Cloning Vectors for Expression of Signaling Proteins

This project has the following goals. A two plasmid system for cloning protein domains ofinterest in E. coli will be developed. Features of this system include the option of in vivo tyrosinephosphorylation 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 ofpurification, the Kemptide for in vitro radiolabeling with cAMP-dependent protein kinase and a factor Xacleavage site for isolating just the domain of interest. The system will be useful for studyingprotein-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 insectcells. The transfer vector contains the human placental alkaline phosphatase signal sequence forsecretion of the recombinant protein. This offers advantages which include simplification of purificationand increased yields of purified product due to decreased proteolysis and increased solubility. Thissystem will make available large amounts of receptor protein for use in structural studies, functionalstudies, including development of therapeutics, and for generation of probes for screening expressionlibraries.