Lu, Yi and Casimiro, Danilo R. and Bren, Kara L. and Richards, John H. and Gray, Harry B. (1993) Structurally engineered cytochromes with unusual ligand-binding properties: Expression of Saccharomyces cerevisiae Met-80 --> Ala iso-1-cytochrome c. Proceedings of the National Academy of Sciences of the United States of America, 90 (24). pp. 11456-11459. ISSN 0027-8424 http://resolver.caltech.edu/CaltechAUTHORS:LUYpnas93
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A strategy has been developed to express and purify a recombinant, nonfunctional axial-ligand mutant of iso-1-cytochrome c (Met-80 --> Ala) in Saccharomyces cerevisiae in quantities necessary for extensive biophysical characterization. It involves coexpressing in the same plasmid (YEp213) the nonfunctional gene with a functional gene copy for complementation in a selective medium. The functional gene encodes a product with an engineered metal-chelating dihistidine site (His-39 and Leu-58 --> His) that enables efficient separation of the two isoforms by immobilized metal-affinity chromatography. The purified Met-80 --> Ala protein possesses a binding site for dioxygen and other exogenous ligands. Absorption spectra of several derivatives of this mutant show striking similarities to those of corresponding derivatives of horse-radish peroxidase, myoglobin, and cytochrome P450. The use of a dual-gene vector for cytochrome c expression together with metal-affinity separation opens the way for the engineering of variants with dramatically altered structural and catalytic properties.
|Additional Information:||Copyright © 1993 by the National Academy of Sciences. Contributed by Harry B. Gray, August 23, 1993. We thank Michael Smith for the yeast iso-1-cytochrome c expression system, Frances Arnold for the metal-chelating cytochrome c gene, Torbjdorn Pascher for providing purified cytochrome P450(cam), and Lynn Williams for the sequence analyses. K.L.B. acknowledges a Kodak fellowship. This work was supported by the National Science Foundation, the National Institutes of Health, and the Arnold and Mabel Beckman Foundation. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.|
|Subject Keywords:||metal-affinity chromatography, protein engineering, site-directed mutagenesis, yeast iso-1-cytochrome-c, electron-transfer, heme ligand, replacement, proteins, semisynthesis, stability, mutants, invivo|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Tony Diaz|
|Deposited On:||09 Nov 2005|
|Last Modified:||26 Dec 2012 08:42|
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