Cloning and expression of carp acetylcholinesterase gene in Pichia pastoris and characterization of the recombinant enzyme
Cloning and expression of carp acetylcholinesterase gene in Pichia pastoris and characterization of the recombinant enzyme
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DOI：
10.1016/j.pep.2008.12.003
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发表时间：
2009-04-01
影响因子：
1.6
通讯作者：
Kera, Yoshio
中科院分区：
文献类型：
--
作者：
Sato, Ryohei;Matsumoto, Toru;Kera, Yoshio
从鲤鱼肌肉组织中克隆了乙酰胆碱酯酶（AChE）基因。该基因全长2368 bp，编码区1902 bp，编码634个氨基酸。推导的氨基酸序列与鱼类AChE序列具有高度的同源性，并具有一些共同的特征，包括C端外显子T编码的T肽。构建了三种酵母表达载体，并将其导入毕赤酵母中。缺失外显子T的鲤鱼AChE基因在细胞外最有效地产生AChE活性。用酵母α因子前原信号序列替换天然信号序列反而降低了产量。培养温度从30 ℃降低到15 ℃，活性生产增加了32.8倍。纯化的去T肽重组AChE在凝胶过滤层析上呈单峰洗脱，分子量约为230 kDa，比活力为4970 U/mg。在SDS-PAGE上，观察到分子量为73、54和22 kDa的三种蛋白。这些蛋白质是N-糖基化的，其N-末端序列表明，后两个可能是由前者产生的蛋白水解裂解在C-末端区域。因此，重组AChE是由三个相同的亚基组成的同源三聚体，分子量为73 kDa。重组酶的最适温度和pH值与纯化的天然酶相当，但动力学参数和对底物抑制和抑制剂的敏感性在它们之间有很大差异。(C)2008年爱思唯尔公司All rights reserved.
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. (C) 2008 Elsevier Inc. All rights reserved.