Expression and purification of SARS coronavirus proteins using SUMO-fusions

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.

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

“…When used as an N-terminal carrier protein during prokaryotic expression, SUMO promotes folding and structural stability, which leads to enhanced functional production compared to untagged protein [48][49][50][51][52][53][54]. Unlike GST and MBP, SUMO does not itself serve as a means for purification of fusion proteins; however, the His 6 in series with the SUMO tag has been established to facilitate purification of fusion proteins.…”

Development and application of an enzyme-linked immunosorbent assay (ELISA) using a soluble recombinant nucleoprotein for the detection of antibodies to avian influenza virus

“…One of the reasons for this result may be the effect of the SUMO peptide fusion in enhancing the solubility of viral recombinant proteins expressed in E. coli (Guerrero et al, 2015;Shafer et al, 1998;Zuo et al, 2005) Recombinant NP epitope prediction confirmed that most antigenic sites of this recombinant protein were conserved compared to the NP of homologous (H4N6) and heterologous (H1N1, H2N2, H3N2, H5N1, H6N7, and H9N2) AIV subtypes. In addition, some amino acid residue stretches predicted as rNP epitopes in this study, such as 71 to 96 and 290 to 353, have been previously confirmed as target epitopes of monoclonal antibodies to NP from AIV subtype H1N1 (Varich and Kaverin, 2004;Yang et al, 2008).…”

“…An approach used to circumvent this limitation is to increase the solubility of expressed recombinant proteins through the use of vectors harboring peptide and tags, such as the small ubiquitin-like modifier (SUMO) peptide (Guerrero et al, 2015;Zuo et al, 2005). Such vectors have been constructed for cloning and expression in E. coli systems, but to date this approach has not been used for the expression of AIV proteins.…”

Enhanced expression of the structural protein of porcine reproductive and respiratory syndrome virus (PRRSV) by SUMO fusion

“…SUMO proteases remove SUMO from proteins by cleaving the C-termini of SUMO (-GGATY) in yeast to the mature form (-GG) or deconjugating it from lysine side chains (Li and Hochstrasser, 1999;2000). Proteins such as SARS virus protein (Zuo et al, 2005), MMP13 (Marblestone et al, 2006), EGF (Su et al, 2006), metallothionein (Huang et al, 2009), and KGF2 (Wu et al, 2009) have been successfully expressed and purified using this fusion strategy. Recently, Liu et al (2008) have created an R64T, R71E double mutant of yeast SUMO (smt3), termed SUMOstar, which is not recognized by native desumoylases .…”