By Peter E. Vaillancourt

ISBN-10: 1617373028

ISBN-13: 9781617373022

Peter E. Vaillancourt provides a set of well known and rising methodologies that reap the benefits of E. coli's skill to speedy and inexpensively show recombinant proteins. The authors specialise in parts of curiosity: using E. coli vectors and traces for creation of natural, sensible protein, and using E. coli as host for the sensible screening of enormous collections of proteins and peptides. one of the state-of-the-art thoughts established are these for fast high-level expression and purification of soluble and useful recombinant protein and people necessary to sensible genomics, proteomics, and protein engineering.

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E. 31F 43 8/6/2002, 3:49 PM 44 Xu and Evans Fig. 1. Purification of a recombinant protein expressed from a C-terminal fusion vector by cleavage at the N-terminus of a modified intein. The C-terminus of a target protein is fused in-frame to the N-terminus of an engineered intein and expressed in E. coli as a fusion protein consisting of the target protein-intein-chitin binding domain. The fusion protein is purified by binding to chitin resin. The target protein is released when a thioester bond formed at the intein N-terminal residue (Cys1) is attacked by a thiol compound (R-SH) such as DTT.

No. 500-0006). 2. Western Blot Analysis 1. 2. 3. 4. 5. 31F 50 SDS-PAGE and SDS-PAGE running buffer. Nitrocellulose (Schleicher & Schuell, cat. no. 10402599). Transfer buffer. Antichitin binding domain rabbit serum (NEB, cat. no. S6654S). Western blot detection system (Cell Signaling Technology, cat. no. 7071 or 7072). 8/6/2002, 3:49 PM Engineered Inteins 51 3. Methods Molecular Cloning: A Laboratory Manual edited by Sambrook et al. (17) is a good source of information for individuals unfamiliar with cloning or expression of foreign proteins in E.

And Bishop, J. M. (1985) Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol. Cell Biol. 5, 3610–3616. 7. , and Schacher, A. (1987) New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J. Chromatogr. 411, 177–184. 8. , and Vaillancourt, P. (2000) Recombinant Renilla reniformis GFP displays low toxicity. Strategies 13, 85–87. 9. , Wong, D. , et al. (2000) Sequence-validated and protein expression-tested human cDNA clones now available.

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E. coli Gene Expression Protocols by Peter E. Vaillancourt

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