Expression, purification, and in vitro activity of an arterivirus main proteinase.
To allow the biochemical and structural characterization of the chymotrypsin-like "main proteinase" (non-structural protein 4; nsp4) of the arterivirus prototype Equine Arteritis Virus (EAV), we developed protocols for the large-scale production of recombinant nsp4 in Escherichia coli. The nsp4 proteinase was expressed either fused to maltose binding protein or carrying a C-terminal hexahistidine tag. Following purification, the nsp4 moiety of MBP-nsp4 was successfully used for structural studies [Barrette-Ng, I.H., Ng, K.K.S., Mark, B.L., van Aken, D., Cherney, M.M., Garen, C, Kolodenko, Y., Gorbalenya, A.E., Snijder, E.J., James, M.N.G, 2002. Structure of arterivirus nsp4-the smallest chymotrypsin-like proteinase with an alpha/beta C-terminal extension and alternate conformations of the oxyanion hole. J. Biol. Chem. 277, 39960-39966]. Furthermore, both forms of the EAV proteinase were shown to be proteolytically active in two different trans-cleavage assays. Recombinant nsp4 cleaved the cognate nsp6/7- and nsp7/8 site in in vitro synthesized substrates. In a synthetic peptide-based activity assay, the potential of the recombinant proteinase to cleave peptides mimicking the P9-P7' residues of six nsp4 cleavage sites was investigated. The peptide representing the EAV nsp7/8 junction was used to optimize the reaction conditions (pH 7.5, 25mM NaCl, 30% glycerol at 30 degrees C), which resulted in a maximum turnover of 15% of this substrate in 4h, using a substrate to enzyme molar ratio of 24:1. The assays described in this study can be used for a more extensive biochemical characterization of the EAV main proteinase, including studies aiming to identify inhibitors of proteolytic activity.
van Aken
Danny
D
Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, LUMC P4-26, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
Benckhuijsen
Willemien E
WE
Drijfhout
Jan W
JW
Wassenaar
Alfred L M
AL
Gorbalenya
Alexander E
AE
Snijder
Eric J
EJ
D000595:Amino Acid Sequence; D018018:Arterivirus / Q000201:enzymology*; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000378:metabolism; D053818:Chymases; D004926:Escherichia coli / Q000378:metabolism; D005990:Glycerol; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D012965:Sodium Chloride; D013379:Substrate Specificity; D013696:Temperature; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; C471213:His-His-His-His-His-His; D058619:Maltose-Binding Proteins; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D012965:Sodium Chloride; D006639:Histidine; D012697:Serine Endopeptidases; D053818:Chymases; D005990:Glycerol