Kim Young-Mog, Ogawa Wakano, Tamai Eiji, Kuroda Teruo, Mizushima Tohru, Tsuchiya Tomofusa
Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan.
J Biochem. 2002 Jul;132(1):71-6. doi: 10.1093/oxfordjournals.jbchem.a003201.
A gene encoding Na(+)/serine symporter (SstT) of Escherichia coli has been cloned and sequenced in our laboratory [Ogawa et al. (1998) J. Bacteriol. 180, 6749-6752]. In an attempt to overproduce the protein and purify it, we first constructed a plasmid pTSTH in which the modified sstT gene (sstT gene with 8 successive codons for His at the 3'-terminus) is located downstream from the trc promoter. Upon induction by IPTG, the His-tagged SstT protein was overproduced (about 15% of total membrane proteins), and showed activity as high as the wild type SstT. The His-tagged SstT was solubilized with octylglucoside and purified to homogeneity using a nickel nitrilotriacetic acid (Ni(2+)-NTA) affinity resin. The N-terminal sequence (20 amino acid residues) of the purified protein showed that the sequence was identical to that deduced from the DNA sequence of the sstT gene and that the initiation methionine was excised. The purified His-tagged SstT was reconstituted into liposomes by the detergent dilution method. Reconstituted proteoliposomes mediated the transport of serine driven by an artificially imposed electrochemical Na(+) gradient. The K(m) and the V(max) values for serine transport with the proteoliposomes were 0.82 microM and 0.37 nmol/min/mg protein, respectively. Serine transport was inhibited by L-threonine, but not by other amino acids. The purified protein was stable for at least 6 months at -80 degrees C.
我们实验室已克隆并测序了编码大肠杆菌钠/丝氨酸同向转运体（SstT）的基因[小川等人（1998年），《细菌学杂志》180卷，6749 - 6752页]。为了过量表达该蛋白并进行纯化，我们首先构建了质粒pTSTH，其中修饰的sstT基因（3'末端带有8个连续组氨酸密码子的sstT基因）位于trc启动子下游。经异丙基-β-D-硫代半乳糖苷（IPTG）诱导后，带有组氨酸标签的SstT蛋白大量表达（约占总膜蛋白的15%），且活性与野生型SstT一样高。带有组氨酸标签的SstT用辛基葡糖苷溶解，并用镍次氮基三乙酸（Ni(2 +)-NTA）亲和树脂纯化至同质。纯化蛋白的N端序列（20个氨基酸残基）表明，该序列与从sstT基因的DNA序列推导的序列相同，且起始甲硫氨酸已被切除。通过去污剂稀释法将纯化的带有组氨酸标签的SstT重组到脂质体中。重组的蛋白脂质体介导了由人工施加的电化学钠梯度驱动的丝氨酸转运。蛋白脂质体转运丝氨酸的米氏常数（K(m)）和最大反应速度（V(max)）值分别为0.82微摩尔和0.37纳摩尔/分钟/毫克蛋白。丝氨酸转运受到L-苏氨酸的抑制，但不受其他氨基酸的抑制。纯化的蛋白在-80℃下至少稳定6个月。
