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University of Southern California

1. Ma, Tao. Expression and purification of different elastin like polypeptides (ELPs) constructs for therapeutic applications.

Degree: MS, Pharmaceutical Sciences, 2015, University of Southern California

There are many rationales for modifying recombinant proteins by using polymers, which include: i) altering permeability/diffusion via controlling their hydrodynamic radius; ii) modulating mediators of controlled release; and iii) improving their avidity to molecular/cellular targets. Many of these strategies require covalent bioconjugation, which are inefficient and may compromise protein activity. An emerging solution to this challenge is to develop gene products that act like polymers. One such polymer is known as the Elastin Like Polypeptides (ELPs). ELPs are ‘protein polymers’ composed of repetitive amino acid sequence derived from the human extracellular matrix protein called tropoelastin. In addition to their high molecular weight, which exceeds 80 kDa, ELPs undergo thermo‐responsive phase separation. ELPs are highly soluble in aqueous solutions below their transition temperature (Tt) and rapidly self‐assemble into viscous coacervate particles above Tt. Tt can be modulated by changing the molecular weight and hydrophobicity of the ELP. In this thesis, ELPs are developed as a carrier for lacritin, which is a novel tear glycoprotein that promotes tear secretion, maintains ocular surface integrity and reduces formation of lymphocytic foci in lacrimal gland. Free lacritin increases the basal tear secretion in rabbits; however, it must be administered frequently. The rationale for exploring fusions of lacritin and ELP is that they may prolong the retention on the anterior segment of the eye. In order to explore this strategy, my thesis has focused on scaling‐up the production of high purity, low endotoxin level material for subsequent evaluation treating in models of aqueous‐deficient dry eye diseases. E. coli was used for expression of the lacritin‐ELP fusion construct and size exclusion chromatography (SEC) was used to separate LV96 from cleavage product. Endotoxin removal was explored using polymyxin chromatography. This thesis represents the first report of scaling‐up production of low endotoxin level lacritin ELP fusions. Advisors/Committee Members: Hamm-Alvarez, Sarah F. (Committee Chair), Mackay, John Andrew (Committee Member), Shen, Wei-Chiang (Committee Member).

Subjects/Keywords: elastin like polypeptides; ELPs; lacritin; protein expression; scaling-up of protein purification; endotoxin removal

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

APA (6th Edition):

Ma, T. (2015). Expression and purification of different elastin like polypeptides (ELPs) constructs for therapeutic applications. (Masters Thesis). University of Southern California. Retrieved from http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll3/id/592829/rec/2685

Chicago Manual of Style (16th Edition):

Ma, Tao. “Expression and purification of different elastin like polypeptides (ELPs) constructs for therapeutic applications.” 2015. Masters Thesis, University of Southern California. Accessed December 12, 2019. http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll3/id/592829/rec/2685.

MLA Handbook (7th Edition):

Ma, Tao. “Expression and purification of different elastin like polypeptides (ELPs) constructs for therapeutic applications.” 2015. Web. 12 Dec 2019.

Vancouver:

Ma T. Expression and purification of different elastin like polypeptides (ELPs) constructs for therapeutic applications. [Internet] [Masters thesis]. University of Southern California; 2015. [cited 2019 Dec 12]. Available from: http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll3/id/592829/rec/2685.

Council of Science Editors:

Ma T. Expression and purification of different elastin like polypeptides (ELPs) constructs for therapeutic applications. [Masters Thesis]. University of Southern California; 2015. Available from: http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll3/id/592829/rec/2685

2. Tang, Mingjie. SYNTHESIS AND CHARACTERIZATION OF pH-RESPONSIVE ELASTIN-LIKE POLYPEPTIDES WITH DIFFERENT CONFIGURATIONS.

Degree: MSin Chemical Engineering, Fenn College of Engineering, 2013, Cleveland State University

Elastin-like polypeptides (ELP) are environmentally responsive polymers that exhibit phase transition behavior in response to various stimuli such as temperature, pH and irradiation. In this work, we focused on pH-responsive ELPs. It has been shown that pH value, configurations of ELPs, ELP concentration, and salt concentration can impact the phase transition behavior of pH-responsive ELPs. Quantitative models have been developed to engineer various linear ELPs, but there has not been any detailed study for engineering ionizable ELP with non-linear configurations.In this study, we designed, synthesized, and characterized pH-responsive ELPs with two different configurations. One is linear ELP, (GVGVPGEGVPGVGVP)12, and the other one is a three-armed star ELP, (GVGVPGEGVPGVGVP)12-foldon. Three ELP-foldon chains fold as a trimer in solutions. These constructs were synthesized by molecular biology techniques and characterized by different methods.A modified model based on previous studies was used to describe the pH-dependent reversible phase transition in response to their solution environment. The model was validated with pH-responsive ELPs with different configurations, i.e., linear and trimer. The phase transition of both ELPs fit this model across a range of pH. These results demonstrate that we are able to rationally design responsive polypeptides with different configurations whose transition can be triggered at a specified pH. Advisors/Committee Members: Holland, Nolan (Advisor).

Subjects/Keywords: Biomedical Engineering; Chemical Engineering; pH-responsive, ELPs

…LIST OF FIGURES Figure 1.1 Mechnisms of phase transition behaviour of ELPs… …amino acids except proline [4]. ELPs exhibit an inverse temperature transition… …ELPs, several studies have been done to explain the mechanism of this unique behavior… …differences in interactions of the water solvent and residues of ELPs. It can be used as a measure… …advantages of ELPs is the inverse temperature transition, as described above. The responsive… 

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

APA (6th Edition):

Tang, M. (2013). SYNTHESIS AND CHARACTERIZATION OF pH-RESPONSIVE ELASTIN-LIKE POLYPEPTIDES WITH DIFFERENT CONFIGURATIONS. (Masters Thesis). Cleveland State University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=csu1376784321

Chicago Manual of Style (16th Edition):

Tang, Mingjie. “SYNTHESIS AND CHARACTERIZATION OF pH-RESPONSIVE ELASTIN-LIKE POLYPEPTIDES WITH DIFFERENT CONFIGURATIONS.” 2013. Masters Thesis, Cleveland State University. Accessed December 12, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=csu1376784321.

MLA Handbook (7th Edition):

Tang, Mingjie. “SYNTHESIS AND CHARACTERIZATION OF pH-RESPONSIVE ELASTIN-LIKE POLYPEPTIDES WITH DIFFERENT CONFIGURATIONS.” 2013. Web. 12 Dec 2019.

Vancouver:

Tang M. SYNTHESIS AND CHARACTERIZATION OF pH-RESPONSIVE ELASTIN-LIKE POLYPEPTIDES WITH DIFFERENT CONFIGURATIONS. [Internet] [Masters thesis]. Cleveland State University; 2013. [cited 2019 Dec 12]. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=csu1376784321.

Council of Science Editors:

Tang M. SYNTHESIS AND CHARACTERIZATION OF pH-RESPONSIVE ELASTIN-LIKE POLYPEPTIDES WITH DIFFERENT CONFIGURATIONS. [Masters Thesis]. Cleveland State University; 2013. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=csu1376784321

3. Kherb, Jaibir. Specific Cation Effects in Biological Systems: Thermodynamic and Spectroscopic Insights.

Degree: 2012, Texas A&M University

Very specific protein-salt interactions are involved in a multitude of biological phenomena such as protein folding/stability, enzymatic activity, and signal transduction events. In this work, we used two very simple, protein-mimic model biopolymers to obtain a better understanding of specific cation effects operating in aqueous protein environments. The two biopolymers used were Elastin-like Polypeptides (ELPs) and poly(N-isopropylacrylamide) (PNIPAM). ELPs are an especially an ideal model system as these polypeptides can be easily genetically engineered to observe the effect of specific amino acid residues and peptide chain length on these salt interactions. Both of these biopolymers are also highly thermoresponsive as their aqueous solutions undergo a hydrophobic collapse/aggregation induced phase transition process above a lower critical solution temperature (LCST). Thermodynamic measurements of these biopolymers were carried out under various salt solution conditions. Additionally, both of these biopolymers are suitable for making surface specific spectroscopic measurements. Vibrational sum frequeny spectroscopy (VSFS), a non-linear interface sensitive spectroscopic technique, was employed here to investigate biologically relevant cation interactions which occur at peptide/protein surfaces. First, the LCST response of a non-polar ELP and a neutral biopolymer, PNIPAM, was investigated in the presence of 12 different alkali, alkaline-earth metal and transition metal chloride salts. Even though the salt interactions for uncharged proteins are dominated by anions, subtle specific cation effects were also observed. The results followed a direct Hofmeister series for cations. Most alkali cations are excluded from the polar amide regions of proteins. More polarizable cations, however, can solvate the hydrophobic moieties and somewhat counter the salting-out effect of the chloride anion. More charged and hydrated ions like lithium and divalent cations showed a weak interaction to the amide moiety through their hydration shell. The role of acidic amino acid residues in inducing cation specificities was investigated using an aspartate-rich ELP system. Both thermodynamic and spectroscopic data conclusively proved that the negative charge on protein surfaces is the main driving force for cation partitioning and specificity under physiological relevant concentration regimes. Apparent binding constants of carboxylate moieties with cations were determined. This is the first quantitative and thoroughly systematic study of such biologically relevant cation-carboxylate interactions prevalent in enzyme active sites and protein surfaces. Advisors/Committee Members: Cremer, Paul S. (advisor), Russell, David H. (committee member), Barondeau, David P. (committee member), Scholtz, J. M. (committee member).

Subjects/Keywords: Cation Hofmeister Series; Vibrational Sum Frequency Spectroscopy; Phase Transition Temperature Data of PNIPAM/ELPs.

…employed in this study. a) PNIPAM. b) ELPs. The orange oval shows the location of guest… …PNIPAM and ELPs, both of which can mimic the real proteins in their structural and chemical… …under a wide variety of experimental conditions. 5 Elastin-like polypeptides (ELPs… …ELPs of varying degree of chain length, hydrophobicity, polarity and structure.29 All… …synthesized ELPs have a –SKGPG leader sequence on the N-terminal and a –WP trailer sequence on the C… 

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

APA (6th Edition):

Kherb, J. (2012). Specific Cation Effects in Biological Systems: Thermodynamic and Spectroscopic Insights. (Thesis). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/ETD-TAMU-2011-12-10443

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):

Kherb, Jaibir. “Specific Cation Effects in Biological Systems: Thermodynamic and Spectroscopic Insights.” 2012. Thesis, Texas A&M University. Accessed December 12, 2019. http://hdl.handle.net/1969.1/ETD-TAMU-2011-12-10443.

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

MLA Handbook (7th Edition):

Kherb, Jaibir. “Specific Cation Effects in Biological Systems: Thermodynamic and Spectroscopic Insights.” 2012. Web. 12 Dec 2019.

Vancouver:

Kherb J. Specific Cation Effects in Biological Systems: Thermodynamic and Spectroscopic Insights. [Internet] [Thesis]. Texas A&M University; 2012. [cited 2019 Dec 12]. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2011-12-10443.

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

Council of Science Editors:

Kherb J. Specific Cation Effects in Biological Systems: Thermodynamic and Spectroscopic Insights. [Thesis]. Texas A&M University; 2012. Available from: http://hdl.handle.net/1969.1/ETD-TAMU-2011-12-10443

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

.