Markmeyer P, Rühlmann A, Englisch U, Cramer F
Max-Planck-Institut für experimentelle Medizin, Abteilung Chemie, Göttingen, F.R.G.
Gene. 1990 Sep 1;93(1):129-34. doi: 10.1016/0378-1119(90)90146-i.
A family of plasmid cloning vectors have been constructed, allowing both the sequencing and mutagenesis of foreign genes and the easy isolation of their expression products via fusion proteins in Escherichia coli. Fusion proteins can be inducibly expressed and isolated by affinity chromatography on APTG-Sepharose. The fusion protein consists of beta-galactosidase at the N-terminus, linked by a collagen 'hinge' region containing blood coagulation factor Xa cleavage site to the foreign protein at the C terminus. The factor Xa cleavage site at the N-terminal side of the foreign protein allows the release of the desired amino acid sequence under mild conditions. A multiple cloning site in all three reading frames and stop codons followed by the strong lambda t0 terminator facilitate simple gene insertions and manipulations. The intergenic region of the phage f1 inserted in both orientations allows the isolation of single-stranded DNA from either plasmid-strand for sequencing and mutagenesis. This vector family has been successfully used for the expression and purification of the isoleucyl-tRNA synthetase from Saccharomyces cerevisiae and the histidyl-tRNA synthetase from E. coli.
已经构建了一个质粒克隆载体家族，它能够对外源基因进行测序和诱变，并能通过在大肠杆菌中形成融合蛋白轻松分离其表达产物。融合蛋白可被诱导表达，并通过在APTG-琼脂糖上进行亲和层析来分离。融合蛋白在N端由β-半乳糖苷酶组成，通过含有凝血因子Xa切割位点的胶原蛋白“铰链”区域与C端的外源蛋白相连。外源蛋白N端一侧的因子Xa切割位点能够在温和条件下释放所需的氨基酸序列。所有三个阅读框中的多克隆位点以及紧随强λ t0终止子之后的终止密码子便于简单的基因插入和操作。以两种方向插入的噬菌体f1的基因间区域使得能够从任一质粒链分离单链DNA用于测序和诱变。这个载体家族已成功用于酿酒酵母异亮氨酰-tRNA合成酶和大肠杆菌组氨酰-tRNA合成酶的表达与纯化。
