Bacterial expression, purification, and in vitro N-myristoylation of HIV-1 p17gag.
The coding region of the N-terminal 17-kDa portion of HIV-1 Pr55gag (p17gag) was cloned into the pET-3c expression vector and was used to overexpress HIV-1 p17gag 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 identify of the purified 17-kDa polypeptide as authentic p17gag. In the presence of myristoyl-CoA and bovine brain N-myristoyl-transferase, p17gag was quantitatively N-myristoylated in vitro with a pseudo-first-order rate constant of 4.7 +/- 1.0 x 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 p17gag. The myristate group in the N-myristoylated p17gag 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.
Burnette
B
B
Laboratory of Biological Chemistry, National Cancer Institute, Bethesda, Maryland 20894.
Kahn
R
R
Glover
C J
CJ
Felsted
R L
RL
D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015683:Gene Products, gag / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D005822:Genetic Vectors; D015488:HIV Antigens / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics*; D007700:Kinetics; D008970:Molecular Weight; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D015488:HIV Antigens; D009227:Myristic Acids; D011994:Recombinant Proteins; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C054858:p17 protein, Human Immunodeficiency Virus Type 1; D019814:Myristic Acid