High-level secretion and purification of recombinant acetylcholinesterase from human cerebral tissue in P. pastoris and identification by chromogenic reaction.
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.
Ma
Xingyuan
X
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China. maxingyuan@hotmail.com
Tan
Jianhua
J
Wei
Dongzhi
D
Zhu
Pin
P
Sun
Manji
M
D000110:Acetylcholinesterase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008957:Models, Genetic; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013687:Telencephalon / Q000201:enzymology*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D000110:Acetylcholinesterase