BSGC Role

Dr. David McKay oversees Component Project VIII to develop soluble overexperssion systems in collaboration with Component Project II. He is directly involved in planning the experiments and establishing the necessary collaborations, both within Stanford and with Berkeley. He also participates directly in many aspects of the work, such as plasmid design and protein crystallization.

Component Project Information

This project focuses on development of improved methodologies for cloning genes and expressing protein domains. Two specific projects will be pursued initially. The first is to develop "cloning by recombination" in S. cerevisiae (yeast) as a facile, high-throughput tool for cloning genes into expression vectors, as an alternative to traditional restriction/ligation cloning. The second is to explore the efficacy of using in vivo selection as a tool for reporting and improving expression behavior of protein domains. The initial system of study will be derived from the E. coli trp operon. Deletion of the first helix of the TrpA protein (TrpA(D18)) severely impairs in vivo TrpA activity and stability; fusing the maltose binding protein (MBP) to the amino terminus of TrpA(D18) "rescues" activity. If pilot experiments demonstrate that the rescue phenomenon is general, i.e. that it only requires a polypeptide which is soluble and well-behaved expressed as a fusion with TrpA(D18), it will be developed as a tool for delineating domain boundaries and improving solution behavior of proteins by in vivo selection.

Rosalind Kim, Component Project Leader, Component II