Research Project
2190387
This Phase I proposal has the following goals. A two plasmid system for cloning protein domains of interest in E. coli will be developed. Features of this system include the option of in vivo tyrosine phosphorylation of the cloned domain and high level T7 RNA polymerase-directed, inducible expression. Domains are cloned as fusions to a tripeptide sequence which contains an epitope tag for ease of purification, the Kemptide for in vitro radiolabeling with cAMP-dependent protein kinase and a factor Xa cleavage site for isolating just the domain of interest. The system will be useful for studying protein-protein interactions, for generating affinity matrices and for cloning interacting signalling proteins. The second objective is to evaluate a novel transfer vector for expression of receptor proteins in insect cells. The transfer vector contains the human placental alkaline phosphatase signal sequence for secretion of the recombinant protein. This offers advantages which include simplification of purification and increased yields of purified product due to decreased proteolysis and increased solubility. This system will make available large amounts of receptor protein for use in structural studies, functional studies, including development of therapeutics, and for generation of probes for screening expression libraries.
