文献类型：期刊文献

英文题名：High-level secretion and purification of recombinant acetylcholinesterase from human cerebral tissue in P. pastoris and identification by chromogenic reaction

作者：Ma, Xingyuan[1]; Tan, Jianhua[2]; Wei, Dongzhi[1]; Zhu, Pin[2]; Sun, Manji[3]

机构：[1] State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China; [2] Gene Engineering Key Laboratory of PLA, Academy of Military Medical Sciences, Changchun 130062, China; [3] Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China

年份：2006

卷号：72

期号：2

起止页码：316

外文期刊名：Applied Microbiology and Biotechnology

收录：EI(收录号：20063510089334)

语种：英文

外文关键词：Affinity chromatography - Chromogenics - Cloning - Ion exchange - Methanol - Tissue

摘要：The gene encoding human cerebral tissue acetylcholinesterase (AChE) was cloned from an 18-week fetal cerebral tissue and expressed in Pichia pastoris. Twenty-two positive transformants were obtained by Mut+/Mut s phenotypes screening in MD/MM medium and polymerase chain reaction amplification, and four recombinant P. pastoris strains that could secrete active AChE at high level were identified by simple and specific development reaction with indoxyl acetate as the chromogenic substrate. In shake-flask culture induced with methanol, the recombinant human AChE (rhAChE) content was about 76% of the total secreted proteins, and rhAChE activity in supernatant was 40 U/ml. The enzyme was purified through anion-exchange and affinity chromatography. Purity of the rhAChE was up to 96% after the simple purification procedure. The enzymatic activity reached 200 U/mg. ? Springer-Verlag 2006.