Construction and application of cloning vector which carries signal sequence of Escherichia coli.

本项目旨在阐明大肠杆菌外膜蛋白之一耐洗涤磷脂酶a （DR-phospholipase a）的定位和加工的分子机制。我们还尝试构建了一个利用DR-磷脂酶a .1信号序列的克隆载体。dr -磷脂酶A前体分子的生化和遗传分析：克隆了dr -磷脂酶A的结构基因pldA。对pldA的体外产物进行了分析，发现其分子量略高于体内产物。在另一项实验中，在编码dr -磷脂酶a信号序列的DNA区域中引入了位点定向突变，插入了一个氨基酸，发现突变的pldA基因产生的酶量较少。这些结果强烈提示dr -磷脂酶a的前体分子的存在，突变的pldA基因产生dr -磷脂酶a的数量较少可能是由于加工步骤效率低下。克隆载体的构建：在编码DR-磷脂酶A信号序列的DNA区域后立即引入含有多个克隆位点的合成DNA片段，然后用三个特征明确的启动子（tac、consensus和lambda PL）分别取代pldA基因的启动子。载体的应用：将合成的人尿胃溶菌酶原编码基因克隆到这些新型克隆载体上。我们检测了携带重组质粒的大肠杆菌菌株对人蛋白的表达。到目前为止，已观察到一种成熟形式的人溶菌酶的产生和积累，但产量很低。必须对表达式进行进一步改进。

The aim of this project was to clarify a molecular mechanism of location and processing of one of E. coli outer membrance protein, detergent resistant phospholipase A (DR-phospholipase A). We also attempted to construct a cloning vector which utilized the signal sequence of DR- phospholipase A.1. Biochemical and genetical analysis of DR-phospholipase A precursor molecule:The structure gene of DR-phosphclipase A, pldA, was cloned. The in vitro product of pldA was analyzed and was found to bear a molecular weight slightly higher than that generated in vivo< In other experiment, a site-directed mutagenesis, which resulted in one amino acid insertgion, was introduced into the DNA region coding for the signal sequence of the DR-phospholipase A. The mutated pldA gene was found to produce less amount of enzyme. These results strongly suggested the existence of a precursor molecule of DR-phospholipase A. Less amount of production of DR-phospholipase A from mutated pldA gene might be due to the inefficient processing step.2. Construction of cloning vector:A synthetic DNA fragment containing multi-cloning sites was introduced immediately after the DNA region which coded for the signal sequence of DR- phospholipase A. The promoter of pldA gene was then replaced by three well-characterized promoters (tac, consensus, and lambda PL), respectively.3. Application of vectors:Synthetic genes coding for fuman urogastron of lysozyme were cloned onto these novel cloning vectors. E. coli strains bearing recombinant plasmids were examined for the expression of human proteins. So far, production and accumulation of a mature form of human lysozyme was observed, however, the yield was low. Further improvement in the expression must be performed.