Cold Shock Vectors for Single Protein Production

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DESCRIPTION (provided by applicant): Is it possible to produce only a single protein of interest in an extremely large amount in E. coli so that structural determination of that protein can be carried out without purification? In the present proposal, we will attempt to develop a novel cold-shock-vector-host system that converts E. coli cells into such a protein-synthesizing machinery dedicated to the production of only a single protein of interest in a very high yield upon cold shock. For the last 12 years, we have been working on a number of proteins specifically induced at extremely high levels upon temperature downshift in E. coli. In particular, the mRNA for CspA, the major cold shock protein in E. coli is induced at a very high level, and is translated in a highly efficient manner so that all cellular ribosomes can be trapped with this mRNA. This effect (called the LACE effect) causes complete cell-growth inhibition as a result of almost complete inhibition of all the other cellular proteins. In the present proposal, using the unique features of the cspA promoter and the cspA mRNA, we will attempt to construct cold-shock vectors for exclusive production of a protein of interest in E. coli. In order to achieve our goal, the host strain will be improved for further stabilization of the mRNA for the cloned gene, higher translation initiation, and prevention of other cellular protein synthesis upon cold shock. In a preliminary result, few eukaryotic proteins that are either produced very poorly or not produced at all using the conventional T7 expression system can be produced at a very high yield using a prototype cold-shock vector system. With the proposed system, protein production is induced only at low temperatures, therefore unstable proteins will be produced in a better efficiency as compared with other systems. Our goal is to achieve more than 90% efficiency for a target protein synthesis upon cold shock and more than 70 % yield for the cloned gene product with respect to the total cellular proteins. Thus, a cloned gene product can be easily purified. Furthermore, a target protein can be specifically labeled with isotopes such as 13-C and 15-N so that NMR spectroscopy can be carried out directly with cell lysates after simply removing insoluble materials by centrifugation without any further purification. The proposed vector-host system will be highly useful not only for structural biology but also for medicinal research involving human proteins, which can not be obtained by conventional expression systems.