Use of recombinant biotinylated aequorin in microtiter and membrane-based assays: purification of recombinant apoaequorin from Escherichia coli.
Use of recombinant biotinylated aequorin in microtiter and membrane-based assays: purification of recombinant apoaequorin from Escherichia coli.
复制标题
重组生物素化水母发光蛋白在微量滴定和基于膜的测定中的应用：从大肠杆菌中纯化重组脱辅基水母发光蛋白。
DOI：
10.1021/bi00120a021
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Smith,DF
中科院分区：
文献类型：
--
作者：
Stults,NL;Stocks,NF;Rivera,H;Gray,J;McCann,RO;O'Kane,D;Cummings,RD;Cormier,MJ;Smith,DF
摘要：Aequorin是从水母水母中分离得到的一种钙依赖的生物发光蛋白。Aequorin基因已被克隆并在大肠杆菌中过表达[Prasher等人(1985)生物化学]。生物群落。[中英文摘要]Re.Commun.126,1259；Prasher等人(1987)生物化学26,1326]。最近，通过将Aequorin基因亚克隆到pRC23载体中，使其表达不受X-PL启动子的控制，获得了更高水平的表达[Cormier等()PhotoChem]。光生物醇。49,509]。从含新重组质粒(PAEQ1)的大肠杆菌中纯化重组Apoquorin3)分别用Sephadex G-100凝胶过滤和DEAE-Sepharose阴离子交换层析两步完成。通常，从100个L发酵培养物中可以获得400-500 mg重组蛋白。在人工合成腔肠荧光素、溶氧和硫醇试剂的作用下，纯化的重组麦冬蛋白可以高产率地转化为麦冬蛋白，其光子产率与天然光蛋白相似。在Dynatech ML1000微板发光仪上，重组水飞蓟素在10~18~10~12摩尔浓度范围内呈良好的线性关系，用生物素衍生化后，其比活损失不大。生物素化的衍生物在冷冻、冷冻干燥或在4℃下储存时都是稳定的。通过使用高亲和力的链霉亲和素/生物素相互作用，将生物素化的木犀草素用作非放射性标记的可行性被应用于各种固相分析形式。建立了一种基于生物素化aequorin和ML1000发光计的微滴定生物发光免疫分析(BLIA)方法，用于检测亚纳克量的神经鞘糖脂(Forsmann抗原)。此外，使用生物素化的aequorin在即时和X射线胶片上分别检测到固定在Western和Southern印迹上的纳克到亚纳克量的蛋白质抗原和DNA。
Revised Manuscript Received October 16, 1991 abstract: Aequorin is a calcium-dependent bioluminescent protein isolated from the hydromedusan Aequorea victoria. The gene for aequorin has been cloned and overexpressed in Escherichia coli [Prasher et al.(1985) Biochem. Biophys. Res. Commun. 126, 1259; Prasher et al.(1987) Biochemistry 26, 1326]. Higher levels of expression have recently been obtained by subcloning aequorin cDNA into the pRC23 plasmid vector such that its expression is undercontrol of the X PL promoter [Cormier etal.(1989) Photochem. Photobiol. 49, 509]. Purification of recombinantapoaequorin from E. coli containing this new recombinant plasmid (pAEQ1. 3) was accomplished by a two-step procedure involving gel filtration and anion-exchange chromatography on Sephadex G-100 and DEAE-Sepharose, respectively. Typically, 400-500 mg of re-combinant protein was obtained from 100 L of fermentation culture. The purified recombinant apoaequorin could be converted to aequorin in high yield upon incubationwith syntheticcoelenterate luciferin, dissolved oxygen, and a thiol reagent with a photon yield similar to the native photoprotein. Detection of recombinant aequorin in the Dynatech ML1000 Microplate luminometer was linear between 10~ 18 and 10~ 12 mol, and little loss of specific activity was observed when the protein was derivatized with biotin. The biotinylated derivative was stable when frozen, lyophilized, or stored at 4 C. The feasibility of using biotinylated aequorin as a nonradioactive tag was established by its application in a variety of solid-phase assay formats using the high-affinity streptavidin/biotininteraction. A microtiter-based bioluminescent immunoassay (BLIA) using biotinylated aequorin and the ML1000 luminometer was developed for the detection of subnanogram amounts of a glycosphingolipid (Forsmann antigen). In addition, nanogram to subnanogram quantities of protein antigens and DNA, immobilized on Western and Southern blots, respectively, were detected on instant and X-ray films using biotinylated aequorin.