Murphy J P, Atkinson T, Hartwell R, Stevens G B, Hinton R J, Goward C R
Division of Biotechnology, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, UK.
Bioseparation. 1992;3(1):63-71.
PCR was used to isolate the gene fragment coding for Protein G' (SpG'), a truncated bacterial cell surface protein from Streptococcus G148 which binds to the Fc region of IgG and expressed in E. coli [Goward et al. (1990) Biochem. J. 267: 171-177]. The PCR primer was designed to change the TTG initiation triplet to ATG and to incorporate it into an NdeI restriction site (CATATG), allowing the gene to be cloned in frame into an NdeI restriction site immediately downstream of a trp promoter. Expression of SpG' was estimated as about 30% total soluble cell protein which compares very favourably to the less than 1% total soluble cell protein obtained from the original system [Goward, et al. (1990) Biochem. J. 267: 171-177]. Homogeneous SpG' was recovered by a single anion-exchange chromatography step on Q-Sepharose FF in a process which avoided use of an affinity adsorbent. Even though SpG' consists of almost identical repetitive domains from amino acid sequence analysis, different proteolytic sensitivity of each domain was observed indicating their structural dissimilarity.
聚合酶链反应（PCR）用于分离编码G'蛋白（SpG'）的基因片段，SpG'是来自G148链球菌的截短型细菌细胞表面蛋白，可与IgG的Fc区域结合，并在大肠杆菌中表达[Goward等人（1990年），《生物化学杂志》267卷：171 - 177页]。设计PCR引物是为了将起始三联体TTG变为ATG，并将其整合到NdeI限制位点（CATATG）中，从而使该基因能够读框克隆到色氨酸启动子下游紧邻的NdeI限制位点中。SpG'的表达量估计约占总可溶性细胞蛋白的30%，与原始系统中获得的不到1%的总可溶性细胞蛋白相比，这是非常有利的[Goward等人（1990年），《生物化学杂志》267卷：171 - 177页]。通过在Q - Sepharose FF上进行单步阴离子交换色谱法回收了均一的SpG'，该过程避免了使用亲和吸附剂。尽管从氨基酸序列分析来看，SpG'由几乎相同的重复结构域组成，但观察到每个结构域的蛋白水解敏感性不同，这表明它们的结构存在差异。
