mediated Transmission.” 2011. Web. 18 Jan 2021.

Vancouver:

Gulersen M. Characterization of NP22 and its Potential Role in NMDA Receptor-mediated Transmission. [Internet] [Masters thesis]. University of Toronto; 2011. [cited 2021 Jan 18]. Available from: http://hdl.handle.net/1807/30617.

Council of Science Editors:

Gulersen M. Characterization of NP22 and its Potential Role in NMDA Receptor-mediated Transmission. [Masters Thesis]. University of Toronto; 2011. Available from: http://hdl.handle.net/1807/30617

Universidade Nova

14. Santana, Sara Daniela Filipe. Magnetic nanoparticles for biocatalysis and bioseparation.

Degree: 2011, Universidade Nova

URL: http://www.rcaap.pt/detail.jsp?id=oai:run.unl.pt:10362/6259

►

Dissertação para obtenção do Grau de Mestre em Biotecnologia

This work aimed to prepare iron oxide magnetic nanoparticles (MNPs) coated with biopolymers and further modified… (more)

Subjects/Keywords: Magnetic nanoparticles; Biopolymers; Enzyme cleavage; Affinity ligands; Purification

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Santana, S. D. F. (2011). Magnetic nanoparticles for biocatalysis and bioseparation. (Thesis). Universidade Nova. Retrieved from http://www.rcaap.pt/detail.jsp?id=oai:run.unl.pt:10362/6259

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Santana, Sara Daniela Filipe. “Magnetic nanoparticles for biocatalysis and bioseparation.” 2011. Thesis, Universidade Nova. Accessed January 18, 2021. http://www.rcaap.pt/detail.jsp?id=oai:run.unl.pt:10362/6259.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Santana, Sara Daniela Filipe. “Magnetic nanoparticles for biocatalysis and bioseparation.” 2011. Web. 18 Jan 2021.

Vancouver:

Santana SDF. Magnetic nanoparticles for biocatalysis and bioseparation. [Internet] [Thesis]. Universidade Nova; 2011. [cited 2021 Jan 18]. Available from: http://www.rcaap.pt/detail.jsp?id=oai:run.unl.pt:10362/6259.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Santana SDF. Magnetic nanoparticles for biocatalysis and bioseparation. [Thesis]. Universidade Nova; 2011. Available from: http://www.rcaap.pt/detail.jsp?id=oai:run.unl.pt:10362/6259

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of Toronto

15. Wheaton, Sarah. Optimization of an Affinity Purification-mass Spectrometry Pipeline and Characterization of the Rub1p and Smt3p Interactomes.

Degree: 2011, University of Toronto

URL: http://hdl.handle.net/1807/27374

►

The ubiquitin-like proteins (Ubls) are small polypeptides that function as post-translational modifications. Modification of a protein with a Ubl can alter its localization, activity and/or… (more)

Subjects/Keywords: Ubiquitin-like Proteins; Affinity Purification-Mass Spectrometry; Dcn1p; Cdc48p; 0307

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Wheaton, S. (2011). Optimization of an Affinity Purification-mass Spectrometry Pipeline and Characterization of the Rub1p and Smt3p Interactomes. (Masters Thesis). University of Toronto. Retrieved from http://hdl.handle.net/1807/27374

Chicago Manual of Style (16^th Edition):

Wheaton, Sarah. “Optimization of an Affinity Purification-mass Spectrometry Pipeline and Characterization of the Rub1p and Smt3p Interactomes.” 2011. Masters Thesis, University of Toronto. Accessed January 18, 2021. http://hdl.handle.net/1807/27374.

MLA Handbook (7^th Edition):

Wheaton, Sarah. “Optimization of an Affinity Purification-mass Spectrometry Pipeline and Characterization of the Rub1p and Smt3p Interactomes.” 2011. Web. 18 Jan 2021.

Vancouver:

Wheaton S. Optimization of an Affinity Purification-mass Spectrometry Pipeline and Characterization of the Rub1p and Smt3p Interactomes. [Internet] [Masters thesis]. University of Toronto; 2011. [cited 2021 Jan 18]. Available from: http://hdl.handle.net/1807/27374.

Council of Science Editors:

Wheaton S. Optimization of an Affinity Purification-mass Spectrometry Pipeline and Characterization of the Rub1p and Smt3p Interactomes. [Masters Thesis]. University of Toronto; 2011. Available from: http://hdl.handle.net/1807/27374

University of Washington

16. McKinney, Bonnie Lewis. Cell-specific transcriptional analysis in complex tissues.

Degree: 2017, University of Washington

URL: http://hdl.handle.net/1773/38194

► Cell-type specific transcriptional analysis can yield important information about the role of a certain cell type in the context of its tissue microenvironment. Previous methods… (more)

Subjects/Keywords: Lung stromal cells; Ribotag; Translating ribosome affinity purification; Pathology; Medicine; pathology

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APA (6^th Edition):

McKinney, B. L. (2017). Cell-specific transcriptional analysis in complex tissues. (Thesis). University of Washington. Retrieved from http://hdl.handle.net/1773/38194

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

McKinney, Bonnie Lewis. “Cell-specific transcriptional analysis in complex tissues.” 2017. Thesis, University of Washington. Accessed January 18, 2021. http://hdl.handle.net/1773/38194.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

McKinney, Bonnie Lewis. “Cell-specific transcriptional analysis in complex tissues.” 2017. Web. 18 Jan 2021.

Vancouver:

McKinney BL. Cell-specific transcriptional analysis in complex tissues. [Internet] [Thesis]. University of Washington; 2017. [cited 2021 Jan 18]. Available from: http://hdl.handle.net/1773/38194.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

McKinney BL. Cell-specific transcriptional analysis in complex tissues. [Thesis]. University of Washington; 2017. Available from: http://hdl.handle.net/1773/38194

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

17. XU DAN. ELECTROSPUN AFFINITY MEMBRANE FOR IMMUNOGLOBULIN G PURIFICATION.

Degree: 2007, National University of Singapore

URL: http://scholarbank.nus.edu.sg/handle/10635/17541

Subjects/Keywords: affinity membrane; protein purification; electrospinning

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APA (6^th Edition):

DAN, X. (2007). ELECTROSPUN AFFINITY MEMBRANE FOR IMMUNOGLOBULIN G PURIFICATION. (Thesis). National University of Singapore. Retrieved from http://scholarbank.nus.edu.sg/handle/10635/17541

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

DAN, XU. “ELECTROSPUN AFFINITY MEMBRANE FOR IMMUNOGLOBULIN G PURIFICATION.” 2007. Thesis, National University of Singapore. Accessed January 18, 2021. http://scholarbank.nus.edu.sg/handle/10635/17541.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

DAN, XU. “ELECTROSPUN AFFINITY MEMBRANE FOR IMMUNOGLOBULIN G PURIFICATION.” 2007. Web. 18 Jan 2021.

Vancouver:

DAN X. ELECTROSPUN AFFINITY MEMBRANE FOR IMMUNOGLOBULIN G PURIFICATION. [Internet] [Thesis]. National University of Singapore; 2007. [cited 2021 Jan 18]. Available from: http://scholarbank.nus.edu.sg/handle/10635/17541.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

DAN X. ELECTROSPUN AFFINITY MEMBRANE FOR IMMUNOGLOBULIN G PURIFICATION. [Thesis]. National University of Singapore; 2007. Available from: http://scholarbank.nus.edu.sg/handle/10635/17541

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Texas Tech University

18. Hamouda, Ayman K. Structural studies with affinity-purified muscle and neuronal nicotinic acetylcholine receptors.

Degree: TTUHSC – Pharmacology and Neuroscience, 2007, Texas Tech University

URL: http://hdl.handle.net/2346/12495

► Nicotinic acetylcholine receptors (nAChRs) are a heterogeneous group of ligand gated ion channels that mediate the function of the endogenous neurotransmitter acetylcholine. A more refined… (more)

▼ Nicotinic acetylcholine receptors (nAChRs) are a heterogeneous group of ligand gated ion channels that mediate the function of the endogenous neurotransmitter acetylcholine. A more refined understanding of the molecular structure of individual neuronal nAChRs is a prerequisite for the development of subtype-selective ligands. Such ligands will be valuable in the understanding and treatment of certain CNS diseases as well as nicotine addiction. Using an acetylcholine derivatized affinity column originally used for purification of Torpedo nAChR, we have purified a4b2 and a4b4 neuronal nAChRs from HEK293 cell lines that stably express a4b2 or a4b4 nAChRs respectively. The lipid-protein interface and the agonist-binding site of purified neuronal nAChRs were directly examined using photoaffinity labeling with [125I]TID and [125I]Epibatidine respectively. The preliminary results of these studies include: 1) [125I]TID photoincorporates into the M4 and M1 transmembrane segments of both a4 and b2 subunits; 2) Within a4M4/b2M4 segments, [125I]TID labeled amino acid residues a4Cys582/b2Cys445 which are identical (in the aligned sequence) to the [125I]TID-labeled residues Cys418 in the lipid exposed face of Torpedo a1M4; 3) [125I]TID also labeled a4Arg572 within a4M4 which is situated in the same position as His408 in the lipid exposed face of Torpedo a1M4; 4) Within a4M1 segment, [125I]TID labeled Cys226, Cys231 and Leu232 which correspond to [125I]TID labeled residues Cys222, Phe227 and Leu228 in the lipid exposed face of Torpedo a1M1. In similar fashion, [125I]TID labeled b2Cys220 and b2Val221 which correspond to [125I]TID labeled Cys222 and Leu223 in the lipid exposed face of Torpedo a1M1. 5) Further studies are needed to determine if [125I]TID photoincorporates into the M2 and M3 transmembrane segments; 6) Photoaffinity labeling of a4b2 and a4b4 nAChRs with [125I]Epibatidine reveals that [125I]Epibatidine photoincorporates specifically into the b4 subunit with no (or insignificant photoincorporation) within a4 and b2 subunits; 7) [125I]Epibatidine photoincorporation ion the a4 subunit was limited to the agonist binding pocket as the majority of labeling was displaceable by addition of nicotine; 8) The site of specific [125I]Epibatidine photincorporation within the b4 subunit was mapped to loop E (b4Val102-Glu131) within the complementary components of the agonist binding site. Advisors/Committee Members: Blanton, Michael P. (Committee Chair), Lombardini, J. Barry (committee member), Popp, R. Lisa (committee member), Pressley, Thomas A. (committee member), Machu, Tina K. (committee member).

Subjects/Keywords: Photolabeling; Lipids; Affinity purification; Nicotine

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Hamouda, A. K. (2007). Structural studies with affinity-purified muscle and neuronal nicotinic acetylcholine receptors. (Thesis). Texas Tech University. Retrieved from http://hdl.handle.net/2346/12495

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Hamouda, Ayman K. “Structural studies with affinity-purified muscle and neuronal nicotinic acetylcholine receptors.” 2007. Thesis, Texas Tech University. Accessed January 18, 2021. http://hdl.handle.net/2346/12495.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Hamouda, Ayman K. “Structural studies with affinity-purified muscle and neuronal nicotinic acetylcholine receptors.” 2007. Web. 18 Jan 2021.

Vancouver:

Hamouda AK. Structural studies with affinity-purified muscle and neuronal nicotinic acetylcholine receptors. [Internet] [Thesis]. Texas Tech University; 2007. [cited 2021 Jan 18]. Available from: http://hdl.handle.net/2346/12495.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Hamouda AK. Structural studies with affinity-purified muscle and neuronal nicotinic acetylcholine receptors. [Thesis]. Texas Tech University; 2007. Available from: http://hdl.handle.net/2346/12495

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Virginia Tech

19. Sines, Brian James. Evaluation of Phenomena that Determine the Performance of Immunoaffinity, Peptide-Based and Ion Exchange Affinity Sorbents.

Degree: PhD, Chemical Engineering, 2000, Virginia Tech

URL: http://hdl.handle.net/10919/29841

► This report presents an evaluation of the design of low-solids content, large-particle diameter beaded cellulose supports for column-mode protein purification. The study presented here optimizes… (more)

Subjects/Keywords: affinity chromatography; protein purification

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APA (6^th Edition):

Sines, B. J. (2000). Evaluation of Phenomena that Determine the Performance of Immunoaffinity, Peptide-Based and Ion Exchange Affinity Sorbents. (Doctoral Dissertation). Virginia Tech. Retrieved from http://hdl.handle.net/10919/29841

Chicago Manual of Style (16^th Edition):

Sines, Brian James. “Evaluation of Phenomena that Determine the Performance of Immunoaffinity, Peptide-Based and Ion Exchange Affinity Sorbents.” 2000. Doctoral Dissertation, Virginia Tech. Accessed January 18, 2021. http://hdl.handle.net/10919/29841.

MLA Handbook (7^th Edition):

Sines, Brian James. “Evaluation of Phenomena that Determine the Performance of Immunoaffinity, Peptide-Based and Ion Exchange Affinity Sorbents.” 2000. Web. 18 Jan 2021.

Vancouver:

Sines BJ. Evaluation of Phenomena that Determine the Performance of Immunoaffinity, Peptide-Based and Ion Exchange Affinity Sorbents. [Internet] [Doctoral dissertation]. Virginia Tech; 2000. [cited 2021 Jan 18]. Available from: http://hdl.handle.net/10919/29841.

Council of Science Editors:

Sines BJ. Evaluation of Phenomena that Determine the Performance of Immunoaffinity, Peptide-Based and Ion Exchange Affinity Sorbents. [Doctoral Dissertation]. Virginia Tech; 2000. Available from: http://hdl.handle.net/10919/29841

20. Lacombe, Andrée-Anne. Étude de la fonction de la protéine RPAP4 et de son association avec l’ARN polymérase II.

Degree: 2015, Université de Montréal

URL: http://hdl.handle.net/1866/12072

Subjects/Keywords: ARN polymérase II; RPAP4/GPN1; RPAP2; Transport nucléocytoplasmique; Biogenèse de l’ARNPII; Interaction protéine-protéine; Purification d’affinité en tandem; Phosphorylation; ERK5; RNA polymerase II; Nucleocytoplasmic transport; RNAPII biogenesis; Protein-protein interaction; Tandem affinity purification; Biology - Molecular / Biologie - Biologie moléculaire (UMI : 0307)

…chromosome TAF TBP-associated factor TAP Tandem affinity purification TBP TATA box-binding… …désoxyribonucléique AP Affinity purification AP-MS Affinity purification coupled with mass spectrometry… …58 2.2.3.2 Expression of TAP-tagged Proteins and Purification of Protein Complexes ... 59… …purification par chromatographie sur DEAE-sephadex. L’ARNPI est responsable de la transcription des…

11 more images…

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APA (6^th Edition):

Lacombe, A. (2015). Étude de la fonction de la protéine RPAP4 et de son association avec l’ARN polymérase II. (Thesis). Université de Montréal. Retrieved from http://hdl.handle.net/1866/12072

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Lacombe, Andrée-Anne. “Étude de la fonction de la protéine RPAP4 et de son association avec l’ARN polymérase II.” 2015. Thesis, Université de Montréal. Accessed January 18, 2021. http://hdl.handle.net/1866/12072.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Lacombe, Andrée-Anne. “Étude de la fonction de la protéine RPAP4 et de son association avec l’ARN polymérase II.” 2015. Web. 18 Jan 2021.

Vancouver:

Lacombe A. Étude de la fonction de la protéine RPAP4 et de son association avec l’ARN polymérase II. [Internet] [Thesis]. Université de Montréal; 2015. [cited 2021 Jan 18]. Available from: http://hdl.handle.net/1866/12072.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Lacombe A. Étude de la fonction de la protéine RPAP4 et de son association avec l’ARN polymérase II. [Thesis]. Université de Montréal; 2015. Available from: http://hdl.handle.net/1866/12072

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Freie Universität Berlin

21. Klass, Kathrin. Proteins interacting with the alpha subunit of the stimulatory G protein, G(alpha)s.

Degree: 2009, Freie Universität Berlin

URL: https://refubium.fu-berlin.de/handle/fub188/10515

► The alpha-subunit of the stimulatory G protein, G(alpha)s, plays a central role in cellular signal transduction. G(alpha)s acts as a transmitter between a number of… (more)

▼ The alpha-subunit of the stimulatory G protein, G(alpha)s, plays a central role in cellular signal transduction. G(alpha)s acts as a transmitter between a number of G protein-coupled receptors (GPCRs) and the effector enzyme adenylyl cyclase (AC) thereby stimulating the production of the second messenger cAMP. As opposed to other G proteins, few effectors other than AC and no regulators of G(alpha)s activity are known to date. In the present study, direct protein-protein interactions of G(alpha)s and novel partners were characterized. Therefore, two methods based on affinity purification were used to isolate the bait protein G(alpha)s together with bound partners from mammalian cell lysates. The isolated protein complexes were analyzed using SDS-gel electrophoresis and mass spectrometry. For the functional characterization of its interaction with novel unknown partners in vitro, recombinant G(alpha)s was used. Unlike bovine G(alpha)s, neither wild type nor C-terminally or internally affinity tagged murine G(alpha)s orthologs could be recovered in sufficient amounts. Therefore, the functional characterization of novel interactors was performed using C-terminally His6 tagged bovine G(alpha)s. Recently, human RGS-PX1 was described as a novel regulator of G(alpha)s activity and therefore, the mouse ortholog was characterized in detail. In functional assays it was demonstrated that neither the mouse nor the human ortholog affects G(alpha)s activity, therefore RGS-PX1 can not be regarded a regulator of G(alpha)s activity. Using the affinity purification methods, 10 specific interactors for inactive G(alpha)s and 29 specific interactors for activated G(alpha)s were identified. Of these proteins, calmodulin, 14-3-3(beta), 14-3-3(gamma) and 14-3-3(zeta) as well as G(gamma)10 and G(gamma)12 were further characterized with regard to their interaction with G(alpha)s. The interaction of G(alpha)s and the 14-3-3-isoforms was not confirmed. Calmodulin was confirmed as a G(alpha)s-interacting protein by means of reciprocal co-purification of the isolated proteins. A functional influence of calmodulin on G(alpha)s-catalysed GTP hydrolysis was not detected. In this study, the interaction of G(alpha)s with G(gamma)10 and G(gamma)12 was demonstrated for the first time by co-purification of these proteins from insect cells where they were heterologously expressed. This provides a valuable basis for future characterization of alternate, non- plasmamembrane localized G(gamma)s signal transduction. Advisors/Committee Members: n (gender), Prof. Dr. W. Rosenthal (firstReferee), Prof. Dr. V. Haucke (furtherReferee).

Subjects/Keywords: Heterotrimere G-Proteine, stimulatorisches G-Protein, Signaltransduktion, Tandem Affinity Purification; RGS; 500 Naturwissenschaften und Mathematik::570 Biowissenschaften; Biologie::572 Biochemie; 500 Naturwissenschaften und Mathematik::570 Biowissenschaften; Biologie::571 Physiologie und verwandte Themen

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Klass, K. (2009). Proteins interacting with the alpha subunit of the stimulatory G protein, G(alpha)s. (Thesis). Freie Universität Berlin. Retrieved from https://refubium.fu-berlin.de/handle/fub188/10515

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Klass, Kathrin. “Proteins interacting with the alpha subunit of the stimulatory G protein, G(alpha)s.” 2009. Thesis, Freie Universität Berlin. Accessed January 18, 2021. https://refubium.fu-berlin.de/handle/fub188/10515.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Klass, Kathrin. “Proteins interacting with the alpha subunit of the stimulatory G protein, G(alpha)s.” 2009. Web. 18 Jan 2021.

Vancouver:

Klass K. Proteins interacting with the alpha subunit of the stimulatory G protein, G(alpha)s. [Internet] [Thesis]. Freie Universität Berlin; 2009. [cited 2021 Jan 18]. Available from: https://refubium.fu-berlin.de/handle/fub188/10515.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Klass K. Proteins interacting with the alpha subunit of the stimulatory G protein, G(alpha)s. [Thesis]. Freie Universität Berlin; 2009. Available from: https://refubium.fu-berlin.de/handle/fub188/10515

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Universitat Politècnica de València

22. Rodríguez Solovey, Leisa Natacha. IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs).

Degree: 2015, Universitat Politècnica de València

URL: http://hdl.handle.net/10251/58862

► [EN] ABSTRACT Abscisic acid (ABA) signaling plays a critical role in regulating root growth and root system architecture. ABA-mediated growth promotion and root tropic response… (more)

▼ [EN] ABSTRACT Abscisic acid (ABA) signaling plays a critical role in regulating root growth and root system architecture. ABA-mediated growth promotion and root tropic response under water stress are key responses for plant survival under limiting water conditions. In this work, we have explored the role of Arabidopsis (Arabidopsis thaliana) PYR/PYL/RCAR receptors (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS) for root ABA signaling. As a result, we discovered that PYL8 plays a nonredundant role for the regulation of root ABA sensitivity. Unexpectedly, given the multigenic nature and partial functional redundancy observed in the PYR/PYL family, the single pyl8 mutant showed reduced sensitivity to ABA-mediated root growth inhibition. This effect was due to the lack of PYL8-mediated inhibition of several clade A phosphatases type 2C (PP2Cs), since PYL8 interacted in vivo with at least five PP2Cs, namely HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 as revealed by tandem affinity purification and mass spectrometry proteomic approaches. Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calciumdependent interactions of PYR/PYL/RCAR ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL/RCAR receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL/RCAR function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL/RCAR-interacting partners that mediate a transient Ca2+-dependent interaction with phospholipid vesicles, which affects PYR/PYL/RCAR subcellular localization and positively regulates ABA signaling.; [ES] RESUMEN La señalización por la hormona vegetal ácido abscísico (ABA) desempeña un papel crítico en la regulación del crecimiento de la raíz y en la arquitectura del sistema radical. La promoción de crecimiento de la raíz en condiciones de estrés hídrico mediada por ABA es clave para la supervivencia de las plantas bajo condiciones limitantes de agua. En este trabajo, hemos… Advisors/Committee Members: González Guzmán, Miguel (advisor), Rodríguez Egea, Pedro Luís (advisor).

Subjects/Keywords: Abscisic acid (ABA); Arabidopsis thaliana, Abiotic Stress; PYRABACTIN RESISTANCE/PYRABACTIN RESISTANCE 1-LIKE/REGULATORY COMPONENT OF ABA RECEPTOR (PYR/PYL/RCAR) ABA receptors; C2-DOMAIN ABA-RELATED PROTEINS (CARs); CLADE A PROTEIN PHOSPHATASE 2C (PP2C); SUCROSE NON-FERMENTING-1 (SNF1)-RELATED PROTEIN KINASE 2 (SNRK2); Protein Binding Targets; Ca2+-Dependent Lipid-Binding Domain; Subcellular Localization; Plasma-membrane binding; Signal transduction; Root ABA Response; Yeast Two-Hybrid; Tandem Affinity Purification (TAP); Crystal Structure; Bimolecular Fluorescence Complementation (BiFC).

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Rodríguez Solovey, L. N. (2015). IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs). (Doctoral Dissertation). Universitat Politècnica de València. Retrieved from http://hdl.handle.net/10251/58862

Chicago Manual of Style (16^th Edition):

Rodríguez Solovey, Leisa Natacha. “IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs). ” 2015. Doctoral Dissertation, Universitat Politècnica de València. Accessed January 18, 2021. http://hdl.handle.net/10251/58862.

MLA Handbook (7^th Edition):

Rodríguez Solovey, Leisa Natacha. “IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs). ” 2015. Web. 18 Jan 2021.

Vancouver:

Rodríguez Solovey LN. IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs). [Internet] [Doctoral dissertation]. Universitat Politècnica de València; 2015. [cited 2021 Jan 18]. Available from: http://hdl.handle.net/10251/58862.

Council of Science Editors:

Rodríguez Solovey LN. IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs). [Doctoral Dissertation]. Universitat Politècnica de València; 2015. Available from: http://hdl.handle.net/10251/58862

23. Morrison, Linda Kay. Partial Purification and Some Properties of Lipase From Pseudomonas Aeruginosa.

Degree: 1979, North Texas State University

URL: https://digital.library.unt.edu/ark:/67531/metadc798171/

► Purification of lipase from Pseudomonas aeruginosa (from both a washed cell suspension and crude culture supernatant as the enzyme source) was performed utilizing affinity chromatography.… (more)

Subjects/Keywords: lipase purification; Pseudomonas aeruginosa; affinity chromatography; Lipase – Purification.; Pseudomonas aeruginosa.; Affinity chromatography.

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APA (6^th Edition):

Morrison, L. K. (1979). Partial Purification and Some Properties of Lipase From Pseudomonas Aeruginosa. (Thesis). North Texas State University. Retrieved from https://digital.library.unt.edu/ark:/67531/metadc798171/

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Morrison, Linda Kay. “Partial Purification and Some Properties of Lipase From Pseudomonas Aeruginosa.” 1979. Thesis, North Texas State University. Accessed January 18, 2021. https://digital.library.unt.edu/ark:/67531/metadc798171/.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Morrison, Linda Kay. “Partial Purification and Some Properties of Lipase From Pseudomonas Aeruginosa.” 1979. Web. 18 Jan 2021.

Vancouver:

Morrison LK. Partial Purification and Some Properties of Lipase From Pseudomonas Aeruginosa. [Internet] [Thesis]. North Texas State University; 1979. [cited 2021 Jan 18]. Available from: https://digital.library.unt.edu/ark:/67531/metadc798171/.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Morrison LK. Partial Purification and Some Properties of Lipase From Pseudomonas Aeruginosa. [Thesis]. North Texas State University; 1979. Available from: https://digital.library.unt.edu/ark:/67531/metadc798171/

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of Rochester

24. Asher, Wesley B. (1984 - ); Bren, Kara. Engineering native and artificial heme c containing proteins for biochemical applications and studies of protein folding.

Degree: PhD, 2012, University of Rochester

URL: http://hdl.handle.net/1802/21222

► Heme c containing proteins are known for their intense colors and essential functions in nature. These proteins contain heme that is covalently bound to the… (more)

▼ Heme c containing proteins are known for their intense colors and essential functions in nature. These proteins contain heme that is covalently bound to the protein. For part of the work in this thesis, we have developed fusion tags that contain heme c, known as heme-tags, that reversibly bind an L-histidine immobilized Sepharose (HIS) chromatography resin for affinity purification of recombinant proteins expressed in Escherichia coli. The heme-tag HIS purification method couples the ease of affinity purification with the convenience of visible detection for protein tracking. In addition, we show that the heme-tag can be used to quantify the protein. Heme is covalently bound to native heme c proteins by several dedicated biogenesis systems that exhibit a variable degree of diversity and substrate specificity. Heme-tagged proteins are produced using a biogenesis system native to E. coli with promiscuous substrate specificity. The biogenesis system that matures native mitochondrial cytochromes c (cyt c) that function in cellular respiration and apoptosis is referred to as cytochrome c heme lyase (CCHL). The substrate specificity of CCHL has been explored, but the details are unclear. In this thesis, we show that CCHL can mature the first 18 N-terminal residues of horse cyt c fused to a non-heme containing protein, which demonstrates that the C-terminal portion of cyt c is unimportant for substrate recognition. This work lends new insight into the substrate specificity of CCHL and provides a new approach for producing artificial heme c proteins that could find use in multiple applications. In addition to developing novel biochemical applications of the heme tag, we study the folding of the important model protein horse cyt c. Protein folding is considered one of the most confounding problems in science, and cyt c has long been a model protein to understand the general principles of folding. Herein, we show that the folding of cyt c is more complex than previously reported using single molecule fluorescence methods. We show evidence of multiple folding intermediates and pathways as the protein folds on its energy landscape.

Subjects/Keywords: Heme-tagged protein; Protein affinity purification; Single molecule FRET; Protein folding; Cytochrome c

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APA (6^th Edition):

Asher, Wesley B. (1984 - ); Bren, K. (2012). Engineering native and artificial heme c containing proteins for biochemical applications and studies of protein folding. (Doctoral Dissertation). University of Rochester. Retrieved from http://hdl.handle.net/1802/21222

Chicago Manual of Style (16^th Edition):

Asher, Wesley B. (1984 - ); Bren, Kara. “Engineering native and artificial heme c containing proteins for biochemical applications and studies of protein folding.” 2012. Doctoral Dissertation, University of Rochester. Accessed January 18, 2021. http://hdl.handle.net/1802/21222.

MLA Handbook (7^th Edition):

Asher, Wesley B. (1984 - ); Bren, Kara. “Engineering native and artificial heme c containing proteins for biochemical applications and studies of protein folding.” 2012. Web. 18 Jan 2021.

Vancouver:

Asher, Wesley B. (1984 - ); Bren K. Engineering native and artificial heme c containing proteins for biochemical applications and studies of protein folding. [Internet] [Doctoral dissertation]. University of Rochester; 2012. [cited 2021 Jan 18]. Available from: http://hdl.handle.net/1802/21222.

Council of Science Editors:

Asher, Wesley B. (1984 - ); Bren K. Engineering native and artificial heme c containing proteins for biochemical applications and studies of protein folding. [Doctoral Dissertation]. University of Rochester; 2012. Available from: http://hdl.handle.net/1802/21222

25. Rossa, Luis Augusto Ferreira. Purificação da gonadotrofina coriônica eqüina, do plasma sanguíneo de éguas prenhes, por cromatografia de afinidade.

Degree: PhD, Reprodução Animal, 2009, University of São Paulo

URL: http://www.teses.usp.br/teses/disponiveis/10/10131/tde-29072009-111402/ ;

►

A Gonadotrofina Coriônica Eqüina (eCG) é produzida pela égua prenhe e tem ação folículo estimulante e luteinizante em animais domésticos não eqüídeos. Um pool formado… (more)

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A Gonadotrofina Coriônica Eqüina (eCG) é produzida pela égua prenhe e tem ação folículo estimulante e luteinizante em animais domésticos não eqüídeos. Um pool formado por plasma de 4 éguas prenhes, com média de 69 dias de gestação, foi purificado em coluna cromatográfica com resina de afinidade Blue Sepharose FF (BS). As frações que adsorveram à resina BS foram purificadas em coluna cromatográfica com resina de afinidade Concanavalina A 4B (ConA). As frações que não adsorveram à resina BS também foram purificadas em coluna cromatográfica com resina de afinidade ConA. O mesmo pool de palsma foi diafiltrado, em cartucho de hemodiálise. O diafiltrado foi aplicado em coluna cromatográfica com resina de afinidade ConA. Atividade biológica (UI/mL) do plasma, do diafiltrado e das frações purificadas foram quantificadas por ensaio biológico com ratas impúbres. As atividades biológicas encontradas no plasma e no plasma diafiltrado foram de 3,63 e 5,14UI/mL, respectivamente. A atividade biológica encontrada nas frações que adsorveram à BS foi de 3,50UI/mL. Não foi encontrarda atividade biológica nas frações que não adsorveram à BS. A atividade biológica contida nas frações que adsorveram à BS e que também adsorveram a ConA foi de 3,65UI/mL. O rendimento do processo cromatográfico onde o plasma foi adsorvido pela BS e pela ConA, foi de 69,52%. Não foi encontrada atividade biológica nas frações obtidas da aplicação do plasma diafiltrado em coluna de ConA. O processo cromatográfico com uso de BS seguido de ConA mostou-se eficaz em purificar a eCG do plasma de éguas prenhes.

The equine Chorionic Gonadotrophin (eCG) is produced by the pregnant mare and has follicle-stimulant and luteinizing actions on non-equine domestic animals. A pool formed by the plasma of 4 pregnant mares (with mean gestation of 69 days) was purified in chromatographic column with Blue-Sepharose FF affinity resin (BS resin). Fractions adsorbed by BS resin were then purified in chromatographic column with Concavalin A 4B affinity resin (ConA resin). The fractions not adsorbed by the BS resin were also purified in chromatographic column with ConA resin. The same plasma pool was dialyzed in hemodialysis cartridge. The dialyzed was applied in chromatographic column with ConA resin. Biological activities (in IU/mL) of the plasma, of the dialyzed and of the purified fractions were quantified in a biological assay with female rats that did not reach puberty. The biological activities found in the plasma and dialyzed were of 3.63 and 5.14 IU/mL, respectively. Fractions that were adsorbed by BS had a biological activity of 3.50 IU/mL. No biological activity was found in fractions that were not adsorbed by BS. Biological activity found in fractions adsorbed by both BS and ConA was of 3.65 IU/mL. When plasma was both adsorbed by BS and ConA, the chromatographic process yield had results of 69.52%. No biological activity was found in the fractions obtained from the administration of dialyzed plasma in ConA column. The BS - followed by ConA -chromatographic process…

Advisors/Committee Members: Madureira, Ed Hoffmann.

Subjects/Keywords: Affinity chromatography; Chorionic gonadotrophin; Cromatografia de afinidade; Eqüines; Eqüinos; Gonadotrofina; Gonadotrofina coriônica; Gonadotrophin; Purificação; Purification

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APA (6^th Edition):

Rossa, L. A. F. (2009). Purificação da gonadotrofina coriônica eqüina, do plasma sanguíneo de éguas prenhes, por cromatografia de afinidade. (Doctoral Dissertation). University of São Paulo. Retrieved from http://www.teses.usp.br/teses/disponiveis/10/10131/tde-29072009-111402/ ;

Chicago Manual of Style (16^th Edition):

Rossa, Luis Augusto Ferreira. “Purificação da gonadotrofina coriônica eqüina, do plasma sanguíneo de éguas prenhes, por cromatografia de afinidade.” 2009. Doctoral Dissertation, University of São Paulo. Accessed January 18, 2021. http://www.teses.usp.br/teses/disponiveis/10/10131/tde-29072009-111402/ ;.

MLA Handbook (7^th Edition):

Rossa, Luis Augusto Ferreira. “Purificação da gonadotrofina coriônica eqüina, do plasma sanguíneo de éguas prenhes, por cromatografia de afinidade.” 2009. Web. 18 Jan 2021.

Vancouver:

Rossa LAF. Purificação da gonadotrofina coriônica eqüina, do plasma sanguíneo de éguas prenhes, por cromatografia de afinidade. [Internet] [Doctoral dissertation]. University of São Paulo; 2009. [cited 2021 Jan 18]. Available from: http://www.teses.usp.br/teses/disponiveis/10/10131/tde-29072009-111402/ ;.

Council of Science Editors:

Rossa LAF. Purificação da gonadotrofina coriônica eqüina, do plasma sanguíneo de éguas prenhes, por cromatografia de afinidade. [Doctoral Dissertation]. University of São Paulo; 2009. Available from: http://www.teses.usp.br/teses/disponiveis/10/10131/tde-29072009-111402/ ;

North Carolina State University

26. Gagnon, Keith Thomas Jr. Archaeal Box C/D sRNP Assembly, Structure and Function.

Degree: PhD, Biochemistry, 2007, North Carolina State University

URL: http://www.lib.ncsu.edu/resolver/1840.16/5947

Subjects/Keywords: affinity purification; RNA remodeling; archaeal; snoRNA

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Gagnon, K. T. J. (2007). Archaeal Box C/D sRNP Assembly, Structure and Function. (Doctoral Dissertation). North Carolina State University. Retrieved from http://www.lib.ncsu.edu/resolver/1840.16/5947

Chicago Manual of Style (16^th Edition):

Gagnon, Keith Thomas Jr. “Archaeal Box C/D sRNP Assembly, Structure and Function.” 2007. Doctoral Dissertation, North Carolina State University. Accessed January 18, 2021. http://www.lib.ncsu.edu/resolver/1840.16/5947.

MLA Handbook (7^th Edition):

Gagnon, Keith Thomas Jr. “Archaeal Box C/D sRNP Assembly, Structure and Function.” 2007. Web. 18 Jan 2021.

Vancouver:

Gagnon KTJ. Archaeal Box C/D sRNP Assembly, Structure and Function. [Internet] [Doctoral dissertation]. North Carolina State University; 2007. [cited 2021 Jan 18]. Available from: http://www.lib.ncsu.edu/resolver/1840.16/5947.

Council of Science Editors:

Gagnon KTJ. Archaeal Box C/D sRNP Assembly, Structure and Function. [Doctoral Dissertation]. North Carolina State University; 2007. Available from: http://www.lib.ncsu.edu/resolver/1840.16/5947

University of Delaware

27. Swartz, Andrew R. Functionalized nanoparticle crosslinking for enhanced affinity precipitation of monoclonal antibodies.

Degree: PhD, University of Delaware, Department of Chemical and Biomolecular Engineering, 2018, University of Delaware

URL: http://udspace.udel.edu/handle/19716/23945

► Recent advances in upstream therapeutic monoclonal antibody (mAb) production have posed significant challenges to the downstream purification platform. Protein A affinity chromatography has been identified… (more)

▼ Recent advances in upstream therapeutic monoclonal antibody (mAb) production have posed significant challenges to the downstream purification platform. Protein A affinity chromatography has been identified as a potential process bottleneck due to limitations in throughput, scale-up, and cost. Affinity precipitation is a promising non-chromatographic alternative because it combines the selectivity of an affinity ligand with the operational benefits of conventional precipitation. After binding to a target protein in solution, phase transition is induced through crosslinking with multivalent affinity ligands (primary effect; multimeric protein only) or using stimuli-responsive affinity ligands (secondary effect; monomer or multimer). Primary effect affinity precipitation is not widely used due to low precipitation yields, but there are several examples of mAb purification using protein A ligands conjugated to a responsive domain of synthetic or natural origin. ☐ Our group has demonstrated the feasibility of using a recombinant antibody binding domain (Z-domain) genetically fused to a temperature responsive elastin-like polypeptide (ELP) biopolymer (Z-ELP) for mAb capture and precipitation. However, the high temperature and salt concentrations and the heating/cooling steps required for reversible Z-ELP phase transition impacted antibody stability and operational efficiency. We proposed a two-part solution: Increase the affinity ligand multivalency and dimension by scaffolding Z-ELP to a nanoparticle using site-specific conjugation. We hypothesized that this would enable primary effect mAb-nanoparticle crosslinking, while also improving ELP precipitation by enlarging the scale of aggregate formation. ☐ The first objective was to engineer and characterize the functionalization of Z-ELP to a self-assembled, thermostable E2 nanocage (Z-ELP-E2), derived from Bacillus stearothermophilus using Staphylococcal Sortase A bioconjugation. We demonstrated a significant decrease in ELP transition temperature and an increase in aggregate size upon antibody binding at isothermal solution conditions. We concluded this was due to multivalent antibody-Z-ELP-E2 interactions that nucleated into a crosslinked network. The second objective was to develop an affinity precipitation process capable of purifying industrial monoclonal antibodies at ambient temperature with minimal added salt. We discovered that the nanoparticles rapidly aggregated upon mixing with mAb cell culture harvests due to multivalent crosslinking into large, insoluble particles. After optimization of key process steps, the affinity precipitation mAb yield and impurity clearance was found to be comparable to Protein A chromatography performance. ☐ The third objective of the research was to establish a platform that can be applied to any target mAb or Fc-containing protein with minimal optimization of process parameters. Z-ELP-E2 affinity precipitation was evaluated using four industrial mAbs (mAbs A–D) and one Fc fusion protein (Fc A) with different molecular properties.… Advisors/Committee Members: Chen, Wilfred.

Subjects/Keywords: Applied sciences; Affinity precipitation; Crosslinking; Elastin-like polypeptide; Monoclonal antibody; Nanoparticle; Purification

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Swartz, A. R. (2018). Functionalized nanoparticle crosslinking for enhanced affinity precipitation of monoclonal antibodies. (Doctoral Dissertation). University of Delaware. Retrieved from http://udspace.udel.edu/handle/19716/23945

Chicago Manual of Style (16^th Edition):

Swartz, Andrew R. “Functionalized nanoparticle crosslinking for enhanced affinity precipitation of monoclonal antibodies.” 2018. Doctoral Dissertation, University of Delaware. Accessed January 18, 2021. http://udspace.udel.edu/handle/19716/23945.

MLA Handbook (7^th Edition):

Swartz, Andrew R. “Functionalized nanoparticle crosslinking for enhanced affinity precipitation of monoclonal antibodies.” 2018. Web. 18 Jan 2021.

Vancouver:

Swartz AR. Functionalized nanoparticle crosslinking for enhanced affinity precipitation of monoclonal antibodies. [Internet] [Doctoral dissertation]. University of Delaware; 2018. [cited 2021 Jan 18]. Available from: http://udspace.udel.edu/handle/19716/23945.

Council of Science Editors:

Swartz AR. Functionalized nanoparticle crosslinking for enhanced affinity precipitation of monoclonal antibodies. [Doctoral Dissertation]. University of Delaware; 2018. Available from: http://udspace.udel.edu/handle/19716/23945

KTH

28. Larsson, Emma. Calcium-dependent affinity ligands for protein purification.

Degree: Biotechnology and Health (CBH), 2020, KTH

URL: http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-278695

►

The rapid growth of the biopharmaceutical industry has led to increasing demands on the protein production process. An important aspect is the yield of… (more)

▼

The rapid growth of the biopharmaceutical industry has led to increasing demands on the protein production process. An important aspect is the yield of functional protein, which can be greatly affected by the choice of downstream purification. Purification based on acidic elution can be an issue for pH-sensitive proteins, since dramatic changes in pH can lead to protein aggregation and loss of function. The harsh, acidic elution conditions used in conventional purification of antibodies by Protein A affinity chromatography can thus be problematic. To address this, a calcium-dependent protein domain, called ZCa, has previously been developed for mild purification of antibodies, with elution close to physiological pH. Presented here are engineered variants of ZCa with novel affinity towards other biotherapeutics, which could also benefit from mild purification. Phage display selection, using a ZCa-based library, was applied to isolate promising ZCa-based binders against antigen-binding fragments, tissue plasminogen activator, and granulocyte colony stimulating factor, yet to be characterized. Additionally, three ZCa-based variants from a previous selection, with affinity for single-chain variable fragments (scFvs), have been identified and characterized. In a purification setup, they were shown to elute the scFv protein at neutral pH in a calcium-dependent manner. The reported results demonstrate that novel affinity can be introduced to the ZCa domain, while maintaining the calcium-dependent behavior that enables gentle purification. This offers a strategy for broadening the range of proteins that can be purified under mild conditions, with the benefit of reducing protein aggregation and thus increasing the yield of functional protein.

Den snabba tillväxten inom bioläkemedelsindustrin har lett till ökade krav på processen för proteinproduktion. En viktig aspekt är utbytet av funktionellt protein, där valet av reningsmetod kan ha stor påverkan. Proteinrening med syraeluering kan utgöra ett problem för pH-känsliga proteiner, då stora förändringar i pH kan leda till aggregering och försämrad funktionalitet. Det låga pH som används för eluering i den traditionella reningen av antikroppar med Protein A-baserad affinitetskromatografi kan därmed vara problematiskt. Som ett svar på detta har en kalciumberoende proteindomän, vid namn ZCa, tidigare utvecklats för mild rening av antikroppar med eluering nära fysiologiskt pH. I det här arbetet presenteras nya varianter av ZCa som modifierats för att binda till andra bioläkemedel, vilka också skulle kunna gynnas av mild proteinrening. Fagdisplay av ett ZCa-baserat bibliotek har applicerats för att isolera lovande ZCa-baserade bindare mot antikroppsfragment (Fab), vävnadsplasminogenaktivator och granulocytkolonistimulerande faktor, vilka ännu inte karaktäriserats. Utöver detta identifierades och karaktäriserades tre ZCa-baserade varianter från en tidigare selektion, med affinitet för enkelkedjiga antikroppsfragment (scFv). Då dessa varianter utvärderades för rening…

Subjects/Keywords: Purification; Affinity chromatography; Protein A; Z domain; Calcium-dependent; ZCa; Elution; Medical Biotechnology; Medicinsk bioteknologi

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APA (6^th Edition):

Larsson, E. (2020). Calcium-dependent affinity ligands for protein purification. (Thesis). KTH. Retrieved from http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-278695

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Larsson, Emma. “Calcium-dependent affinity ligands for protein purification.” 2020. Thesis, KTH. Accessed January 18, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-278695.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Larsson, Emma. “Calcium-dependent affinity ligands for protein purification.” 2020. Web. 18 Jan 2021.

Vancouver:

Larsson E. Calcium-dependent affinity ligands for protein purification. [Internet] [Thesis]. KTH; 2020. [cited 2021 Jan 18]. Available from: http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-278695.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Larsson E. Calcium-dependent affinity ligands for protein purification. [Thesis]. KTH; 2020. Available from: http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-278695

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of Toronto

29. Kean, Michelle. Structure-function Analysis of the STRIPAK Complex.

Degree: 2013, University of Toronto

URL: http://hdl.handle.net/1807/68949

►

Reversible phosphorylation events regulate critical aspects of biology. Kinases and phosphatases interact not only with their substrates but also with regulatory subunits and scaffolds. In… (more)

▼

Reversible phosphorylation events regulate critical aspects of biology. Kinases and phosphatases interact not only with their substrates but also with regulatory subunits and scaffolds. In recent years, affinity purification coupled to mass spectrometry (AP-MS) has proven to be a powerful tool to identify protein-protein interactions (PPIs) involving kinases and phosphatases. Using AP-MS we discovered a novel-signaling complex, which we termed STRIPAK (for STRiatin Interacting Phosphatase and Kinase). The STRIPAK complex contains a protein phosphatase 2A (PP2A) holoenzyme and a family of Ste20 kinases (the GCK-III kinases), as well as the protein MOB4, a member of the MOB family of proteins, which are known kinase regulators. Interestingly the protein CCM3, one of three proteins mutated in familial cases of cerebral cavernous malformations (CCMs), was also found to reside within the STRIPAK complex. My project focused on the structural and functional characterization of STRIPAK. I mapped direct interactions and interacting regions on these proteins and determined that the PP2A substrate binding subunit (striatin) and CCM3 act as adapter molecules to bridge the kinase and phosphatase subunits of STRIPAK. I also characterized the functional role for STRIPAK components in Golgi positioning, and demonstrated that depletion of CCM3 or striatin affects GCK-III kinase and Golgi polarization in an opposite manner. Further AP-MS experiments revealed that CCM3 and the GCK-III kinases reside within at least two distinct protein complexes, either with STRIPAK or with the protein CCM2, which is also mutated in familial cases of CCM disease, and that CCM3 is found in complex with CCM1 and CCM2, thus revealing a possible common pathogenesis for CCM proteins in CCM disease. To gain insight into the role of MOB4 within STRIPAK, I characterized the entire MOB family and their known binding partners, the NDR/LATS kinases, using AP-MS. These studies identified many new MOB and kinase interactors, including a family of guanine nucleotide exchange factors and protein phosphatase 6 (PP6).

PhD

Advisors/Committee Members: Gingras, Anne-Claude, Sicheri, Frank, Molecular and Medical Genetics.

Subjects/Keywords: STRIPAK; CCM3; Cerebral Cavernous Malformations; PP2A; MOB proteins; Affinity Purification-Mass Spectrometry; 0307

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APA (6^th Edition):

Kean, M. (2013). Structure-function Analysis of the STRIPAK Complex. (Doctoral Dissertation). University of Toronto. Retrieved from http://hdl.handle.net/1807/68949

Chicago Manual of Style (16^th Edition):

Kean, Michelle. “Structure-function Analysis of the STRIPAK Complex.” 2013. Doctoral Dissertation, University of Toronto. Accessed January 18, 2021. http://hdl.handle.net/1807/68949.

MLA Handbook (7^th Edition):

Kean, Michelle. “Structure-function Analysis of the STRIPAK Complex.” 2013. Web. 18 Jan 2021.

Vancouver:

Kean M. Structure-function Analysis of the STRIPAK Complex. [Internet] [Doctoral dissertation]. University of Toronto; 2013. [cited 2021 Jan 18]. Available from: http://hdl.handle.net/1807/68949.

Council of Science Editors:

Kean M. Structure-function Analysis of the STRIPAK Complex. [Doctoral Dissertation]. University of Toronto; 2013. Available from: http://hdl.handle.net/1807/68949

University of Iowa

30. Molinarolo, Steven. Biochemical techniques for the study of voltage-gated sodium channel auxiliary subunits.

Degree: PhD, Molecular Physiology and Biophysics, 2018, University of Iowa

URL: https://ir.uiowa.edu/etd/6217

► Voltage-gated sodium channels auxiliary subunits evolutionary emerged nearly 500 million years ago during the Cambrian explosion. These subunits alter one the most important ion… (more)

Subjects/Keywords: Co-affinity purification; Ion channels; Protein-protein interaction; Voltage-gated sodium channel auxiliary subunits; Biophysics

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Molinarolo, S. (2018). Biochemical techniques for the study of voltage-gated sodium channel auxiliary subunits. (Doctoral Dissertation). University of Iowa. Retrieved from https://ir.uiowa.edu/etd/6217

Chicago Manual of Style (16^th Edition):

Molinarolo, Steven. “Biochemical techniques for the study of voltage-gated sodium channel auxiliary subunits.” 2018. Doctoral Dissertation, University of Iowa. Accessed January 18, 2021. https://ir.uiowa.edu/etd/6217.

MLA Handbook (7^th Edition):

Molinarolo, Steven. “Biochemical techniques for the study of voltage-gated sodium channel auxiliary subunits.” 2018. Web. 18 Jan 2021.

Vancouver:

Molinarolo S. Biochemical techniques for the study of voltage-gated sodium channel auxiliary subunits. [Internet] [Doctoral dissertation]. University of Iowa; 2018. [cited 2021 Jan 18]. Available from: https://ir.uiowa.edu/etd/6217.

Council of Science Editors:

Molinarolo S. Biochemical techniques for the study of voltage-gated sodium channel auxiliary subunits. [Doctoral Dissertation]. University of Iowa; 2018. Available from: https://ir.uiowa.edu/etd/6217

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Open Access Theses and Dissertations