Bacterial expression, purification, and in vitro N-myristoylation of HIV-1 pl7 gag

Abstract

The coding region of the N-terminal 17-kDa portion of HIV-1 Pr55 gag (p17 gag ) was cloned into the pET-3c expression vector and was used to overexpress HIV-1 p17 gag in Escherichia coli. Induction of the transformed bacteria caused the accumulation of a 17-kDa polypeptide in the soluble cell fraction which was released by sonication in hypotonic nondetergent buffer. The 17-kDa polypeptide was purified by ammonium sulfate precipitation and successive chromatography on G-75 Sephadex, DEAE-Sephacel, and S-Sephadex. The final product was purified 12-fold with about a 16% recovery from the original soluble cell lysate and was judged to be 97+% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blotting with two different antibodies confirmed the identity of the purified 17-kDa polypeptide as authentic p17 gag . In the presence of myristoyl-CoA and bovine brain N-myristoyl-transferase, p17 gag was quantitatively N-myristoylated in vitro with a pseudo-first-order rate constant of 4.7 ± 1.0 × 10 −3 min −1, but with only about 3% of the catalytic efficiency of N-myristoylation of a 16-residue peptide homologous to the N-terminus of p17 gag . The myristate group in the N-myristoylated p17 gag was stable to treatment with detergent and hydroxylamine consistent with a covalent N-acyl-amide linkage. The N-myristoylglycyl linkage was confirmed by partial acid hydrolysis and identification of the p-nitrobenzylazlactone derivative of the resulting N-myristoylglycine by high-performance liquid chromatography.