High-level synthesis of recombinant HIV-1 protease and the recovery of active enzyme from inclusion bodies.
A complete chemical synthesis and assembly of genes for the production of human immunodeficiency virus type-I protease (HIV-PR) and its precursors are described. The T7 expression system was used to produce high levels of active HIV-PR and its precursors in Escherichia coli inclusion bodies. The gene encoding the open reading frames of HIV-PR was expressed in E. coli as a 10-kDa protein, while the genes encoding HIV-PR precursors were expressed as larger proteins, which were partially processed in E. coli to the 10-kDa form. These processing events are autoproteolytic, since a single-base mutation, changing the active-site aspartic acid to glycine, completely abolished the conversion. HIV-PR can be released with 8 M urea from washed cellular inclusion bodies, resulting in a preparation with few bacterial host proteins. After refolding, this preparation contains no nonspecific protease or peptidase activities. The recombinant HIV-PR isolated from inclusion bodies cleaves HIV-PR substrates specifically with a specific activity comparable to column-purified HIV-PR.
Cheng
Y S
YS
Central Research and Development Department, E.I. duPont de Nemours and Co., Wilmington, DE 19880-0328.
McGowan
M H
MH
Kettner
C A
CA
Schloss
J V
JV
Erickson-Viitanen
S
S
Yin
F H
FH
D000595:Amino Acid Sequence; D001483:Base Sequence; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D015684:Gene Products, pol / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005813:Genes, Synthetic; D005814:Genes, Viral; D016333:HIV Protease; D015497:HIV-1 / Q000201:enzymology*; D002479:Inclusion Bodies / Q000201:enzymology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
D004792:Enzyme Precursors; D015684:Gene Products, pol; D011994:Recombinant Proteins; D010450:Endopeptidases; D016333:HIV Protease