Ghatge M, Mawal Y, Gaikwad S, Deshpande V
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Appl Biochem Biotechnol. 1991 Oct;31(1):11-20. doi: 10.1007/BF02922121.
A procedure was developed to purify glucose/xylose isomerase from cell extract of Streptomyces sp. NCIM 2730 using immunoaffinity chromatography. High-titer polyclonal antibodies were raised in rabbit using electrophoretically homogeneous glucose/xylose isomerase as an antigen. The specificity of antibodies was confirmed by double immunodiffusion, rocket electrophoresis, and Western-blot ELISA, which revealed the presence of a single immunoreactive protein with an Mr of 40,000. The antibodies recognized 2-3 antigenic determinants/mol of enzyme and were found to partially neutralize the enzymatic activity in an immunotitration experiment. The affinity gel was prepared by coupling antibodies at pH 10.0 to divinyl sulfone-activated Sepharose CL-4B. The glucose/xylose isomerase purified by immunoaffinity chromatography yielded 75% recovery with a single enzymatically active protein band on gel electrophoresis and showed specific activity of 16 U/mg. The crossreaction of the antibodies with glucose isomerase from other actinomycetes indicated that they share common epitopes.
开发了一种使用免疫亲和色谱法从链霉菌属NCIM 2730的细胞提取物中纯化葡萄糖/木糖异构酶的方法。以电泳纯的葡萄糖/木糖异构酶为抗原，在兔体内制备了高效价的多克隆抗体。通过双向免疫扩散、火箭电泳和Western印迹ELISA证实了抗体的特异性，结果显示存在一种Mr为40,000的单一免疫反应性蛋白。抗体识别每摩尔酶2 - 3个抗原决定簇，并且在免疫滴定实验中发现其能部分中和酶活性。通过在pH 10.0条件下将抗体偶联到二乙烯砜活化的琼脂糖凝胶CL - 4B上制备亲和凝胶。经免疫亲和色谱法纯化的葡萄糖/木糖异构酶回收率为75%，在凝胶电泳上呈现单一的酶活性蛋白条带，比活性为16 U/mg。抗体与其他放线菌的葡萄糖异构酶的交叉反应表明它们具有共同的表位。
