从一种多元醇反应性单克隆抗体中快速纯化甲苯双加氧酶的加氧酶组分。
Rapid purification of the oxygenase component of toluene dioxygenase from a polyol-responsive monoclonal antibody.
作者信息
Lynch N A, Jiang H, Gibson D T
机构信息
Department of Microbiology, College of Medicine University of Iowa, Iowa City, USA.
出版信息
Appl Environ Microbiol. 1996 Jun;62(6):2133-7. doi: 10.1128/aem.62.6.2133-2137.1996.
Abstract
A monoclonal antibody designated 302 beta that is specific for the beta subunit of the oxygenase component (ISPTOL) of toluene dioxygenase from Pseudomonas putida F1 was used to prepare an immunoaffinity column. ISPTOL in cell extracts of Escherichia coli JM109(pDTG611) bound to the column, and an enzyme-linked immunosorbent elution-screening assay with different combinations of polyols and kosmotropic anions was used to determine the conditions necessary for recovery of active enzyme. Elution from an 8-ml antibody column with 50 mM 2-(N-morpholino)ethanesulfonate buffer (pH 6.8) containing 50% ethylene glycol, 1.0 M ammonium sulfate, 1.0 mM dithiothreitol, and 0.2 mM ferrous ammonium sulfate gave approximately 2 mg of ISPTOL with a specific activity that was more than 300 times the specific activity previously obtained.
摘要
一种名为302β的单克隆抗体，它对恶臭假单胞菌F1甲苯双加氧酶的加氧酶组分（ISPTOL）的β亚基具有特异性，被用于制备免疫亲和柱。大肠杆菌JM109(pDTG611)细胞提取物中的ISPTOL与该柱结合，并且使用不同组合的多元醇和促溶剂阴离子进行酶联免疫吸附洗脱筛选试验，以确定回收活性酶所需的条件。用含有50%乙二醇、1.0 M硫酸铵、1.0 mM二硫苏糖醇和0.2 mM硫酸亚铁铵的50 mM 2-(N-吗啉代)乙磺酸盐缓冲液（pH 6.8）从8 ml抗体柱上洗脱，得到约2 mg的ISPTOL，其比活性比之前获得的比活性高300倍以上。
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