Enhanced Expression and Purification of Membrane Proteins by SUMO Fusion in Escherichia coli

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV) membrane protein and 5-lipoxygenase-activating protein (FLAP) are among a large number of membrane proteins that are poorly expressed when traditional expression systems and methods are employed. Therefore to efficiently express difficult membrane proteins, molecular biologists will have to develop novel or innovative expression systems. To this end, we have expressed the SARS-CoV M and FLAP proteins in Escherichia coli by utilizing a novel gene fusion expression system that takes advantage of the natural chaperoning properties of the SUMO (small ubiquitin-related modifier) tag. These chaperoning properties facilitate proper protein folding, which enhances the solubility and biological activity of the purified protein. In addition to these advantages, we found that SUMO Protease 1, can cleave the SUMO fusion high specificity to generate native protein. Herein, we demonstrate that the expression of FLAP and SARS-CoV membrane proteins are greatly enhanced by SUMO fusions in E. coli.

Expression and purification of antimicrobial peptide buforin IIb in Escherichia coli

“…Our result suggested that Trx fusion partner was a superior fusion tag in enhancing buforin IIb expression solubility. Our result was contrary to a previous report that also suggested that subtraction purification with affinity chromatography was particularly useful for the production of small peptides, as they were easily lost during purification with non-affinity chromatographic methods (Loll 2003;Zuo et al 2005). Besides, because of the toxicity and the high cost of IPTG, lactose is a promising inducer in the lacpromotor based expression system.…”

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.…”

Enhanced protein expression in mammalian cells using engineered SUMO fusions: Secreted phospholipase A2

“…Fusion to SUMO has been shown to enhance the expression of recombinant proteins in Escherichia coli (Malakhov et al 2004;Butt et al 2005;Zuo et al 2005;Marblestone et al 2006;Panavas et al 2008), while fusion to hSUMO3 led to similar enhancement in E. coli (data not shown) and Pichia pastoris (R.J. Peroutka, D. Roy, J. Strickler, and T.R. Butt, in prep.).…”

“…Paradoxically, lysine residues in target proteins that are SUMOylated are protected from ubiquitylation, while SUMOylation of proteins can also serve as a signal for ubiquitylation (Prudden et al 2007). Thus, nature has evolved two pathways that have remarkably similar mechanical and biochemical features, yet have very distinct roles in cellular physiology (Johnson 2004).Fusion to SUMO has been shown to enhance the expression of recombinant proteins in Escherichia coli (Malakhov et al 2004;Butt et al 2005;Zuo et al 2005;Marblestone et al 2006;Panavas et al 2008), while fusion to hSUMO3 led to similar enhancement in E. coli (data not shown) and Pichia pastoris (R.J. Peroutka, D. Roy, J. Strickler, and T.R. Butt, in prep.).…”