McKay, Component Project Leader
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.
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.
Kim, Component Project Leader, Component II