Artificial genetic selection for an efficient translation initiation site for expression of human RACK1 gene in Escherichia coli.
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).
Zhelyabovskaya
Olga B
OB
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow 117997, Russia.
Berlin
Yuri A
YA
Birikh
Klara R
KR
D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D014176:Protein Biosynthesis; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012641:Selection, Genetic
D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins