Expression and purification of recombinant full-length NS3 protease-helicase from a new variant of Hepatitis C virus

Poliakov, Anton (author): Uppsala universitet,Institutionen för naturvetenskaplig biokemi

Shuman, Cynthia F (author): Uppsala universitet,Institutionen för naturvetenskaplig biokemi

Stenberg, Gun (author): Uppsala universitet,Institutionen för naturvetenskaplig biokemi

Danielson, U Helena (author): Uppsala universitet,Institutionen för naturvetenskaplig biokemi

(creator_code:org_t)

2002
2002
English.
In: Protein Expression and Purification. - 1046-5928 .- 1096-0279. ; 25:3, s. 363-371

Related links:: http://www.ncbi.nlm....; show more...; https://urn.kb.se/re...; https://doi.org/10.1...; show less...

Abstract Subject headings

Viral mRNA extracted from the serum of a patient infected with HCV strain I a was used for cloning, expression, and purification of full-length Hepatitis C NS3 protein. Sequencing of the protease gene identified the virus to be a new variant closely related to strain H77, differing in 15 out of 631 amino acids in the NS3 protein, none of which were predicted to be directly involved in catalysis, binding of substrate, or cofactor. A pBAD expression system was used to express the enzyme with an N-terminal tag in Escherichia coli. Purification from the soluble cellular fraction was achieved by Ni2(+)-IMAC and PolyU Sepharose affinity chromatography. The dependence of the proteolytic activity of the full-length NS3 protein on ionic strength, glycerol concentration, and a peptide corresponding to the activating region of NS4A was analyzed and used to design an activity assay that is suitable for inhibition studies. The kinetic constants (k(cat) and K-M) for catalysis and the inhibitory potencies (IC50 and K-i) of five product-based hexapeptide inhibitors were comparable to those reported for the truncated NS3 protein. Detailed kinetic and inhibition studies using this variant of full-length NS3 can increase the understanding of the enzymatic characteristics of NS3, reveal the importance of the substituted amino acids and the significance of the genetic variability for design of effective inhibitors of the virus, and is thus of relevance for drug discovery.

By the author/editor: Poliakov, Anton; Hubatsch, Ina; Shuman, Cynthia ...; Stenberg, Gun; Danielson, U Hel ...

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