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PEDS Advance Access published online on February 9, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzm002
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

A new application of the yeast two-hybrid system in protein engineering

Andreas Bichet1, Frank Hannemann1, Matthias Rekowski1 and Rita Bernhardt1,2

1 FR 8.3—Biochemie, Universität des Saarlandes, D-66041 Saarbrücken, Germany

2 To whom correspondence should be addressed. E-mail: ritabern{at}mx.uni-saarland.de

Cytochromes P450 are involved in the biosynthesis of steroid hormones in mitochondria of the adrenal gland. The electrons required for these reactions are provided via a redox chain consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx). A prerequisite for a fast and efficient electron transfer as well as high catalytic activity is the formation of functional complexes between the different redox partners. To improve the protein–protein interactions by directed evolution, we developed a new in vivo selection system. This high-throughput screening method is based on the yeast two-hybrid system. It enables a background-free screening for increased protein–protein interactions between stable and functional species including cofactor-containing proteins (FAD, [2Fe–2S], heme). The method was successfully applied for the directed evolution of Adx and selected variants were analyzed biochemically and biophysically. All analyzed proteins exhibit typical characteristics of [2Fe–2S]-cluster-type ferredoxins. Adx-dependent substrate conversion assays with different cytochromes demonstrated that the improved ability of the mutants to form complexes results in an enhanced catalytic efficiency of the cytochrome P450 system.

Keywords: cytochromes P450/directed evolution/ferredoxins/high-throughput screening method/two-hybrid system

Received September 4, 2006; revised December 7, 2006; accepted December 15, 2006.


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