Construction of an Rb gene expression plasmid.

We obtained an 844 bp Bg1II fragment from an Rb cDNA clone and inserted it into the expression vector pWR-13 to construct an Rb gene expression plasmid. When the Rb Bg1II fragment was fused in-frame into pWR-13, it was operated by a Lac Z promoter and produced a fusion protein which consisted of expressed Rb protein and a small peptide from Lac Z. The recombinants were transformed into E. coli with the CaCl2 method, screened by in situ hybridization, and restriction mapped. Total cellular protein of transformed clones was analyzed by SDS-PAGE and Commassie blue staining. The sense clones showed a unique band at 28,000. On Western blot, this band specifically reacted with 125I-labelled antibody against synthetic Rb peptide. This protein comprised more than 5% of total bacterial protein.