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.

Formation of Soluble Recombinant Proteins in Escherichia Coli is Favored by Lower Growth Temperature

Abstract: Human interferon–α2 (IFN–α2), an 18 kD, acidic protein, human interferon–γ (IFN–γ), a 17 kD, basic protein, and the interferon–induced murine protein Mx (72 kD) were all found in aggregates when produced in genetically engineered strains of E. coli grown at 37°C. However, at a growth temperature of 23–30°C, 30–90% of the recombinant protein was soluble. The temperature effect was not directly dependent on the concentration of the protein and was observed for several E. coli strains and for different plasmid constructions. Lysates of non–transformed E. coli grown at either temperature rendered initially soluble human recombinant IFN–α2 insoluble at 37° but not at 0° or 30°C. Insolubilization was not abolished by nuclease treatment, and may involve sulfhydryl group shuffling, as sulfhydryl reducing agents added to a mock lysate gave a similar temperature dependent precipitation.

Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA.

Abstract: In bacteria, genetic recombination is catalysed by RecA protein, the product of the recA gene. A human gene that shares homology with Escherichia coli recA (and its yeast homologue RAD51) has been cloned from a testis cDNA library, and its 37 kDa product (hRad51) purified to homogeneity. The human Rad51 protein binds to single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using a topological assay, we demonstrate that hRad51 underwinds duplex DNA, in a reaction dependent upon the presence of ATP or its non-hydrolysable analogue ATP gamma S. Complexes formed with single- and double-stranded DNA have been observed by electron microscopy following negative staining. With nicked duplex DNA, hRad51 forms helical nucleoprotein filaments which exhibit the striated appearance characteristic of RecA or yeast Rad51 filaments. Contour length measurements indicate that the DNA is underwound and extended within the nucleoprotein complex. In contrast to yeast Rad51 protein, human Rad51 forms filaments with single-stranded DNA in the presence of ATP/ATP gamma S. These resemble the inactive form of the RecA filament which is observed in the absence of a nucleotide cofactor.

Escherichia coli promoter sequences predict in vitro RNA polymerase selectivity

Abstract: We describe a simple algorithm for computing a homology score for Escherichia coli promoters based on DNA sequence alone. The homology score was related to 31 values, measured in vitro, of RNA polymerase selectivity, which we define as the product KBk2, the apparent second order rate constant for open complex formation. We found that promoter strength could be predicted to within a factor of +/-4.1 in KBk2 over a range of 10(4) in the same parameter. The quantitative evaluation was linked to an automated (Apple II) procedure for searching and evaluating possible promoters in DNA sequence files.

Synthesis in E. coli of a polypeptide with human leukocyte interferon activity

Abstract: Double-stranded cDNA prepared from the 12S fraction of poly(A) RNA from interferon (IF)-producing human leukocytes was cloned in Escherichia coli using the pBR322 vector. One of the resulting clones had a 910-base pair insert which could hybridise to IF mRNA and was responsible for the production of a polypeptide with biological IF activity. Up to 10,000 units IF activity per g of cells was obtained from some clones.