Goward C R, Murphy J P, Atkinson T, Barstow D A
Division of Biotechnology, PHLS Centre for Applied Microbiology and Research, Salisbury, Wilts, U.K.
Biochem J. 1990 Apr 1;267(1):171-7. doi: 10.1042/bj2670171.
The gene for Protein G from Streptococcus strain G148 was cloned and expressed in Escherichia coli. The regions on the gene corresponding to the albumin-binding domains and the Fab-binding region were then deleted by site-directed mutagenesis. The translation of regions corresponding to the cell-wall- and membrane-binding domains was prevented by introduction of stop codons upstream of these domains. This recombinant DNA sequence codes for a protein (G') that contains repetitive regions and that binds only the Fc portion of IgG, analogously to Protein A. Translation of the sequence produces a protein with an Mr of about 20,000. The nucleotide sequence differs from those published previously [Guss, Eliasson, Olsson, Uhlén, Frej, Jornvall, Flock & Lindberg (1986) EMBO J. 5, 1567-1575; Olsson, Eliasson, Guss, Nilsson, Hellman, Lindberg & Uhlén (1987) Eur. J. Biochem. 168, 319-324]. The protein can be substantially purified on a large scale by chromatography on IgG-Sepharose 4B. Homogeneous Protein G' can be prepared by anion-exchange f.p.l.c. on Mono Q HR. This Protein G' has a pI of 4.19 and SDS/PAGE gives an apparent anomalous Mr of 35,000.
来自链球菌菌株G148的蛋白G基因被克隆并在大肠杆菌中表达。然后通过定点诱变删除该基因上与白蛋白结合结构域和Fab结合区域相对应的区域。通过在这些结构域上游引入终止密码子来阻止与细胞壁和膜结合结构域相对应区域的翻译。这种重组DNA序列编码一种蛋白质（G'），它含有重复区域，并且只与IgG的Fc部分结合，类似于蛋白A。该序列的翻译产生一种Mr约为20,000的蛋白质。其核苷酸序列与先前发表的序列不同[古斯、埃利亚松、奥尔松、于伦、弗雷伊、约恩瓦尔、弗洛克和林德伯格（1986年）《欧洲分子生物学组织杂志》5，1567 - 1575；奥尔松、埃利亚松、古斯、尼尔松、赫尔曼、林德伯格和于伦（1987年）《欧洲生物化学杂志》168，319 - 324]。该蛋白质可以通过在IgG - Sepharose 4B上进行色谱法大规模纯化。通过在Mono Q HR上进行阴离子交换快速蛋白质液相色谱法可以制备均一的蛋白G'。这种蛋白G'的pI为4.19，SDS/PAGE给出的表观异常Mr为35,000。
