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.
Key words: 5-lipoxygenase activating protein (FLAP), membrane protein expression, Nickel affinity purification, SARS-CoV membrane protein, SUMO fusion
Abbreviations
- FLAP
5-lipoxygenase-activating protein
- FPLC
Fast Performance Liquid Chromatography
- IPTG
isopropyl-β-d-thiogalactopyranoside
- M protein of SARS-CoV
membrane protein of SARS coronavirus
- Ni-NTA
nickel-nitrilotriacetic acid
- PMSF
phenylmethylsulfonyl fluoride
- SARS
Severe Acute Respiratory Syndrome
- SARS-CoV
SARS coronavirus
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