A Vector Suite for the Overexpression and Purification of Tagged Outer Membrane, Periplasmic, and Secreted Proteins in E. coli
Outer membrane and secreted proteins in Gram-negative bacteria constitute a high percentage of virulence factors that are critical in disease initiation and progression. Despite their importance, it is often difficult to study these proteins due to challenges with expression and purification. Here we present a suite of vectors for the inducible expression of N-terminally 6His-tagged outer membrane, periplasmic, and secreted proteins in E. coli and show this system to be capable of producing milligram quantities of pure protein for downstream functional and structural analysis. This system can not only be used to purify recombinant virulence factors for structural and functional studies but can also be used to create gain-of-function E. coli for use in phenotypic screens, and examples of each are provided herein.
KeywordsOmpA TamA SufI Periplasm Protein secretion Sec apparatus Twin-arginine translocation Membrane protein Protein purification Protein expression
Research was supported by start-up funding from the Department of Biochemistry at Virginia Tech, the Institute for Critical Technology and Applied Science (ICTAS) at Virginia Tech, and the National Institute for Food and Agriculture.
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