Project Grant R43GM140633

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 A SELECTION SYSTEM FOR EVOLVING TAGS THAT ENABLE FOLDING OF CYTOKINES IN E. COLI AND FACILE PURIFICATION OF THE TAG-FREE, NATIVE PROTEIN. SELECTION IS BASED ON THE DISCOVERY THAT UNFOLDED PROTEINS RESTRICT GROWTH OF THE E. COLI HOST CELL WHEN CO-EXPRESSED WITH AN ENGINEERED RESTRICTION PROTEASE. A TAG THAT FACILITATES THE FOLDING OF AN OTHERWISE POORLY-FOLDED PROTEIN RESCUES GROWTH. USING THE SELECTION METHOD, WE WILL DEVELOP TAGS ENABLING PURIFICATION OF KEY CYTOKINES NEEDED TO CREATE DEFINED, SYNTHETIC CELL CULTURE MEDIA. OUR APPROACH EMPLOYS RANDOM MUTAGENESIS OF A TANDEM TAG OF PROTEIN G DOMAINS (GA-GB) AND SELECTION USING A RESTRICTION PROTEASE SYSTEM THAT WE WILL DEVELOP. WE WILL EVOLVE FOLDING TAGS FOR FOUR TARGETS AND EXPAND THE ENVELOP OF RECOMBINANT PROTEINS THAT CAN BE PURIFIED IN E COLI. SPECIFIC AIMS ARE: CONSTRUCT PLASMIDS FOR CO- EXPRESSION OF GA-GB-TARGET PROTEINS WITH THE RESTRICTION PROTEASE; CONSTRUCT RANDOM LIBRARIES OF GA-GB- TARGET PROTEIN; SELECT FOR ESCAPE MUTANTS; ANALYZE OF MUTATIONS THAT ALLOW ESCAPE; PURIFY PROTEINS USING NEWLY-EVOLVED TAGS; TEST PHYSICAL AND BIOLOGICAL PROPERTIES OF PURIFIED PROTEINS. FEASIBILITY WILL BE DETERMINED BY WHETHER WE CAN EVOLVE EFFECTIVE FOLDING TAGS FOR ALL FOUR TARGETS. AN EFFECTIVE FOLDING TAG IS DEFINED AS ONE THAT ALLOWS PURIFICATION OF =50MG OF NATIVE, BIOLOGICALLY-ACTIVE CYTOKINE PER L OF CULTURE. AT THE END OF PHASE I WE WILL HAVE DEVELOPED METHODS FOR IDENTIFYING EFFECTIVE FOLDING TAGS FOR TWO MAJOR STRUCTURAL CLASSES OF CYTOKINES. WE WILL ALSO HAVE LEARNED WHETHER EVOLVED FOLDING TAGS FOR A STRUCTURAL CLASS OF CYTOKINE ARE SIMILAR. THIS KNOWLEDGE WILL SIMPLIFY IDENTIFICATION OF FOLDING TAGS FOR NEW CYTOKINE TARGETS AS WELL AS OTHER HIGH-VALUE PROTEINS. IN PHASE II WE WOULD EVOLVE TAGS FOR 17 MAJOR CYTOKINES USED IN CELL CULTURE.