Protein engineering by cDNA recombination in yeasts: shuffling of mammalian cytochrome P-450 functions.
We have constructed, in the yeast Saccharomyces cerevisiae, a mosaic assembly of genes by in vivo recombination of partially homologous sequences. The approach was tested on cDNAs encoding functionally distinct mammalian cytochromes P-450 (P-450). The selection for recombinant cDNAs used the transformation of yeast cells, which required the recircularization of a linearized plasmid by recombination of two partially homologous cDNAs. Libraries of mosaic genes with bipartite or tripartite structures were generated by intramolecular and intermolecular recombination events. The presence of yeast promoter and terminator sequences on the flanking sides of the recombined cDNAs has allowed the synthesis of encoded mosaic proteins. A library of yeast clones producing recombinant mouse P-450 P1 and rabbit P-450 LM4 was screened using functional criteria to identify chimeras with shuffled substrate specificity. Restriction mapping of mosaic genes, biochemical analysis of the synthesized proteins, comparison of chimeric enzymes, and the alignment of sequences with bacterial P-450 camphor hydroxylase of known three-dimensional structure, all suggest that the P-450 P1 amino acid residues 203-238 play a major role in the control of cytochrome activity toward carcinogenic polycyclic aromatic hydrocarbons. Similar approaches to structure-function analysis are believed to be applicable to other protein families.
Pompon
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Centre de Génétique Moléculaire, C.N.R.S. Laboratoire propre Associé à l'Université Pierre et Marie Curie, Gif-sur-Yvette, France.
Nicolas
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A
D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004274:DNA, Recombinant / Q000032:analysis*; D015870:Gene Expression; D015723:Gene Library; D009030:Mosaicism; D010957:Plasmids; D015202:Protein Engineering / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D003577:Cytochrome P-450 Enzyme System