Expression of the murine leukemia virus protease in fusion with maltose-binding protein in Escherichia coli.
The protease of murine leukemia virus (MLV) was cloned into pMal-c2 vector, expressed in fusion with maltose-binding protein (MBP), and purified to homogeneity after Factor Xa cleavage of the chimeric protein. Substantial degradation of the fusion protein was observed during expression, which severely diminished the yield. The degree of degradation of the fusion protein was even more pronounced when a single-chain form of the MLV protease was cloned after the gene coding for MBP. To increase the yield, a hexahistidine tag with an additional Factor Xa cleavage site was cloned after the protease and nickel chelate affinity chromatography was used as the first purification step. The modified procedure resulted in substantially higher yield as compared to the original procedure. The degradation of hexahistidine-tagged active site mutant MLV protease was very low and comparable to that obtained with hexahistidine-tagged MBP, but purified MLV protease alone was not able to degrade purified MBP, suggesting that during expression the active MLV protease may activate bacterial proteases which appear to be responsible for the degradation of the fusion proteins.
Fehér
Anita
A
Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.
Boross
Péter
P
Sperka
Tamás
T
Oroszlan
Stephen
S
Tözsér
József
J
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D009052:Leukemia Virus, Murine / Q000201:enzymology* / Q000235:genetics; D058619:Maltose-Binding Proteins; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases