Purification and characterization of the mouse mammary tumor virus protease expressed in Escherichia coli.
The mouse mammary tumor virus (MMTV) protease gene was cloned into pGEX-2T, an Escherichia coli expression vector containing the glutathione S-transferase coding region of Schistosoma japonicum. The chimeric protein was formed by fusion of the glutathione S-transferase with a hexapeptide which contains a thrombin cleavage site, followed by the MMTV protease. Affinity chromatography on a glutathione-Sepharose 4B column was used to isolate the chimeric protein. After thrombin cleavage, the glutathione S-transferase and the protease were separated by gel filtration chromatography on a Sephadex G-75 column. The overall yield of the protease purification procedure was about 1 mg of protease/liter of culture, and the specific activity was 380 pmol/min.micrograms of enzyme. Like other retroviral proteases, the MMTV enzyme was active as a dimer, showed maximum activity at pH between 4 and 6, and could be inhibited by pepstatin A and a phosphinic acid derivative HIV-1 protease inhibitor. Enzymatic characterization of this protease reveals its broad specificity, showing a clear preference for the oligopeptide substrate mimicking the cleavage site at the amino-terminal end of the capsid protein (kcat/Km = 9725.5 M-1.s-1). The chimeric protein was also an active dimer and showed a similar Km (17 microM) for such an oligopeptide, although its kcat was about 10 times smaller. Autocatalytic processing of the MMTV protease was observed after expression of clones containing the natural cleavage site, as it occurs at the amino-terminal end of the viral protease, instead of the thrombin-sensitive sequence.
Menéndez-Arias
L
L
Laboratory of Molecular Virology and Carcinogenesis, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702-1201.
Young
M
M
Oroszlan
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular / Q000379:methods; D010450:Endopeptidases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005814:Genes, Viral; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D008324:Mammary Tumor Virus, Mouse / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D009994:Osmolar Concentration; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012549:Schistosoma japonicum / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D000906:Antibodies; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D010450:Endopeptidases