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.

译文

肾性肾病(RLBP)的Ca2触发的萤光素结合蛋白是载脂蛋白(apoRLBP)和腔肠素(萤光素)的非共价复合物。已经用合成的寡核苷酸通过组装PCR方法合成了具有552个核苷酸的编码apoRLBP的基因,并且组氨酸标记的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,该程序从80ml培养的细胞中产生18.2mg重组apoRLBP,纯度大于95%。通过在二硫苏糖醇存在下与腔肠素一起孵育,将纯化的apoRLBP转化为RLBP,与天然RLBP的吸收光谱数据相比,重组RLBP的纯度估计超过95%。当RLBP与Ca2混合时,腔肠素与RLBP分离,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Bis-腔肠素的半合成RLBP。

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