Purification, reconstitution, and characterization of Na+/serine symporter, SstT, of Escherichia coli
Purification, reconstitution, and characterization of Na+/serine symporter, SstT, of Escherichia coli
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DOI：
10.1093/oxfordjournals.jbchem.a003201
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发表时间：
2002-07-01
影响因子：
2.7
通讯作者：
Tsuchiya, T
中科院分区：
文献类型：
--
作者：
Kim, YM;Ogawa, W;Tsuchiya, T
编码大肠杆菌 Na+/丝氨酸同向转运体 (SstT) 的基因已在我们的实验室被克隆和测序 [Okawa 等人，2017]。 (1998)细菌学杂志。 180、6749-6752]。为了过量生产该蛋白质并纯化它，我们首先构建了一个质粒 pTSTH，其中修饰的 sstT 基因（3' 端有 8 个连续的组氨酸密码子的 sstT 基因）位于 trc 启动子的下游。在 IPTG 诱导下，His 标记的 SstT 蛋白过量产生（约占总膜蛋白的 15%），并且表现出与野生型 SstT 一样高的活性。 His 标记的 SstT 用辛基葡萄糖苷溶解，并使用次氮基三乙酸镍 (Ni2+-NTA) 亲和树脂纯化至均质。纯化蛋白的N端序列(20个氨基酸残基)显示该序列与从sstT基因的DNA序列推导的序列相同，并且起始甲硫氨酸被切除。将纯化的His标签SstT通过去污剂稀释法重构为脂质体。重组蛋白脂质体介导由人工施加的电化学 Na+ 梯度驱动的丝氨酸转运。蛋白脂质体丝氨酸转运的 K-m 和 V-max 值分别为 0.82 muM 和 0.37 nmol/min/mg 蛋白质。丝氨酸转运被 L-苏氨酸抑制，但不被其他氨基酸抑制。纯化的蛋白质在-80℃下稳定至少6个月。
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 (Ni2+-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 muM 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 -80degreesC.