Summary auto-generated
This study describes the successful expression and purification of the hepatitis C virus (HCV) NS3 serine protease domain (NS3181) in yeast (Saccharomyces cerevisiae) as a histidine-tagged fusion protein. The researchers used immobilized metal affinity chromatography to efficiently purify the active enzyme from soluble cell lysates, achieving approximately 90% purity with yields of 1-0.5 mg per liter of culture. Using in vitro transcription-translation generated substrates, they characterized the protease's substrate specificity and identified a previously unknown cleavage site within the NS5A region, occurring after Thr-2172. This site contains a conserved recognition motif similar to other known cleavage sites. The purified protease was tested against various protease inhibitors; while metalloprotease and aspartic protease inhibitors showed no effect, certain serine protease inhibitors like TPCK and elastinal demonstrated inhibitory activity. The findings suggest the NS3 protease has a chymotrypsin or elastase-like fold, though its active site geometry differs from previously characterized serine proteases. This work provides a reliable method for producing recombinant NS3 protease, facilitating development of anti-hepatitis C therapeutics.
Key findings
- HCV NS3181 protease domain was successfully expressed in yeast and purified to >90% purity using metal-affinity chromatography, yielding 1-0.5 mg per liter of culture
- A previously unobserved cleavage site within NS5A (after Thr-2172) was identified; this site is conserved among HCV strains and contains a consensus recognition motif
- The NS3 protease exhibits substrate specificity that can be modulated by an NS4A peptide cofactor, affecting trans-cleavage patterns at different polyprotein junction sites
- Selective serine protease inhibitors (TPCK, elastinal) inhibited protease activity, while common serine and metalloprotease inhibitors showed no effect, suggesting a unique active site geometry
- The purified protease is suitable for structural studies and inhibitor screening for anti-HCV drug development
This summary was generated automatically from the article PDF and is not part of the original publication. Refer to the PDF for the authoritative text.
Abstract
W Markland, RA Petrillo, M Fitzgibbon, T Fox, R McCarrick, T McQuaid, JR Fulghum, W Chen, MA Fleming, JA Thomson and SP Chambers
Vertex Pharmaceuticals Inc., Cambridge, MA 02139-4242, USA.
cDNA encoding the putative core of the hepatitis C virus NS3 serine protease domain (residues 1-181 of NS3; NS3 (181)) was expressed as an N-terminally (His)6-tagged fusion protein in Saccharomyces cerevisiae. NS3 (181) protease activity was found in soluble cell lysates, and the N-terminal metal-chelating domain facilitated the efficient purification of active enzyme, using immobilized metal affinity chromatography. The purified NS3(181), protease activity was characterized by assaying the trans-cleavage of in vitro transcription- translation generated substrates, and subsequently a previously unobserved cleavage site within the NS5A region was identified. The inhibitory effect of known protease inhibitors was also examined. It is hoped that availability of this method for the expression and purification of the NS3(181) protease will facilitate the development of anti-hepatitis C therapies.