Expression and purification of a two-component flaviviral proteinase resistant to autocleavage at the NS2B-NS3 junction region.
Regulated proteolysis of the polyprotein precursor of West Nile virus (WNV) by the essential NS2B-NS3(pro)tease, a promising drug target for WNV inhibitors, is required for the propagation of infectious virions. Structural and drug design studies, however, require pilot-scale quantities of a pure and catalytically active WNV protease that is resistant to self-proteolysis. Autolytic cleavage at the NS2B-NS3 boundary leads to individual, non-covalently associated, NS2B and NS3 domains, together with residual amounts of the intact NS2B-NS3, in the NS2B-NS3pro samples. We modified the cleavage site sequence of the NS2B-NS3 junction region and then developed expression and purification procedures to prepare a covalently linked, single-chain, NS2B-NS3pro K48A mutant construct. This construct exhibits high stability and functional activity and is thus well suited for the follow-up purification and structural and drug design studies.
Shiryaev
Sergey A
SA
Inflammatory and Infectious Disease Center, Burnham Institute for Medical Research, La Jolla, CA 92037, USA.
Aleshin
Alexander E
AE
Ratnikov
Boris I
BI
Smith
Jeffrey W
JW
Liddington
Robert C
RC
Strongin
Alex Y
AY
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014902:West Nile virus / Q000201:enzymology* / Q000235:genetics; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D010450:Endopeptidases; D010447:Peptide Hydrolases; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases