Krebs M P, Spudich E N, Spudich J L
Department of Microbiology & Molecular Genetics, University of Texas-Medical School Health Science Center, Houston 77030, USA.
Protein Expr Purif. 1995 Dec;6(6):780-8. doi: 10.1006/prep.1995.0009.
We have used Ni(2+)-affinity chromatography as a rapid and efficient method to purify a sensory rhodopsin I (SR-I) derivative containing six consecutive histidine residues at its C-terminus (His-tagged SR-I). The protein was expressed in Halobacterium salinarium by integrating the corresponding gene at the chromosomal bacterioopsin locus under the control of the bacterioopsin promoter. His-tagged SR-I retains native SR-I photochemical reactions in purified membranes and phototaxis signaling function in vivo. Immobilized Ni(2+)-affinity chromatography of membranes solubilized in 1% layryl maltoside provides a single-step purification of the protein to electrophoretic homogeneity (> or = 90% pure). The procedure yields 1.7 mg pure photoactive protein/liter of culture (60% efficiency). This yield combined with engineered overproduction of the protein provides at least 120-fold greater amounts than that of a previously reported multistep purification procedure, permitting structural and biochemical analysis previously not feasible. The purified protein in lauryl maltoside at pH 5.3 exhibits a visible absorption maximum at 587 nm characteristic of SR-I. Spectrometric titration reveals an alkaline-induced species at 550 nm previously observed with transducer-free SR-I in native membranes. A previously unreported structured absorption band at 400 nm, consistent with a deprotonated Schiff base, forms with the same pKa as the 550-nm species. His-tagged SR-I reconstituted into phosphatidylglycerol proteoliposomes retains properties of transducer-free SR-I in native membranes: its flash-induced absorption difference spectrum is identical, its photochemical reaction cycle kinetics show a similar pH dependence, and it forms a photoactive 550-nm species under alkaline conditions. These results indicate His-tagged SR-I reconstituted in proteoliposomes is suitable for analyzing SR-I interaction with its transducer protein in vitro.
我们使用镍离子亲和层析法作为一种快速有效的方法，来纯化一种在其C末端含有六个连续组氨酸残基的感官视紫红质I（SR-I）衍生物（His标签化的SR-I）。通过将相应基因整合到嗜盐菌紫膜蛋白基因座的染色体上，并在紫膜蛋白启动子的控制下，该蛋白在盐生盐杆菌中表达。His标签化的SR-I在纯化的膜中保留了天然SR-I的光化学反应，并在体内保留了趋光信号功能。用1%月桂基麦芽糖苷溶解的膜进行固定化镍离子亲和层析，可将该蛋白一步纯化至电泳纯（纯度≥90%）。该方法每升培养物可产生1.7毫克纯的光活性蛋白（效率为60%）。该产量与工程化过量生产的蛋白相结合，比之前报道的多步纯化方法产量至少高120倍，使得之前无法进行的结构和生化分析成为可能。在pH 5.3的月桂基麦芽糖苷中的纯化蛋白在587 nm处呈现出SR-I特有的可见吸收最大值。光谱滴定显示在550 nm处有一个碱性诱导的物种，这与天然膜中无转导蛋白的SR-I先前观察到的情况一致。一个先前未报道的在400 nm处的结构化吸收带，与去质子化的席夫碱一致，与550 nm物种具有相同的pKa。重组到磷脂酰甘油蛋白脂质体中的His标签化SR-I保留了天然膜中无转导蛋白的SR-I的特性：其闪光诱导的吸收差异光谱相同，其光化学反应循环动力学显示出相似的pH依赖性，并且在碱性条件下形成一个光活性的550 nm物种。这些结果表明，重组在蛋白脂质体中的His标签化SR-I适用于体外分析SR-I与其转导蛋白的相互作用。
