title: High-level synthesis of recombinant HIV-1 protease and the recovery of active enzyme from inclusion bodies.
abstract: 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.
authors.affiliation: Central Research and Development Department, E.I. duPont de Nemours and Co., Wilmington, DE 19880-0328.
authors.forename: Y S
authors.initials: YS
authors.lastname: Cheng
authors.forename: M H
authors.initials: MH
authors.lastname: McGowan
authors.forename: C A
authors.initials: CA
authors.lastname: Kettner
authors.forename: J V
authors.initials: JV
authors.lastname: Schloss
authors.forename: S
authors.initials: S
authors.lastname: Erickson-Viitanen
authors.forename: F H
authors.initials: FH
authors.lastname: Yin
mesh_terms: 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
chemical_list: D004792:Enzyme Precursors; D015684:Gene Products, pol; D011994:Recombinant Proteins; D010450:Endopeptidases; D016333:HIV Protease