Potomac Affinity Proteins, LLC

Selection System for Identifying Protein-specific Folding Tags That Enable Purification of Native Cytokines From E. Coli - Project Summary: Our Overall Objective Is to Develop and Test Tags That Enable Folding of Growth Factors in E. Coli and Facilitate Purification of the Tag-free, Native Protein. Numerous Growth Factors Are Needed for Cell Culture, But Most Are Difficult to Express and Costly to Purify. This Is Because Many Eukaryotic Proteins, Particularly Those With Multiple Disulfide Bonds, Do Not Fold Properly in E. Coli and Are Difficult and Expensive to Fold in Vitro. in Phase I We Developed a Method to Identify Dual-function Tags That Guide the Formation of Native Disulfide Bonds in E. Coli and Then Bind Tightly to the Processing Protease and Direct Tag Removal in the Purification Phase. We Showed That One or More of These Tags Work for Not Only the Original Target Set But Also for Other Growth Factors Representing All Major Structural Classes. in Phase Ii We Will Use the Wealth of Empirical Data From Phase I to Gain an Understanding of Structural and Energetic Characteristics of Successful Tags. We Will Then Design Tags for the Remaining Growth Factors Most Used in Cell Culture. Designed Tags Will Be Tested for Their Ability to Promote Native Structure and Facilitate Purification. This Procedure Should Be Applicable to Any Protein That Cannot Be Recombinantly Expressed in Native Form With Standard Tags and Overcome Long-standing Challenges That Have Limited Progress in Many Areas of Biology and Medicine. We Will Also Develop a Scalable Process for Producing Pre-gmp Quality Growth Factors and Validate Physical Properties and Biological Activity. Gram Qualities of Validated TGFB1, Vegfa, and IGF1 Will Be Produced in Phase Ii. Other Growth Factors Will Enter the Production Pipeline in Phases Based on Their Market Share and Profit Margin. This Will Expand Commercial Opportunities for Low-cost Growth Factor Production and Enable Economical Manufacturing of Synthetic Cell Culture Media.