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Miami University
1.
New, Christopher Paul.
Analysis of Tha4 Function and Organization in Chloroplast
Twin Arginine Transport.
Degree: PhD, Cell, Molecular and Structural Biology
(CMSB), 2020, Miami University
URL: http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538 
► The chloroplast Twin Arginine Translocase (cpTAT) system transports fully folded proteins across the thylakoid membrane in plant cells using only energy derived from the proton…
(more)
▼ The chloroplast Twin
Arginine Translocase (cpTAT)
system transports fully folded proteins across the thylakoid
membrane in plant cells using only energy derived from the proton
motive force (PMF). Three membrane bound component proteins:
cpTatC, Hcf106, and Tha4 function together in a transient manner to
accomplish transport. However, clear mechanistic details of this
process remain elusive such as how cpTAT utilizes energy stored in
the PMF or how the individual component proteins interact during
each step of transport. In addition, prior structural
characterization of (cp)TAT proteins used truncated versions of the
components. This dissertation describes work to develop methods to
purify full-length Hcf106 for biophysical characterization.
Additionally, this dissertation details the work to determine the
function of a membrane embedded glutamate in the Tha4 transmembrane
helix (TMH).A series of purification trials were carried out to
isolate Hcf106 fused to maltose binding protein (MBP) by the
recognition sequence of tobacco etch virus protease (TEVp). Fusion
protein and protease were expressed in and purified from E. coli
using
affinity chromatography. Multiple parameters and additives
were tested during optimization of TEVp proteolysis reactions with
MBP-Hcf106. TEVp and free MBP were separated from un-cleaved
MBP-Hcf106 and free Hcf106 by
affinity and size exclusion
chromatography. Although TEVp and free MBP were removed after an
optimized proteolysis reaction, free Hcf106 showed its recalcitrant
nature through resistance of separation from un-cleaved MBP-Hcf106
by size exclusion chromatography in several detergent and buffer
conditions.To better understand the role of the membrane embedded
Tha4 glutamate 10 (E10), Tha4 variants with glutamate to alanine
(E10A) or glutamate to aspartate (E10D) substitutions were used to
complement loss of cpTAT function in thylakoid membranes.
Sequential glutamate substitutions in the TMH of Tha4 variant E10A
were unable to restore transport while aspartate substitutions were
mildly able to complement loss of function. Furthermore,
organization between three structural regions in Tha4 E10/A/D
variants was determined by disulfide crosslinking during various
transport conditions. Tha4 E10/A/D variant oligomer formation was
enhanced in the presence of functional precursor with and without
PMF present. An increase in TMH hydrophobicity by alanine
substitution was shown to increase Tha4 stability in isolated
thylakoid membranes and to promote tighter packing interactions
between adjacent Tha4 monomers. The interaction data was then used
to develop a model of how Tha4 E10/A/D variant tetramers pack and
reorganize in the presence of precursor.
Advisors/Committee Members: Dabney-Smith, Carole (Advisor), Page, Rick (Committee Chair).
Subjects/Keywords: Biochemistry; Cellular Biology; Plant Biology; Molecular Biology; chloroplast twin arginine transport; protein transport; cpTAT; TAT; Tha4; Hcf106; protein purification; maltose binding protein affinity chromatography; oligomer formation; complementation; transmembrane domain hydrophobicity
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APA (6th Edition):
New, C. P. (2020). Analysis of Tha4 Function and Organization in Chloroplast
Twin Arginine Transport. (Doctoral Dissertation). Miami University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538
Chicago Manual of Style (16th Edition):
New, Christopher Paul. “Analysis of Tha4 Function and Organization in Chloroplast
Twin Arginine Transport.” 2020. Doctoral Dissertation, Miami University. Accessed January 18, 2021.
http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538.
MLA Handbook (7th Edition):
New, Christopher Paul. “Analysis of Tha4 Function and Organization in Chloroplast
Twin Arginine Transport.” 2020. Web. 18 Jan 2021.
Vancouver:
New CP. Analysis of Tha4 Function and Organization in Chloroplast
Twin Arginine Transport. [Internet] [Doctoral dissertation]. Miami University; 2020. [cited 2021 Jan 18].
Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538.
Council of Science Editors:
New CP. Analysis of Tha4 Function and Organization in Chloroplast
Twin Arginine Transport. [Doctoral Dissertation]. Miami University; 2020. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538
2.
Chenette, Heather.
High-Productivity Membrane Adsorbers: Polymer Surface-Modification Studies for Ion-Exchange and Affinity Bioseparations.
Degree: PhD, Chemical Engineering, 2014, Clemson University
URL: https://tigerprints.clemson.edu/all_dissertations/1319
► This Dissertation centers on the surface-modification of macroporous membranes to make them selective adsorbers for different proteins, and the analysis of the performance of…
(more)
▼ This Dissertation centers on the surface-modification of macroporous membranes to make them selective adsorbers for different proteins, and the analysis of the performance of these membranes relative to existing technology. Traditional chromatographic separations for the isolation and purification of proteins implement a column packed with resin beads or gel media that contain specific binding ligands on their exposed surface area. The productivity of this process is balanced by the effective use of the binding sites within the column and the speed at which the separation can take place, in addition to the need to maintain sufficient protein purity and bioactivity. Because of the nature of the densely packed columns and, in the case of resin columns, the limited access to the binding sites internally located within the resin, the operating speed of this separation process may be constrained by mass-transfer and pressure limitations. Other constraints include the time-intensive measures taken to properly pack the columns, and the challenges associated with scaling chromatography columns to industrial-sized processes. Because of the excellent selectivity, chromatography processes are the workhorse for biopharmaceuticals drugs, and other plant- and animal- based protein products. Thus, there are many markets that could benefit by improvements to this technology. My strategy focused on modifying porous membranes with surface-initiated atom transfer radical polymerization (ATRP) to grow polymer chains containing functional groups that target three different protein-ligand interactions for three different types of chromatography: cation-exchange, carbohydrate
affinity, and
Arginine-specific
affinity chromatography. Although each of these types of separation has different challenges and different possibilities for impact among their unique applications, they all have the common need for a stationary phase platform with the potential for fast separations and specific interactions. The common approach used in these studies, which is using membrane technology for chromatographic applications and using ATRP as a surface modification technique, will be introduced and supported by a brief review in Chapter 1. The specific approaches to address the unique challenges and motivations of each study system are given in the introduction sections of the respective Dissertation chapters. Chapter 2 describes my work to develop cation-exchange membranes. I discuss the polymer growth kinetics and characterization of the membrane surface. I also present an analysis of productivity, which measures the mass of protein that can bind to the stationary phase per volume of stationary phase adsorbing material per time. Surprisingly and despite its importance, this performance measure was not described in previous literature. Because of the significantly shorter residence time necessary for binding to occur, the productivity of these cation-exchange membrane adsorbers (300 mg/mL/min) is nearly two orders of magnitude higher than the productivity…
Advisors/Committee Members: Dr. Scott M. Husson, Dr. Charles Gooding, Dr. Douglas E. Hirt, Dr. Igor Luzinov.
Subjects/Keywords: arginine affinity; biotherapeutics; chromatography; concanavalin A; membrane adsorber; protein purfication; Chemical Engineering; Materials Science and Engineering; Polymer Science
…83
4. AFFINITY MEMBRANE ADSORBERS FOR BINDING
ARGININE-RICH PROTEINS… …have a strong affinity for Arg-rich proteins
is the “Arginine tweezer” formed by polymeric… …affinity chromatography medium is used for protein
capture, when the primary objective is to… …affinity resins are used as the selective material,
with a cost that accounts for over half of… …non-affinity chromatography media, Protein A resins cost
almost an order of magnitude more…
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APA ·
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APA (6th Edition):
Chenette, H. (2014). High-Productivity Membrane Adsorbers: Polymer Surface-Modification Studies for Ion-Exchange and Affinity Bioseparations. (Doctoral Dissertation). Clemson University. Retrieved from https://tigerprints.clemson.edu/all_dissertations/1319
Chicago Manual of Style (16th Edition):
Chenette, Heather. “High-Productivity Membrane Adsorbers: Polymer Surface-Modification Studies for Ion-Exchange and Affinity Bioseparations.” 2014. Doctoral Dissertation, Clemson University. Accessed January 18, 2021.
https://tigerprints.clemson.edu/all_dissertations/1319.
MLA Handbook (7th Edition):
Chenette, Heather. “High-Productivity Membrane Adsorbers: Polymer Surface-Modification Studies for Ion-Exchange and Affinity Bioseparations.” 2014. Web. 18 Jan 2021.
Vancouver:
Chenette H. High-Productivity Membrane Adsorbers: Polymer Surface-Modification Studies for Ion-Exchange and Affinity Bioseparations. [Internet] [Doctoral dissertation]. Clemson University; 2014. [cited 2021 Jan 18].
Available from: https://tigerprints.clemson.edu/all_dissertations/1319.
Council of Science Editors:
Chenette H. High-Productivity Membrane Adsorbers: Polymer Surface-Modification Studies for Ion-Exchange and Affinity Bioseparations. [Doctoral Dissertation]. Clemson University; 2014. Available from: https://tigerprints.clemson.edu/all_dissertations/1319
3.
Ferreira, Soraia Nunes.
Desenvolvimento de novos suportes cromatográficos para purificação do DNA plasmídeo.
Degree: 2014, RCAAP
URL: https://www.rcaap.pt/detail.jsp?id=oai:ubibliorum.ubi.pt:10400.6/6098
► Nos últimos anos, a aplicação de DNA plasmídeo (pDNA) em terapia génica ou vacinas de DNA tem-se revelado uma ferramenta com potencial aplicação no tratamento…
(more)
▼ Nos últimos anos, a aplicação de DNA plasmídeo (pDNA) em terapia génica ou vacinas de DNA
tem-se revelado uma ferramenta com potencial aplicação no tratamento de inúmeras
doenças. Uma vez que esta abordagem recorre a vetores não-virais, é mais segura em termos
de ativação de oncogenes e apresenta uma diminuição de reações do sistema imunitário.
Deste modo, a indústria farmacêutica procura obter pDNA em maiores quantidades e isento
de contaminantes (RNA, DNA genómico, endotoxinas, proteínas). Uma técnica que permite
purificar o pDNA é a cromatografia de afinidade (AC). Esta metodologia baseia-se em
interações específicas entre ligandos e o pDNA, permitindo a eliminação de alguns passos de
purificação. A utilização de aminoácidos como ligandos de afinidade revelou ser uma
abordagem promissora para purificar ácidos nucleicos, pois baseia-se na bioespecificidade
aminoácido-nucleótido. A L-histidina e a L-arginina permitiram isolar a isoforma
superenrolada (sc) do pDNA, indicando a presença de interações específicas entre o pDNA e os
aminoácidos. Neste trabalho são apresentados os resultados de três ligandos, a L-tirosina e os
dipeptídeos L-tirosina-L-tirosina e L-tirosina-L-arginina. Os dipeptídeos foram sintetizados de
acordo com o método clássico de síntese química, que envolve a proteção do grupo amina e
do grupo carboxílico de cada aminoácido, e posterior formação da ligação peptídica e por fim
desproteção usando TFA (ácido trifluoracético). De seguida, os ligandos foram imobilizados na
Sepharose CL-6B através da ativação dos grupos epóxi com o reagente 1,4-butanediol
diglicidil éter. O suporte foi caracterizado por HR-MAS (Ressonância Magnética de Alta
Resolução de Ângulo Mágico) RMN. A técnica de STD-RMN (Diferença da Transferência de
saturação por ressonância magnética nuclear) permitiu identificar os grupos preferenciais dos
5´-mononucleótidos que interagem com os suportes Sepharose-L-tirosina, Sepharose-Ltirosina-
L-tirosina e Sepharose-L-tirosina-L-arginina. Utilizando a técnica de RPS (Ressonância
Plasmónica de Superfície), foi analisada a afinidade de cada um dos ligandos em estudo em
relação a cada uma das isoformas de pVAX1-LacZ a diferentes temperaturas (T=10°C e 25°C)
e usando diferentes tampões (Tris-HCl 10 mM e Hepes ácido 10 mM). Desta forma, a utilização
das técnicas STD-RMN e SPR permitiu fazer o screening das interações dos suportes-5’-
mononucleótidos e determinar a afinidade do complexo ligando: pDNA, para futura aplicação
em AC.
In the last years, the use of plasmid DNA (pDNA) in gene therapy or DNA vaccines has been
proved to be a tool with potential application in the treatment of diseases. Since this
approach uses non-viral vectors, it is safer in terms of oncogene activation and it presents a
decrease of the immune system response.
In this way, the pharmaceutical industry needs to obtain pDNA in larger quantities and free of
contaminants (RNA, genomic DNA, endotoxins, proteins). A technique that allows purifying
pDNA is affinity chromatography (AC). This method is based in specific…
Advisors/Committee Members: Cruz, Carla Patrícia Alves Freire Madeira, Sousa, Fani Pereira de.
Subjects/Keywords: Affinity Chromatography; L-Tyrosine; L-Tyrosine-L-Arginine; L-Tyrosine-L-Tyrosine; Plasmid Dna; Pvax1-Lacz; Spr; Std-Nmr; Domínio/Área Científica::Engenharia e Tecnologia:: Outras Engenharias e Tecnologias
Record Details
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APA ·
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MLA ·
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CSE |
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to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Ferreira, S. N. (2014). Desenvolvimento de novos suportes cromatográficos para purificação do DNA plasmídeo. (Thesis). RCAAP. Retrieved from https://www.rcaap.pt/detail.jsp?id=oai:ubibliorum.ubi.pt:10400.6/6098
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Ferreira, Soraia Nunes. “Desenvolvimento de novos suportes cromatográficos para purificação do DNA plasmídeo.” 2014. Thesis, RCAAP. Accessed January 18, 2021.
https://www.rcaap.pt/detail.jsp?id=oai:ubibliorum.ubi.pt:10400.6/6098.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Ferreira, Soraia Nunes. “Desenvolvimento de novos suportes cromatográficos para purificação do DNA plasmídeo.” 2014. Web. 18 Jan 2021.
Vancouver:
Ferreira SN. Desenvolvimento de novos suportes cromatográficos para purificação do DNA plasmídeo. [Internet] [Thesis]. RCAAP; 2014. [cited 2021 Jan 18].
Available from: https://www.rcaap.pt/detail.jsp?id=oai:ubibliorum.ubi.pt:10400.6/6098.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Ferreira SN. Desenvolvimento de novos suportes cromatográficos para purificação do DNA plasmídeo. [Thesis]. RCAAP; 2014. Available from: https://www.rcaap.pt/detail.jsp?id=oai:ubibliorum.ubi.pt:10400.6/6098
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of St. Andrews
4.
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)
▼ 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 fluorescent protein (CFP) tag or a tandem
affinity purification (TAP) tag. By infecting cells with the recombinant adenovirus expressing CFP tagged E1B55k, localisation of the E1B55k protein during infection could be studied. Generation of the recombinant viruses expressing TAP tagged E1B55k would allow purification of the E1B55k protein and any cellular or viral proteins that interacted with E1B55k. By infecting different cell types and harvesting at set time points during infection, the proteins interacting with E1B55k at different time points during infection could be identified. The E1B55k protein is modified by small ubiquitin modifying protein 1 (SUMO-1) at lysine residue 104 within the SUMO consensus sequence, and sumoylation can be abolished by mutating the lysine residue to an
arginine (Endter et al, 2001). Therefore in order to study the effect of SUMOylation of E1B55k, on adenovirus infection, CFP and TAP tagged recombinant viruses in which the E1B55k protein was mutated to contain an unmodificable
arginine at postion 104 were also generated.
Stable cell lines expressing TAP tagged wild type E1B55k, and E1B55k in which lysine residue 104 had been mutated to an
arginine were also generated as an alternative method to study the interacting proteins of E1B55k.
Four different isoforms of SUMO have been identified, named SUMO1-4. Stable cell lines expressing SUMO-2 were infected with wild type adenovirus type 5 and harvested at set time points in order to determine if any adenoviral proteins were modified by SUMO-2.
Advisors/Committee Members: Botting, Catherine (advisor), Hay, Ronald T (advisor).
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 ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th 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 (16th 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 (7th 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
. 
Open Access Theses and Dissertations
