Cold Shock Vectors for Single Protein Production

描述(申请人提供)：是否有可能在大肠杆菌中大量生产一种感兴趣的单一蛋白质，从而无需纯化即可对该蛋白质进行结构测定？在目前的方案中，我们将尝试开发一种新型的冷休克载体宿主系统，将大肠杆菌细胞转化为这样一种蛋白质合成机器，专门用于在冷休克时以非常高的产量生产单一感兴趣的蛋白质。在过去的12年里，我们一直在研究一些蛋白质，这些蛋白质在大肠杆菌中温度下降时，特别是在极高的水平上诱导出来的。特别是，大肠杆菌中主要的冷休克蛋白CSPA的mRNA在非常高的水平被诱导，并以一种高效的方式被翻译，从而使所有的细胞核糖体都能被这种mRNA捕获。这种效应(称为花边效应)由于几乎完全抑制了所有其他细胞蛋白质，从而导致完全抑制细胞生长。在本方案中，利用CSPA启动子和CSPA mRNA的独特功能，我们将尝试构建冷休克载体，以在大肠杆菌中独家生产目的蛋白。为了达到我们的目标，将对宿主菌株进行改进，以进一步稳定克隆基因的mRNA，提高翻译起始值，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量生产出很少使用传统T7表达系统产生的或者根本不产生的真核蛋白。与其他系统相比，该系统只在较低的温度下才能诱导蛋白质的产生，因此产生不稳定的蛋白质的效率更高。我们的目标是在冷休克时达到90%以上的目标蛋白合成效率，以及克隆的基因产物相对于细胞总蛋白的70%以上的产率。因此，克隆的基因产物可以很容易地提纯。此外，目标蛋白可以专门标记13-C和15-N等同位素，这样只需通过离心法去除不溶物质而无需进一步纯化，即可直接用细胞裂解产物进行核磁共振波谱分析。所提出的载体-宿主系统不仅在结构生物学方面非常有用，而且在涉及传统表达系统无法获得的人类蛋白质的医学研究中也将非常有用。

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.