Moran G R, Entsch B
Department of Biochemistry, Microbiology and Nutrition, University of New England, Armidale, NSW, Australia.
Protein Expr Purif. 1995 Apr;6(2):164-8. doi: 10.1006/prep.1995.1020.
We report a PCR deletion mutagenesis method for the exact positioning of a foreign gene (pobA) in the lac operon of an expression plasmid in place of the lacZ protein code. This method requires the synthesis of four oligonucleotides and three PCR reactions to delete unwanted bases and retain the nucleotide sequence naturally found between the lac promoter and the protein code. The engineered plasmid results in the production of at least 40% of the cellular protein as the foreign polypeptide. In the example presented the expression of the protein is high even with a substantial difference in codon usage between the host (Escherichia coli) and a foreign gene from Pseudomonas aeruginosa. Some of the polypeptide produced has the ame properties as native protein and is easily purified. The remainder is present as insoluble inclusion bodies. This method of plasmid refinement may be applicable to the expression of many proteins.
我们报道了一种PCR缺失诱变方法，用于将外源基因（pobA）精确地定位到表达质粒的乳糖操纵子中，以取代乳糖Z蛋白编码。该方法需要合成四条寡核苷酸并进行三轮PCR反应，以删除不需要的碱基，并保留乳糖启动子和蛋白编码之间天然存在的核苷酸序列。构建的质粒能够产生至少40%的细胞蛋白作为外源多肽。在给出的例子中，即使宿主（大肠杆菌）和来自铜绿假单胞菌的外源基因在密码子使用上存在显著差异，该蛋白的表达量仍然很高。产生的一些多肽具有与天然蛋白相同的性质，并且易于纯化。其余的则以不溶性包涵体的形式存在。这种质粒优化方法可能适用于多种蛋白质的表达。
