Using standard recombinant DNA and yeast transformation techniques, a number of expression vectors have been constructed in order to get the regulated overproduction of sis-related proteins in Saccharomyces cerevisiae. Both retroviral (v-sis) and human (c-sis) cDNA sequences have been used for these constructs. Independently of the type of promoter used, it seems that only extremely low levels of the sis protein accumulate in the yeast cells. Thus, sis proteins are not detectable by radioimmunoprecipitation in labeled yeast cell lysates. Under certain conditions, vectors based on the promoter and processing signals of the yeast mating-type alpha structural gene seem to release sis-related proteins into the culture medium. Therefore, at the present time efforts are concentrated in the detection of the proteins by mitogenic assays on quiescent NIH/3T3 and other PDGF-responsive cells. The inducible lambda bacteriophage-derived promoter PL is currently being used in pBR322-based vectors to attempt the regulated overproduction of the gp70 protein product of the v-fgr oncogene, a member of the tyrosine-kinase family, in Escherichia coli. For this purpose, either the entire v-fgr coding region or portions of it encompassing its three distinguishable domains (gag, actin and tyrosine kinase-related) have been used to construct the expression systems. Radioimmunoprecipitation and western-blot techniques are being used for detection of the protein species synthesized by the bacterial cells.
