High-throughput production of recombinant antigens for mouse KIAA proteins in Escherichia coli: computational allocation of possible antigenic regions, and construction of expression plasmids of glutathione-S-transferase-fused antigens by an in vitro recombination-assisted method.
High-throughput production of recombinant antigens for mouse KIAA proteins in Escherichia coli: computational allocation of possible antigenic regions, and construction of expression plasmids of glutathione-S-transferase-fused antigens by an in vitro recombination-assisted method.
复制标题
在大肠杆菌中高通量生产小鼠 KIAA 蛋白重组抗原：计算分配可能的抗原区域，并通过体外重组辅助方法构建谷胱甘肽-S-转移酶融合抗原的表达质粒。
作者：
Y. Hara;K. Shimada;Hiroshi Kohga;O. Ohara;H. Koga
自2001年底以来，我们已经进行了一个项目，以分离和确定小鼠cDNA克隆的完整序列，这些克隆编码与人KIAA蛋白相对应的多肽。为了实现该项目的最终目标，以澄清KIAA基因的生物学功能，我们已经设定了基于其序列信息的小鼠KIAA基因产物的抗体的生产作为下一个重要阶段。作为第一步，我们开发了一个高通量系统，利用鸟枪克隆在整个测序小鼠KIAA cDNA。该系统由以下三部分组成：（1）利用新开发的浏览器系统筛选出适合于抗原生产的编码区的鸟枪克隆;（2）通过体外重组辅助法将所选鸟枪克隆的蛋白编码序列转移到96孔板的表达载体中，并在大肠杆菌中表达谷胱甘肽S-转移酶融合蛋白;和（3）在小规模培养中初步评估重组抗原的溶解度，然后根据其溶解度使用谷胱甘肽亲和珠或从聚丙烯酰胺凝胶回收进行大规模生产和纯化。使用这些系统，我们成功地生产和纯化了400种抗原，用于生产mKIAA抗体。
Since the end of 2001, we have conducted a project to isolate and determine entire sequences of mouse cDNA clones which encode the polypeptides corresponding to human KIAA proteins. Towards the ultimate goal of this project to clarify the biological functions of KIAA genes, we have set production of antibodies against mouse KIAA gene products based on their sequence information as the next important stage. As the first step, we developed a high-throughput system utilizing shotgun clones generated during entire sequencing of mouse KIAA cDNAs. The system consists of the following three parts: (1) Shotgun clones encoding regions suitable for production of antigens were selected using a newly developed browser system; (2) the protein-coding sequences of the selected shotgun clones were transferred into an expression vector by in vitro recombination-assisted method in a 96-well format, and expressed as glutathione S-transferase fusion proteins in Escherichia coli; and (3) the solubility of the recombinant antigens were preliminarily assessed in a small-scale culture and then large-scale production and purification was performed using glutathione-affinity beads or retrieval from polyacrylamide gels depending on their solubility. Using these systems, we successfully produced and purified 400 antigens for production of mKIAA antibodies to date.