Cloning and expression of carp acetylcholinesterase gene in Pichia pastoris and characterization of the recombinant enzyme.
The gene encoding acetylcholinesterase (AChE) was cloned from common carp muscle tissue. The full-length cDNA was 2368 bp that contains a coding region of 1902 bp, corresponding to a protein of 634 amino acids. The deduced amino acid sequence showed a significant homology with those of ichthyic AChEs and several common features among them, including T peptide encoded by exon T in the C-terminus. Three yeast expression vectors were constructed and introduced into the yeast Pichia pastoris. The transformant harboring carp AChE gene lacking exon T most effectively produced AChE activity extracellularly. The replacement of the native signal sequence with the yeast alpha-factor prepro signal sequence rather decreased the production. A decrease in cultivation temperature from 30 to 15 degrees C increased the activity production 32.8-fold. The purified recombinant AChE lacking T peptide, eluted as a single peak with a molecular mass of about 230 kDa on the gel filtration chromatography, exhibited the specific activity of 4970 U/mg. On the SDS-PAGE, three proteins with molecular masses of 73, 54, and 22 kDa were observed. These proteins were N-glycosylated, and their N-terminal sequence showed that the latter two were produced from the former probably by proteolytic cleavage at the C-terminal region. Thus, the recombinant AChE is homotrimer of three identical subunits with 73 kDa. The optimal temperature and pH of the recombinant were comparable to those of the native enzyme purified previously, but the values of kinetic parameters and the sensitivities to substrate inhibition and inhibitors were considerably different between them.
Sato
Ryohei
R
Laboratory of Environmental Biochemistry, Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan.
Matsumoto
Toru
T
Hidaka
Norio
N
Imai
Yasuko
Y
Abe
Katsumasa
K
Takahashi
Shouji
S
Yamada
Ryo-Hei
RH
Kera
Yoshio
Y
D000110:Acetylcholinesterase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002347:Carps / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D029941:Fish Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D018628:Gene Dosage; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D018076:DNA, Complementary; D029941:Fish Proteins; D011993:Recombinant Fusion Proteins; D000110:Acetylcholinesterase