This project has the following goals. A two pla< mid 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 simplifica ion 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.Awardee's statement of the potential commercial applications of the research:This vector system for cloning receptor binding domains and other signalling proteins will be of value to a large number of customers studying hormone receptor-mediated signalling, mechanisms of malignant transformation, anti-tumor drug development, and normal cell growth and development. The availability of large amounts of receptor, receptor domain, and intracellular signalling proteins will enable these groups to carry out structural and functional studies not otherwise possible.National Institute of General Medical Sciences (NIGMS)
