Inouye Satoshi
Yokohama Research Center, Chisso Corporation, 5-1 Okawa, Yokohama 236-8605, Japan.
Protein Expr Purif. 2007 Mar;52(1):66-73. doi: 10.1016/j.pep.2006.07.028. Epub 2006 Aug 17.
The Ca2+-triggered luciferin-binding protein of Renilla reniformis (RLBP) is a non-covalent complex of apoprotein (apoRLBP) and coelenterazine (luciferin). The gene encoding apoRLBP with 552 nucleotides has been synthesized by assembly PCR methods with synthetic oligonucleotides, and the histidine-tagged apoRLBP expressed as a soluble form in the periplasmic space of Escherichia coli cells. The apoRLBP was purified by nickel chelate chromatography and the procedure yielded 18.2mg of recombinant apoRLBP from 80 ml of cultured cells with purity greater than 95%. The purified apoRLBP was converted to RLBP by incubation with coelenterazine in the presence of dithiothreitol and the purity of recombinant RLBP was estimated to be over 95% by comparison with the absorption spectral data of native RLBP. When RLBP mixed with Ca2+, coelenterazine was dissociated from RLBP and was utilized for the luminescence reaction of Renilla luciferase. Also semi-synthetic RLBPs with h-, e-, and Bis-coelenterazines were prepared and characterized.
海肾（Renilla reniformis）的Ca2+触发的荧光素结合蛋白（RLBP）是脱辅基蛋白（apoRLBP）和腔肠素（荧光素）的非共价复合物。通过用合成寡核苷酸的组装PCR方法合成了编码具有552个核苷酸的apoRLBP的基因，并且组氨酸标签化的apoRLBP在大肠杆菌细胞的周质空间中以可溶形式表达。通过镍螯合层析纯化apoRLBP，该方法从80 ml培养细胞中产生了18.2 mg重组apoRLBP，纯度大于95%。通过在二硫苏糖醇存在下与腔肠素孵育，将纯化的apoRLBP转化为RLBP，通过与天然RLBP的吸收光谱数据比较，重组RLBP的纯度估计超过95%。当RLBP与Ca2+混合时，腔肠素从RLBP中解离出来，并用于海肾荧光素酶的发光反应。还制备并表征了具有h-、e-和双腔肠素的半合成RLBP。
