Chemical synthesis and expression of the HIV-1 protease gene in E. coli.
The 297bp HIV-1 protease gene was constructed from five discrete synthetic fragments and expressed in E. coli. A soluble protein product of 11.5 Kd was detected by immunoblotting using protease specific antisera. A quantitative assay system, utilizing a synthetic nonapeptide spanning the cleavage site between p17-p24 in the gag polyprotein, was used to measure the specific protease activity in crude extracts. The protease hydrolyzed tyrosyl-proline bonds with an approximate specific activity of 43 pmoles/min/micrograms of total protein. The chemical synthesis of the protease gene and it's expression provides a feasible method for rapid mutant analysis, important for structure-function studies and rational design of potential inhibitors.
Louis
J M
JM
Division of Cancer Biology and Diagnosis, National Cancer Institute, Bethesda, Maryland 20892.
Wondrak
E M
EM
Copeland
T D
TD
Smith
C A
CA
Mora
P T
PT
Oroszlan
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000138:chemical synthesis* / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005786:Gene Expression Regulation; D015683:Gene Products, gag; D016333:HIV Protease; D007700:Kinetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010446:Peptide Fragments / Q000378:metabolism; D010957:Plasmids; D011498:Protein Precursors / Q000378:metabolism; D012191:Retroviridae Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014169:Transformation, Bacterial
D015683:Gene Products, gag; D010446:Peptide Fragments; D011498:Protein Precursors; D012191:Retroviridae Proteins; D004247:DNA; D010450:Endopeptidases; D016333:HIV Protease