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Published online 19 March 2004

Nucleic Acids Research, 2004, Vol. 32, No. 5 e52
Oxford University Press

Artificial genetic selection for an efficient translation initiation site for expression of human RACK1 gene in Escherichia coli

Olga B. Zhelyabovskaya, Yuri A. Berlin and Klara R. Birikh*

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow 117997, Russia

*To whom correspondence should be addressed. Tel: +7 095 3364200; Fax: +7 095 3357103; Email: kbirikh{at}ibch.ru
This work is dedicated to the memory of our teacher Professor Yuri Berlin, who died on November 23, 2001

Received January 20, 2004; Revised February 19, 2004; Accepted February 27, 2004

In bacterial expression systems, translation initiation is usually the rate limiting and the least predictable stage of protein synthesis. Efficiency of a translation initiation site can vary dramatically depending on the sequence context. This is why many standard expression vectors provide very poor expression levels of some genes. This notion persuaded us to develop an artificial genetic selection protocol, which allows one to find for a given target gene an individual efficient ribosome binding site from a random pool. In order to create Darwinian pressure necessary for the genetic selection, we designed a system based on translational coupling, in which microorganism survival in the presence of antibiotic depends on expression of the target gene, while putting no special requirements on this gene. Using this system we obtained superproducing constructs for the human protein RACK1 (receptor for activated C kinase).


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