An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of lambda repressor.
An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of lambda repressor.
复制标题
工程化的亚基间二硫键增强了 lambda 阻遏物 N 末端结构域的稳定性和 DNA 结合。
DOI：
10.1021/bi00368a024
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Pabo,CO
中科院分区：
文献类型：
--
作者：
Sauer,RT;Hehir,K;Stearman,RS;Weiss,MA;Jeitler-Nilsson,A;Suchanek,EG;Pabo,CO
材料和方法构建和诱变。质粒pLac 1 -102编码包含X阻遏物的前102个残基的蛋白片段。为了构建该质粒，我们使用了来自pKB 280的412个碱基对的EcoRI/Rstfl片段，其含有IacUV 5启动子和X阻遏物的N-末端区域（Backman和Ptashne，1978）。在填充//mdIII位点的5 ′突出端以产生平端后，将该片段连接到pBR 322的EcoRI/HindIII骨架中。这些片段的平端连接在第102位残基的密码子后产生一个琥珀终止密码子，从而指导N端阻遏物片段的合成。对于寡核苷酸定向诱变，将来自pLacl-102的小Eco-Rl/BamHl片段克隆到噬菌体M13 mp8的多接头中（Messing & Vierer，1982）。在单独的实验中，通过使用具有单碱基错配的反义引物：5 '-CGCTTCACACATCTC-3'和5 '-CATCTCGCAGATTTC-3'，将Tyr-88-* Cys和Tyr-85-* Cys突变引入M13中的阻遏物编码序列。通过标准程序进行退火、第二链合成、连接、转染和噬斑杂交（Zoller & Smith，1983; Carter等人，1984年）。对于每种诱变，通过与标记引物杂交选择几种候选物，通过双脱氧程序测序（桑格等人，1977），并发现与野生型（Sauer，1978）相比仅含有预期的Cys-88或Cys-85置换。将这些突变的N-末端结构域及其相关的lac启动子重新克隆到pBR 322的EcoR 1/BamH 1骨架中，产生与pLac 1 -102具有相同结构的突变质粒。将带有Cys-88突变的质粒命名为pRS 30，将带有Cys-85突变的质粒命名为pRS 40。在蛋白纯化之前，将野生型和突变型1-102基因克隆到杂合trp-lac启动子（Plac）的转录控制下。对于这些构建，将来自pLacl-102或突变衍生物的482-残基Mspl片段克隆到质粒pEA 300的Clal位点中。这种克隆策略与Amann et al.（1983）完整的X阻遏物。含有
Materials and MethodsConstructions and Mutagenesis. Plasmid pLacl-102 en-codes a protein fragment comprising the first 102 residues of X repressor. To construct this plasmid, we used the 412 base pair EcoRI/Rstfl fragment, which contains the/acUV5 promoter and the N-terminal region of X repressor, from pKB280 (Backman & Ptashne, 1978). This fragment was ligated into the EcoRl/Hindlll backbone of pBR322 after filling in the 5'overhang of the//mdlll site to create a blunt end. The blunt ligation of these fragments creates an amber termination codon after thecodon for residue 102 andthus directs synthesis of an N-terminal repressor fragment. For oligonucleotide-directed mutagenesis, the small Eco-Rl/BamHl fragment from pLacl-102 was cloned into the polylinker of phage M13mp8 (Messing & Vierer, 1982). In separate experiments, the Tyr-88—* Cys and Tyr-85-* Cys mutations were introduced into the repressor coding sequence in M13 by using antisense primerswith single-base mis-matches; 5'-CGCTTCACACATCTC-3' and 5'-CATCTCGCAGATTTC-3'. Annealing, second-strandsynthesis, ligation, transfection, and plaque hybridization were performed by standard procedures (Zoller & Smith, 1983; Carter et al., 1984). For each mutagenesis, several candidates were chosen by hybridization with labeled primers, sequenced by the dideoxy procedure (Sanger et al., 1977), and found when compared with wild type (Sauer, 1978) to contain only the expected Cys-88 or Cys-85 substitutions. These mutant N-terminal domains and their associated lac promoters were recloned into the EcoRl/BamHl backbone of pBR322, gen-erating mutant plasmids with the same structure as pLacl-102. The plasmid bearing the Cys-88 mutation was designated pRS30, and that bearing the Cys-85 change was designated pRS40.Prior to protein purification, the wild-type and mutant 1-102 genes were cloned under transcriptional control of the hybrid trp-lac promoter (Plac). For these constructions, the 482-residue Mspl fragment from pLacl-102 or mutant derivatives was cloned into the Clal site of plasmid pEA300. This cloning strategy is completely analogousto that described by Amann et al.(1983) forintact X repressor. Plasmids containing the