Yeast mitochondrial phosphate transport protein expressed in Escherichia coli. Site-directed mutations at threonine-43 and at a similar location in the second tandem repeat (isoleucine-141).
Yeast mitochondrial phosphate transport protein expressed in Escherichia coli. Site-directed mutations at threonine-43 and at a similar location in the second tandem repeat (isoleucine-141).
复制标题
在大肠杆菌中表达的酵母线粒体磷酸转运蛋白。
DOI：
10.1021/bi00198a001
复制
发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Briggs,C
中科院分区：
文献类型：
--
作者：
Wohlrab,H;Briggs,C
材料和方法突变PTP质粒的制备。突变型PTP完全按照所述制备（Phelps & Wohlrab，1991）。然而，在本实验中，质粒pNYHM 131（图1）与表1所示的寡核苷酸引物一起使用。用E.转化大肠杆菌DH 5a，其转化效率明显高于表达菌株BL 21（DE 3）。PCR构建体及其亚克隆位点在双链载体中完全测序。一种新的方法被用于通过包括Klenow片段来消除DNA测序反应中的假停止（Briggs，未发表的观察结果）。遵循测序酶版本2.0（US Biochemicals）的方案。在核苷酸试剂盒中获得试剂，用于用Sequenase T7 DNA聚合酶和7-deaza-dGTP（US Biochemicals）测序。通过与ddNTPs（3 μ L总体积）温育终止延伸反应后，将0.5 μ L Klenow混合物（1 mM dNTPs，0.6单位/μ L Klenow片段）与每个反应混合。
MATERIALS AND METHODSPreparation of Mutant PTP Plasmids. Mutant PTPs were prepared exactly as described (Phelps & Wohlrab, 1991). However, in the present experiments plasmid pNYHM131 (Figure 1) was used with oligonucleotide primers as indicated in Table 1. Manipulations of the plasmid were carried out with E. coli strain DH5a, which showed a much higher transformation efficiency than the expression strain BL21 (DE3). The PCRconstruct as well as its subcloning sites was completely sequenced in the double-stranded vector. A novel method was used to eliminate falsestops in the DNA sequencing reactions by including Klenow fragment (Briggs, unpublished observation). The protocol for Sequenase version 2.0 (US Biochemicals) was followed. Reagents were obtained in a nucleotide kit for sequencing with Sequenase T7 DNA polymerase and 7-deaza-dGTP (US Biochemicals). After extension reactions were stopped byincubation with ddNTPs (3-/UL total volume), 0.5/nL of Klenow cocktail (1 mM dNTPs, 0.6 unit/jiL Klenow fragment) was mixed with each reaction.