Abstract
Severe acute respiratory syndrome coronavirus (SARS-CoV) proteins belong to a large group of proteins that is difficult to express in traditional expression systems. The ability to express and purify SARS-CoV proteins in large quantities is critical for basic research and for development of pharmaceutical agents. The work reported here demonstrates: (1) fusion of SUMO (small ubiquitin-related modifier), a 100 amino acid polypeptide, to the N-termini of SARS-CoV proteins dramatically enhances expression in Escherichia coli cells and (2) 6x His-tagged SUMO-fusions facilitate rapid purification of the viral proteins on a large scale. We have exploited the natural chaperoning properties of SUMO to develop an expression system suitable for proteins that cannot be expressed by traditional methodologies. A unique feature of the system is the SUMO tag, which enhances expression, facilitates purification, and can be efficiently cleaved by a SUMO-specific protease to generate native protein with a desired N-terminus. We have purified various SARS-CoV proteins under either native or denaturing conditions. These purified proteins have been used to generate highly specific polyclonal antibodies. Our study suggests that the SUMO-fusion technology will be useful for enhancing expression and purification of the viral proteins for structural and functional studies as well as for therapeutic uses.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Coronavirus 3C Proteases
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Coronavirus Nucleocapsid Proteins
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Cysteine Endopeptidases / biosynthesis
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Cysteine Endopeptidases / genetics
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Cysteine Endopeptidases / isolation & purification
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Escherichia coli / genetics
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Gene Expression / genetics*
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Genetic Vectors / genetics
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Histidine / genetics
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Membrane Glycoproteins / biosynthesis
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Membrane Glycoproteins / genetics
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Membrane Glycoproteins / isolation & purification
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Nucleocapsid Proteins / biosynthesis
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Nucleocapsid Proteins / genetics
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Nucleocapsid Proteins / isolation & purification
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Peptide Hydrolases / metabolism
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Recombinant Fusion Proteins / biosynthesis*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Severe acute respiratory syndrome-related coronavirus / genetics*
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Small Ubiquitin-Related Modifier Proteins / genetics*
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Spike Glycoprotein, Coronavirus
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Viral Envelope Proteins / biosynthesis
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Viral Envelope Proteins / genetics
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Viral Envelope Proteins / isolation & purification
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Viral Proteins / genetics*
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Viral Proteins / isolation & purification
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Viral Proteins / metabolism
Substances
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Coronavirus Nucleocapsid Proteins
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Membrane Glycoproteins
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Nucleocapsid Proteins
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Recombinant Fusion Proteins
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Small Ubiquitin-Related Modifier Proteins
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Spike Glycoprotein, Coronavirus
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Viral Envelope Proteins
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Viral Proteins
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spike glycoprotein, SARS-CoV
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polyhistidine
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Histidine
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Peptide Hydrolases
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Cysteine Endopeptidases
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Coronavirus 3C Proteases