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Journal of Bacteriology, January 2003, p. 475-481, Vol. 185, No. 2
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.2.475-481.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Combinatorial Redesign of the DNA Binding Specificity of a Prokaryotic Helix-Turn-Helix Repressor

Katja Fromknecht,1,{dagger} Pia D. Vogel,1,{ddagger} and John G. Wise1,2*

Fachbereich Chemie, Universität Kaiserslautern, D-67653 Kaiserslautern, Germany,1 Gene Products, L.L.C., Dallas, Texas 75275-03762

Received 3 June 2002/ Accepted 18 October 2002

Redesign of the bacteriophage 434 Cro repressor was accomplished by using an in vivo genetic screening system to identify new variants that specifically bound previously unrecognized DNA sequences. Site-directed, combinatorial mutagenesis of the 434 Cro helix-turn-helix (HTH) motif generated libraries of new variants which were screened for binding to new target sequences. Multiple mutations of 434 Cro that functionally converted wild-type (wt) 434 Cro DNA binding-sequence specificity to that of a {lambda} bacteriophage-specific repressor were identified. The libraries contained variations within the HTH sequence at only three positions. In vivo and in vitro analysis of several of the identified 434 Cro variants showed that the relatively few changes in the recognition helix of the HTH motif of 434 Cro resulted in specific and tight binding of the target DNA sequences. For the best 434 Cro variant identified, an apparent Kd for {lambda} OR3 of 1 nM was observed. In competition experiments, this Cro variant was observed to be highly selective. We conclude that functional 434 Cro repressor variants with new DNA binding specificities can be generated from wt 434 Cro by mutating just the recognition helix. Important characteristics of the screening system responsible for the successful identifications are discussed. Application of the techniques presented here may allow the identification of DNA binding protein variants that functionally affect DNA regulatory sequences important in disease and industrial and biotechnological processes.

* Corresponding author. Mailing address: Gene Products, L.L.C., 6501 Airline Rd., Dallas, TX 75275-0376. Phone: (972) 816-2802. Fax: (214) 768-3955. E-mail: jwise{at}gene-products.com.

{dagger} Present address: MWG Biotech AG, D-85560 Ebersberg, Germany.

{ddagger} Present address: Department of Biological Sciences, Southern Methodist University, Dallas TX 75275.

Journal of Bacteriology, January 2003, p. 475-481, Vol. 185, No. 2
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.2.475-481.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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