This proposal addresses challenges in eukaryotic protein expression, solublization, stablization and crystallization. We will accomplish this by integrating early assessment measurements of protein quality and quantity into an existing robust mammalian cell expression platform. This approach integrates use of a novel high-throughput self-interaction chromatography system (HT-SIC) that rapidly measures second virial coefficients for the membrane protein mixed with a specially designed panel of additives. An artificial neural network analyzes the experimentally derived second virial coefficient data and performs in silico predictions of novel solution conditions that improve protein solubility and homogeneity. The HT-SIC system also enables informed adjustments to solution conditions that alter protein-protein interactions such that the probability of producing high-quality crystals is improved. Achieving the specific aims of this proposal will provide the research community with significant advancements toward a cost effective, knowledge-based approach to express, purify, stabilize and crystallize membrane proteins. Target proteins include two ion channel proteins (epithelial sodium channel, ENaC and cystic fibrosis transmembrane regulator protein, CFTR), two GPCRs (chemokine receptor-1, CCR1 and sphingosine- 1 phosphate receptor, S1P) and growth hormone receptor, GHR. Structures of these proteins would contribute significantly to our understanding of their biological mechanism of action and role in several important diseases including cancer (colon, breast and prostate), diabetes, cystic fibrosis, growth anomalies, immune system disorders, hypertension, sepsis and the flu. For each IMP, we have established collaborations with biochemists/biologists with a long-standing interest in and experience working with each target protein and its protein interactome.
This proposal is directed at the development of novel protocols to produce, purify, stabilize and crystallize integral membrane proteins. This will be accomplished using several innovative approaches and technologies. The proteins targeted are directly associated with human disease including cancer (colon, breast and prostate), diabetes, cystic fibrosis, growth anomalies, immune system disorders, hypertension, sepsis and the flu.
|Hildebrandt, Ellen; Khazanov, Netaly; Kappes, John C et al. (2016) Specific stabilization of CFTR by phosphatidylserine. Biochim Biophys Acta 1859:289-293|
|Reddy, Bharat G; Dai, Qun; McNicholas, Carmel M et al. (2016) Expression and purification of the alpha subunit of the epithelial sodium channel, ENaC. Protein Expr Purif 117:67-75|
|Hildebrandt, Ellen; Ding, Haitao; Mulky, Alok et al. (2015) A stable human-cell system overexpressing cystic fibrosis transmembrane conductance regulator recombinant protein at the cell surface. Mol Biotechnol 57:391-405|
|Go, Eden P; Herschhorn, Alon; Gu, Christopher et al. (2015) Comparative Analysis of the Glycosylation Profiles of Membrane-Anchored HIV-1 Envelope Glycoprotein Trimers and Soluble gp140. J Virol 89:8245-57|
|Hildebrandt, Ellen; Zhang, Qinghai; Cant, Natasha et al. (2014) A survey of detergents for the purification of stable, active human cystic fibrosis transmembrane conductance regulator (CFTR). Biochim Biophys Acta 1838:2825-37|
|Johnson, David H; Wilson, W William; DeLucas, Lawrence J (2014) Protein solubilization: a novel approach. J Chromatogr B Analyt Technol Biomed Life Sci 971:99-106|
|Wilson, William W; Delucas, Lawrence J (2014) Applications of the second virial coefficient: protein crystallization and solubility. Acta Crystallogr F Struct Biol Commun 70:543-54|
|Lazrak, Ahmed; Fu, Lianwu; Bali, Vedrana et al. (2013) The silent codon change I507-ATC->ATT contributes to the severity of the Î”F508 CFTR channel dysfunction. FASEB J 27:4630-45|
|Ramani, Vishnu C; Pruett, Pamela S; Thompson, Camilla A et al. (2012) Heparan sulfate chains of syndecan-1 regulate ectodomain shedding. J Biol Chem 287:9952-61|
|McClure, Michelle; DeLucas, Lawrence J; Wilson, Landon et al. (2012) Purification of CFTR for mass spectrometry analysis: identification of palmitoylation and other post-translational modifications. Protein Eng Des Sel 25:7-14|