title: An 11-kDa form of human immunodeficiency virus protease expressed in Escherichia coli is sufficient for enzymatic activity.
abstract: In order to define the protease domain of human immunodeficiency virus 1, various regions of the pol open reading frame were cloned and expressed in Escherichia coli. Antiserum directed against the conserved retroviral protease active site was used to identify pol precursor and processed species containing the presumed protease domain. The smallest product that accumulates is about 11 kDa as measured by NaDodSO4/PAGE. This size agrees with that predicted from the presence in this region of two Phe-Pro sequences, which is one of the cleavage sites recognized by HIV protease. DNA encoding only the predicted 11-kDa protein was cloned, bypassing the need for autoprocessing, and the protein was expressed to a high level in E. coli. This form is active as demonstrated by its ability to specifically cleave protease-deficient pol protein in vivo in E. coli. Extracts of E. coli containing the 11-kDa protease also process human immunodeficiency virus gag substrates in vitro. These results demonstrate that the 11-kDa protease is sufficient for enzymatic activity and are consistent with a major role for this form in virus maturation.
authors.affiliation: Department of Molecular Genetics, Hoffmann-La Roche, Nutley, NJ 07110.
authors.forename: M C
authors.initials: MC
authors.lastname: Graves
authors.forename: J J
authors.initials: JJ
authors.lastname: Lim
authors.forename: E P
authors.initials: EP
authors.lastname: Heimer
authors.forename: R A
authors.initials: RA
authors.lastname: Kramer
mesh_terms: D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D006678:HIV / Q000201:enzymology*; D016333:HIV Protease; D008970:Molecular Weight; D011498:Protein Precursors / Q000378:metabolism; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D011498:Protein Precursors; D014764:Viral Proteins; D010450:Endopeptidases; D016333:HIV Protease