PMID:20440843
| Citation |
Mansell, TJ, Linderman, SW, Fisher, AC and DeLisa, MP (2010) A rapid protein folding assay for the bacterial periplasm. Protein Sci. 19:1079-90 |
|---|---|
| Abstract |
An array of genetic screens and selections has been developed for reporting protein folding and solubility in the cytoplasm of living cells. However, there are currently no analogous folding assays for the bacterial periplasm, despite the significance of this compartment for the expression of recombinant proteins, especially those requiring important posttranslational modifications (e.g., disulfide bond formation). Here, we describe an engineered genetic selection for monitoring protein folding in the periplasmic compartment of Escherichia coli cells. In this approach, target proteins are sandwiched between an N-terminal signal recognition particle (SRP)-dependent signal peptide and a C-terminal selectable marker, TEM-1 beta-lactamase. The resulting chimeras are localized to the periplasmic space via the cotranslational SRP pathway. Using a panel of native and heterologous proteins, we demonstrate that the folding efficiency of various target proteins correlates directly with in vivo beta-lactamase activity and thus resistance to ampicillin. We also show that this reporter is useful for the discovery of extrinsic periplasmic factors (e.g., chaperones) that affect protein folding and for obtaining folding-enhanced proteins via directed evolution. Collectively, these data demonstrate that our periplasmic folding reporter is a powerful tool for screening and engineering protein folding in a manner that does not require any structural or functional information about the target protein. |
| Links |
PubMed PMC2868249 Online version:10.1002/pro.388 |
| Keywords |
Amino Acid Sequence; Ampicillin Resistance/genetics; Bacterial Proteins/metabolism; Blotting, Western; Colony Count, Microbial; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Models, Biological; Molecular Sequence Data; Oxidation-Reduction; Periplasm/metabolism; Protein Disulfide-Isomerases/genetics; Protein Disulfide-Isomerases/metabolism; Protein Engineering/methods; Protein Folding; Protein Multimerization; Signal Recognition Particle/genetics; Signal Recognition Particle/metabolism; Solubility; beta-Lactamases/genetics; beta-Lactamases/metabolism |
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