High-level expression of self-processed HIV-1 protease in Escherichia coli using a synthetic gene.
A synthetic gene coding for HIV-1 protease (PR) has been constructed and a system for its efficient expression in E. coli has been established: PR is synthesized as a fusion protein with E. coli dihydrofolate reductase under the control of a bacteriophage T7 promoter. The synthetic gene was constructed to enable rapid construction of defined mutants by restriction fragment replacement. A set of mutants has been constructed which may facilitate elucidation of the mechanism of PR self-cleavage from polyprotein precursors. We have demonstrated that the C-terminal residue (Phe99 in the native sequence) of the processing intermediate is absolutely required for subsequent cleavage at the N-terminal cleavage site. The potential structural role of this residue is discussed with reference to the recently published HIV-1 PR structure.
Hostomsky
Z
Z
Agouron Pharmaceuticals Inc., La Jolla, CA 92037.
Appelt
K
K
Ogden
R C
RC
D001483:Base Sequence; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005796:Genes; D005813:Genes, Synthetic; D005814:Genes, Viral; D014644:Genetic Variation; D016333:HIV Protease; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D015183:Restriction Mapping
D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; D016333:HIV Protease