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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Dec 15;90(24):11456–11459. doi: 10.1073/pnas.90.24.11456

Structurally engineered cytochromes with unusual ligand-binding properties: expression of Saccharomyces cerevisiae Met-80-->Ala iso-1-cytochrome c.

Y Lu 1, D R Casimiro 1, K L Bren 1, J H Richards 1, H B Gray 1
PMCID: PMC48002  PMID: 8265573

Abstract

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 horseradish 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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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