Escherichia coli

About: Escherichia coli is a research topic. Over the lifetime, 59041 publications have been published within this topic receiving 2050337 citations. The topic is also known as: E. coli & E coli jdj.

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

Abstract: 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. 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. 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.

Expression and regulation of Escherichia coli lacZ gene fusions in mammalian cells.

Abstract: Gene fusions between the Escherichia coli lacZ gene and DNA segments containing the simian virus 40 early promoter or the mouse mammary tumor virus (MMTV) promoter direct the synthesis of functional beta-galactosidase in Cos 7 monkey cells and mouse Ltk-cells. Enzymatic activity was measured either 72 h after transfection or in stable transformants. The sensitive beta-galactosidase assay was used to measure gene expression and to optimize the efficiency of DNA-mediated transfection. Glucocorticoids stimulated the production of beta-galactosidase when lacZ was fused to the hormonally regulated MMTV promoter.

High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes

Abstract: Many streptomycetes, including S. coelicolor A3(2), possess a potent methyl-specific restriction system which can present an effective barrier to the introduction of heterologous DNA. We have compared the efficiency of intergeneric conjugal transfer of different types of plasmids to S. coelicolor and S. lividans 66 using two E. coli donors: the standard, methylation proficient strain S17-1. and the methylation deficient donor, ET12567(pUB307). We demonstrate that the methylation deficient donor can yield > 104-fold more S. coelicolor exconjugants than the standard donor. In the case of pSET152 derivatives, which integrate into the host chromosome by site-specific recombination, up to 10% of streptomycete spores in the conjugation mixture inherit the plasmid. The conjugation procedure is efficient enough to obtain exconjugants with ‘suicide’ delivery plasmids and therefore provides a simple route for conducting gene disruptions in methyl DNA-restricting streptomycetes, and possibly other bacteria.

Polynucleotide phosphorylase and ribonuclease II are required for cell viability and mRNA turnover in Escherichia coli K-12

Abstract: The isolation of a temperature-sensitive allele of RNase II (rnb) by in vitro mutagenesis has permitted the demonstration that RNase II and polynucleotide phosphorylase (PNPase) are required for cell viability and mRNA turnover in Escherichia coli. Double-mutant strains carrying the pnp-7 and rnb-500 alleles (PNPase deficient and RNase II thermolabile) ceased growing in Luria broth within 30 min after shift to the nonpermissive temperature. Cessation of growth was accompanied by an accumulation of mRNA fragments 100-1500 nucleotides long. In contrast, single-mutant and wild-type control strains grew normally at the nonpermissive temperature and did not accumulate mRNA. No significant changes in rRNA patterns were observed in any of the strains.