SHuffle, a novel <it>Escherichia coli</it> protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm

• Main Authors: Lobstein Julie, Emrich Charlie A, Jeans Chris, Faulkner Melinda, Riggs Paul, Berkmen Mehmet
• Format: Article
• Language: English
• Published: BMC 2012-05-01
• Series: Microbial Cell Factories
• Subjects: SHuffle; Protein expression strain; Disulfide bond formation; Disulfide bond isomerization; DsbC; <it>trxB</it>; <it>gor</it>; <it>ahpC</it>*
• Online Access: http://www.microbialcellfactories.com/content/11/1/56

<p>Abstract</p> <p>Background</p> <p>Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in <it>Escherichia coli</it> is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood.</p> <p>Results</p> <p>We have engineered a new <it>E. coli</it> protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the <it>trxB gor</it> suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the <it>E. coli</it> B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins.</p> <p>Conclusions</p> <p>This work is the first extensive characterization of the <it>trxB gor</it> suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using SHuffle strains.</p>