TOOLS FOR THE PRODUCTION AND PURIFICATION OF FULL-LENGTH, N-TERMINAL OR C-TERMINAL P-32 LABELED PROTEIN, APPLIED TO HIV-1 GAG AND REV

We have constructed two new vectors for the production of foreign prot eins in Escherichia coli. The vectors, pGEX-GTH and pET-HTG, produce p rotein fused to glutathione S-transferase (GST) at the N- and C-termin i, respectively, allowing one-step purification on glutathione-Sepharo se. Furthermore, they carry the recognition sequence (RRASV) for the c atalytic subunit of cAMP-dependent heart muscle kinase (HMK) at the te rminus distal to the GST tag, enabling specific P-32 labeling in vitro . By positioning the GST and HMK sequences at opposite ends of the int roduced gene, only full-length fusion protein becomes radiolabeled aft er purification. Avoiding the labeling of shorter fusion protein speci es, often observed in bacterial expression of foreign genes, is partic ularly important for a number of different purposes, including protein mobility shift analysis and protein footprinting technology.