Proteins E. coli

Walkup, L.K. Kogoma, T. (1989). E. coli proteins inducible by oxidative stress mediated by the superoxide radical. J. Bacteriol. 171, 1476-1484. 

M. T. Record, Jr., E. S. Courtenay, S. Caley, and H. J. Guttman, Biophysical compensation mechanisms buffering E. coli protein—nucleic acid interactions against changing environments,... 

Chong, B. E. Wall, D. B. Lubman, D. M. Flynn, S. J. Rapid profiling of E. coli proteins up to 500 kDa from whole cell lysates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Comm. Mass Spectrom. 1997,11,1900-1908. 

Tang, Q., Harrata, A. K., Lee, C. S. (1997). Two-dimensional analysis of recombinant E. coli proteins using capillary isoelectric focusing electrospray ionization mass spectrometry. Anal. Chem. 69(16), 3177-3182. 

Chemicals, E. coli proteins, fermentation products, foreign DNA)... 

In a pioneering attempt, Sjostrom et al. (1987) performed a multivariate data analysis on the N-terminal signal peptides of E. coli proteins. 

There are many examples of ELIS As used for detecting host cell impurities in the literature. Pauly et al.12 developed an ELISA to detect impurities in erythropoietin that had a detection limit of around 0.05 ng/ml. SDS polyacrylamide gel and Western blot analysis were used to confirm the spectrum of proteins detected and to demonstrate the specificity of the antibody preparation. Anicetti et al.14 describe an assay for the detection of E. coli proteins in recombinant DNA-derived human growth hormone. Whitmire and Eaton15 report on an immuno-ligand assay for quantitation of process-specific E. coli host cell contaminant proteins in a recombinant bovine somatotropin. 

The Western blot method is often used in the analysis of host cell impurities. It can be used to identify a recurring impurity. O Keefe et al. used a Western blot to identify an E. coli protein impurity in the preparation of the recombinant fibroblast growth factor (aFGF).29 By using specific antisera to the E. coli host cell proteins, they were able to isolate the impurity and determine its N-terminus amino acid sequence to confirm its identity. Antibodies could be used to determine the concentration of this impurity in sample preparations. 

Because the sequencing of DNA has become so straightforward, genes of several ribosomal proteins have been sequenced to allow an independent determination of the primary sequence of some of the proteins (Post et ai, 1979 Olins and Nomura, 1981). These studies have confirmed the sequences previously elucidated by protein chemical techniques. In the case of SI (the largest of all E. coli proteins), the combination of amino acid- and nucleotide-sequence determinations was used to provide the sequence (Schnier et ai, 1982). 

Gateway cloning technology is a modification of the recombination system of phage k (Walhout et ah, 2000 Hartley et ah, 2000). The Gateway system utilizes a minimum set of components of the k system for in vitro transfer of DNA, namely the k integrase protein, k excisionase, the E. coli protein... 

Table 2.1 Common E. coli protein expression strains and their features...

PCR and in vitro recombination reactions are quite simple and straightforward for generating multiple expression plasmids in parallel, e.g., in a 96-well plate see Fig. 3a). The first preliminary expression experiment was done to evaluate the production level of each GST-fused protein. In this step, we compared the staining patterns of E. coli proteins harboring expression plasmids with the patterns of proteins harboring empty vectors on sodium dodecyle sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under the same culture conditions see Fig. 3b). In addition to... 

Originally, recombinant human GH (hGH) was expressed with an additional methionine at the N-terminal because all of the amino-acid sequences of E. coli proteins begin with an S-adenosyl methionine. 

An unusual cysteine sequence motif together with atypical EPR and Mossbauer properties has been reported earlier for a [Fe2S2] protein denoted FhuF. It is an iron-regulated E. coli protein which is probably involved in the reduction of ferric iron in ferrioxamine B.191 In a recent study the authors could provide evidence for the mixed valence state in the FhuF protein to be capable of... 

In subunit R2 of ribonucleotide reductase there is a tyrosyl radical (Y ) in close proximity to a di-iron cluster.100 In the protein from E. coli the EPR signal from Y can be observed up to room temperature. However, in the protein from yeast the Y signal broadens above 15 K and is not observable above about 60 K. Saturation recovery measurements at 140 GHz showed that at 60 K the spin-lattice relaxation rates for the Y signal in the yeast protein were about 2 orders of magnitude faster than for the E. coli protein. The temperature dependence of the relaxation enhancement was consistent with the activation energy for the first excited state of the di-iron cluster, so the relaxation enhancement was attributed to interaction with the di-iron cluster. Relaxation enhancements measured at 140 GHz showed little orientation dependence so the enhancement was assigned to isotropic exchange, which is different from the orientation-dependent dipolar interaction observed for the E. coli protein.100... 

Mitra et al. (2005) Structure of the E. coli protein-conducting channel bound to a translating ribosome. Nature 438 318-324... 

A closely related E. coli protein is a 79-kDa multifunctional enzyme that catalyzes four different reactions of fatty acid oxidation (Chapter 17). The amino-terminal region contains the enoyl hydratase activity.32 A quite different enzyme catalyzes dehydration of thioesters of (3-hydroxyacids such as 3-hydroxydecanoyl-acyl carrier protein (see Eq. 21-2) to both form and isomerize enoyl-ACP derivatives during synthesis of unsaturated fatty acids by E. coli. Again, a glutamate side chain is the catalytic base but an imidazole group of histidine has also been implicated.33 This enzyme is inhibited irreversibly by the N-acetylcysteamine thioester of 3-decynoic acids (Eq. 13-8). This was one of the first enzyme-activated inhibitors to be studied.34... 

Rho and other termination factors. Termination proteins can also react with specific regions of DNA or of an RNA transcript to terminate transcription.183 The best known termination factor is the rho protein a hexamer of 45-kDa subunits. It interacts with transcripts at specific termination sequences, which are often C-rich, and in a process accompanied by hydrolysis of ATP causes release of both RNA and the polymerase from the DNA.192193 Additional E. coli proteins, products of genes nus A and nus G, cooperate with the rho factor at some termination sequences.194-196c The rho hexamer is a helicase that moves along the RNA transcript in the 5 —> 3 direction driven by ATP hydrolysis. If it locates an appropriate termination signal, it may utilize its helicase activity to uncoil the DNA-RNA hybrid segment within the transcription bubble (Fig. 28-4).197 198b... 

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