Cold Shock Vectors for Single Protein Production

描述（由申请人提供）：是否可能在大肠杆菌中以极大的量仅产生单一的目的蛋白质。大肠杆菌，使结构测定的蛋白质可以进行没有纯化？在目前的建议中，我们将尝试开发一种新的冷休克载体宿主系统，将E。大肠杆菌细胞转化为这样的蛋白质合成机器，该机器致力于在冷休克时以非常高的产量仅生产单一的感兴趣的蛋白质。在过去的12年里，我们一直致力于研究在E.杆菌特别是大肠杆菌中主要的冷休克蛋白CspA的mRNA表达。在大肠杆菌中，这种mRNA以非常高的水平被诱导，并以高效的方式被翻译，使得所有细胞的核糖体都可以被这种mRNA捕获。这种效应（称为LACE效应）由于几乎完全抑制所有其他细胞蛋白质而导致完全细胞生长抑制。在本研究中，我们将利用cspA启动子和cspA mRNA的独特功能，尝试构建冷休克载体，在大肠杆菌中专门生产目的蛋白。杆菌为了实现我们的目标，将改进宿主菌株以进一步稳定克隆基因的mRNA，更高的翻译起始，并防止冷休克时其他细胞蛋白质的合成。在初步结果中，使用原型冷休克载体系统可以以非常高的产率产生很少的真核蛋白，所述真核蛋白使用常规T7表达系统产生非常差或根本不产生。利用所提出的系统，蛋白质生产仅在低温下诱导，因此与其他系统相比，将以更好的效率生产不稳定的蛋白质。我们的目标是在冷休克后实现靶蛋白合成的90%以上的效率和克隆的基因产物相对于总细胞蛋白的70%以上的产率。因此，可以容易地纯化克隆的基因产物。此外，可以用同位素如13-C和15-N特异性标记靶蛋白，使得可以在通过离心简单地除去不溶性物质后直接用细胞裂解物进行NMR光谱分析，而无需任何进一步纯化。所提出的载体-宿主系统不仅对结构生物学非常有用，而且对涉及人类蛋白质的医学研究也非常有用，这是通过常规表达系统无法获得的。

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.