Expression and purification of a two-component flaviviral proteinase resistant to autocleavage at the NS2B-NS3 junction region |
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Authors: | Shiryaev Sergey A Aleshin Alexander E Ratnikov Boris I Smith Jeffrey W Liddington Robert C Strongin Alex Y |
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Institution: | aInflammatory and Infectious Disease Center, Burnham Institute for Medical Research, La Jolla, CA 92037, USA |
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Abstract: | 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. |
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Keywords: | West Nile virus Flaviviruses NS2B– NS3 Proteolysis Polyprotein processing Mutagenesis Autoproteolysis |
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