Production of authentic SARS-CoV M(pro) with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction

Xiaoyu Xue; Haitao Yang; Wei Shen; Qi Zhao; Jun Li; Kailin Yang; Cheng Chen; Yinghua Jin; Mark Bartlam; Zihe Rao

doi:10.1016/j.jmb.2006.11.073

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Production of authentic SARS-CoV M(pro) with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction

Journal article

Open access

Peer reviewed

Production of authentic SARS-CoV M(pro) with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction

Xiaoyu Xue, Haitao Yang, Wei Shen, Qi Zhao, Jun Li, Kailin Yang, Cheng Chen, Yinghua Jin, Mark Bartlam and Zihe Rao

Journal of molecular biology, Vol.366(3), pp.965-975

02/23/2007

DOI: 10.1016/j.jmb.2006.11.073

PMCID: PMC7094453

PMID: 17189639

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Abstract

The viral proteases have proven to be the most selective and useful for removing the fusion tags in fusion protein expression systems. As a key enzyme in the viral life-cycle, the main protease (M(pro)) is most attractive for drug design targeting the SARS coronavirus (SARS-CoV), the etiological agent responsible for the outbreak of severe acute respiratory syndrome (SARS) in 2003. In this study, SARS-CoV M(pro) was used to specifically remove the GST tag in a new fusion protein expression system. We report a new method to produce wild-type (WT) SARS-CoV M(pro) with authentic N and C termini, and compare the activity of WT protease with those of three different types of SARS-CoV M(pro) with additional residues at the N or C terminus. Our results show that additional residues at the N terminus, but not at the C terminus, of M(pro) are detrimental to enzyme activity. To explain this, the crystal structures of WT SARS-CoV M(pro) and its complex with a Michael acceptor inhibitor were determined to 1.6 Angstroms and 1.95 Angstroms resolution respectively. These crystal structures reveal that the first residue of this protease is important for sustaining the substrate-binding pocket and inhibitor binding. This study suggests that SARS-CoV M(pro) could serve as a new tag-cleavage endopeptidase for protein overproduction, and the WT SARS-CoV M(pro) is more appropriate for mechanistic characterization and inhibitor design.

Calbindins

Endopeptidases - metabolism

Gene Expression

Genetic Vectors

Glutathione Transferase - metabolism

Recombinant Fusion Proteins - metabolism

S100 Calcium Binding Protein G - isolation & purification

SARS Virus

Viral Matrix Proteins - antagonists & inhibitors

Viral Matrix Proteins - biosynthesis

Viral Matrix Proteins - chemistry

Viral Matrix Proteins - metabolism

Viral Proteins - biosynthesis

Details

Title: Subtitle: Production of authentic SARS-CoV M(pro) with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction
Creators: Xiaoyu Xue - Institute of Biophysics
Haitao Yang - Institute of Biophysics
Wei Shen
Qi Zhao - Tsinghua University
Jun Li - Tsinghua University
Kailin Yang - Tsinghua University
Cheng Chen - Tsinghua University
Yinghua Jin - Tsinghua University
Mark Bartlam - Institute of Biophysics
Zihe Rao - Tsinghua University
Resource Type: Journal article
Publication Details: Journal of molecular biology, Vol.366(3), pp.965-975
DOI: 10.1016/j.jmb.2006.11.073
PMID: 17189639
PMCID: PMC7094453
NLM abbreviation: J Mol Biol
ISSN: 0022-2836
eISSN: 1089-8638
Publisher: ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD; LONDON
Language: English
Date published: 02/23/2007
Academic Unit: Radiation Oncology
Record Identifier: 9984696707402771

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