Moloney murine leukemia virus protease: bacterial expression and characterization of the purified enzyme.
The Moloney murine leukemia virus (Mo-MuLV) protease has been cloned into the prokaryotic expression vector pGEX-2T, expressed in fusion with the glutathione S-transferase from Schistosoma japonicum, and purified to apparent homogeneity after thrombin cleavage of the chimeric protein. The purified protease showed maximum activity at pH 6.0 and was inhibited by several aspartyl protease inhibitors, found to be active toward the human immunodeficiency virus-1 (HIV-1) protease. Peptides representing maturation cleavage sites in Gag and Gag-Pol polyproteins were accurately cleaved by the recombinant protease, and kinetic parameters have been determined. In addition, oligopeptides mimicking the cleavage site found in the transmembrane protein and leading to the formation of p15E and p2E were also hydrolyzed at the expected position. The Mo-MuLV protease appears to be more closely related to the HIV-1 protease than to the mouse mammary tumor virus enzyme, based on its substrate specificity and sensitivity to aspartyl protease inhibitors.
Laboratory of Molecular Virology and Carcinogenesis, NCI-Frederick Cancer Research and Development Center, Maryland 21702-1201.
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Menéndez-Arias
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Gotte
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Oroszlan
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D015685:Fusion Proteins, gag-pol / Q000378:metabolism; D015686:Gene Products, env / Q000378:metabolism; D015683:Gene Products, gag / Q000378:metabolism; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology* / Q000235:genetics; D010446:Peptide Fragments / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity
D015685:Fusion Proteins, gag-pol; D015686:Gene Products, env; D015683:Gene Products, gag; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D016282:Aspartic Acid Endopeptidases; C083201:protease, Moloney murine leukemia virus