Feng G, Winkler M E
University of Texas, Houston Medical School, USA.
Biotechniques. 1995 Dec;19(6):956-65.
The MutS, MutH and MutL proteins mediate methyl-directed-mismatch (MDM) repair in Escherichia coli and Salmonella typhimurium. These proteins have been developed into powerful tools for screening genomes for polymorphisms and detecting and localizing mutations. In an ongoing study of the regulation of MDM repair, we developed one-step schemes to purify the E. coli MutS, MutH and MutL proteins fused to a polyhistidine (His6) affinity tag. The E. coli K-12 mutS+, mutH+ and mutL+ genes were cloned from the Clarke-Carbon plasmid or Kohara-phage library into expression vector pET-15b, which allows fusion to the His6 affinity tag. Each of the resulting recombinant plasmids complemented the corresponding mutHLS mutation in the Cupples-Miller CC106 mutator tester strain, indicating that the His6-MutHLS fusion proteins were individually functional in vivo. The His6-MutHLS proteins were separately purified by variations of batch binding to Ni(2+)-chelation affinity resin. The yield of purified His6-MutHLS proteins from these procedures was 0.4-0.6 mg from 40 mL of induced culture. The binding properties of one-step-purified His6-MutS protein were characterized further. His6-MutS exhibited the same mismatch-binding activity and specificity as native MutS in side-by-side bandshift assays. These constructs and purification methods should be useful to laboratories wishing to apply or develop MutS-mismatch mapping and to other applications or studies of the E. coli MutHLS repair proteins.
MutS、MutH和MutL蛋白介导大肠杆菌和鼠伤寒沙门氏菌中的甲基定向错配（MDM）修复。这些蛋白质已发展成为用于筛选基因组多态性以及检测和定位突变的强大工具。在一项正在进行的MDM修复调控研究中，我们开发了一步法方案来纯化与多组氨酸（His6）亲和标签融合的大肠杆菌MutS、MutH和MutL蛋白。将大肠杆菌K-12 mutS+、mutH+和mutL+基因从克拉克-卡本质粒或小原噬菌体文库克隆到表达载体pET-15b中，该载体允许与His6亲和标签融合。每个所得的重组质粒都能互补Cupples-Miller CC106诱变测试菌株中相应的mutHLS突变体，这表明His6-MutHLS融合蛋白在体内各自具有功能。通过与Ni(2+)螯合亲和树脂进行批量结合的不同方法分别纯化His6-MutHLS蛋白。从这些步骤中纯化得到的His6-MutHLS蛋白的产量为每40 mL诱导培养物0.4 - 0.6 mg。对一步纯化的His6-MutS蛋白的结合特性进行了进一步表征。在并排的带移分析中，His6-MutS表现出与天然MutS相同的错配结合活性和特异性。这些构建体和纯化方法对于希望应用或开发MutS错配图谱的实验室以及对大肠杆菌MutHLS修复蛋白的其他应用或研究应该是有用的。
