Effect of oxidized and reduced forms of Escherichia coli DsbC on protein refolding.
ABSTRACT DsbC, which catalyzes disulfide isomerization, was overproduced in the periplasm of Escherichia coli and purified from the periplasmic fraction by osmotic shock and anion-exchange chromatography. The active site of the purified DsbC was found to be an oxidized form (ox-DsbC) which could be converted to the reduced form (red-DsbC) by the addition of dithiothreitol. The effect of ox- and red-DsbC on the refolding of chemically denatured and reduced proteins with different numbers of disulfide bonds and free cysteine-thiol groups was investigated. Ox-DsbC facilitated the refolding of proteins with multiple disulfide bonds in both oxidative and reductive environments, while red-DsbC facilitated refolding only in the former. On the other hand, only red-DsbC facilitated the refolding of proteins with multiple free cysteine-thiol groups but either form of DsbC did not facilitate the refolding of proteins with only one cysteine-thiol group. It is therefore important to choose the form which suits the properties of the protein. Holo-chaperonin from Thermus thermophilus and DsbC demonstrated a synergistic effect on protein refolding.
- [Show abstract] [Hide abstract]
ABSTRACT: Due to its small size and intense luminescent signal, Gaussia princeps luciferase (GLuc) is attractive as a potential imaging agent in both cell culture and small animal research models. However, recombinant GLuc production using in vivo techniques has only produced small quantities of active luciferase, likely due to five disulfide bonds being required for full activity. Cell-free biology provides the freedom to control both the catalyst and chemical compositions in biological reactions, and we capitalized on this to produce large amounts of highly active GLuc in cell-free reactions. Active yields were improved by mutating the cell extract source strain to reduce proteolysis, adjusting reaction conditions to enhance oxidative protein folding, further activating energy metabolism, and encouraging post-translational activation. This cell-free protein synthesis procedure produced 412mug/mL of purified GLuc, relative to 5mug/mL isolated for intracellular Escherichia coli expression. The cell-free product had a specific activity of 4.2x10(24)photons/s/mol, the highest reported activity for any characterized luciferase.Metabolic Engineering 01/2008; 10(3-4):187-200. · 6.86 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: BACKGROUND: Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli 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. RESULTS: We have engineered a new E. coli 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 trxB gor 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 E. coli 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. CONCLUSIONS: This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using SHuffle strains.Microbial Cell Factories 05/2012; 11(1):56. · 3.31 Impact Factor