Expression and characterization of recombinant Locusta migratoria manilensis acetylcholinesterase 1 in Pichia pastoris.
The acetylcholinesterase 1 from Locusta migratoria manilensis (LmAChE1) was successfully expressed in methylotrophic yeast Pichia pastoris KM71. The maximum expression of recombinant LmAChE1 (reLmAChE1) was achieved after 9 days of induction at 2.5% methanol. The reLmAChE1 was first precipitated with ammonium sulfate (50% saturation) and then was purified with nickel affinity chromatography. The enzyme was purified 3.2×10(3)-fold with a yield of 68% and a specific activity of 8.1 U/mg. The purified reLmAChE1 exhibited highest activity at 30°C in 100 mM phosphate buffer (pH 7.4), and its activity could be inhibited by eserine sulfate and pentan-3-one-dibromide (BW284c51). Substrate specificity analysis showed that the purified reLmAChE1 preferred acetylthiocholine (ATC) and propionylthiocholine (BTC) rather than butyrylthiocholine (BTC). When ATC was used as substrate, the K(m) and V(max) values for the reLmAChE1 were 24.8 μM and 9.5 μmol/min/mg, respectively.
Genetic Engineering Research Center, Bioengineering College, Chongqing University, Chongqing 400030, PR China.
Xiaoxia
X
Zhou
Yuxian
Y
Xia
D000110:Acetylcholinesterase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D002800:Cholinesterase Inhibitors; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D048188:Locusta migratoria / Q000201:enzymology* / Q000235:genetics; D000432:Methanol; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013696:Temperature
D002800:Cholinesterase Inhibitors; D011994:Recombinant Proteins; D000110:Acetylcholinesterase; D000432:Methanol