蚓激酶基因的克隆及在毕赤酵母中的表达

以赤子爱胜蚓(Eisenia fetida)体内的总RNA为模板,通过RT-PCR方法扩增含自身信号肽的蚓激酶基因F238,将其克隆到pUCm-T载体上,并进行测序。GenBank登录号为:DQ202401。测序结果表明基因全长为738bp,共编码245个氨基酸,包括7个氨基酸的信号肽序列和238个氨基酸的成熟肽序列。与粉正蚓(Lumbricus rubellus)F-III-2相比,核苷酸与氨基酸序列的同源性均为99%,仅存在2个碱基的差异,导致2个氨基酸的突变。通过生物信息学方法对蛋白质的理化及结构特性进行分析预测,F238的等电点为4.61,含有11个半胱氨酸,形成3个二硫键。蛋白质分子主要由β折叠组成,具有丝氨酸活性中心,属丝氨酸蛋白酶超家族胰蛋白酶类。以重组质粒pUCm-T-F238为模板,通过PCR方法扩增去信号肽的蚓激酶基因F238-m,构建毕赤酵母(Pichia pastoris)表达载体pPIC9-F238-m,将其线性化后用电穿孔法导入酵母宿主菌GS115中。在MM和MD平板上筛选表型,经甲醇诱导后,SDS-PAGE分析显示表达产物的分子量为28kDa左右,纤维平板法测定活力最高可达100U/mL。

Cloning and expression of lumbrokinase gene in Pichia pastoris

Lumbrokinase gene F238 was amplified by RT-PCR from the total RNA of earthworm (Eisenia fetida). The gene including signal peptide sequence was inserted into pUCm-T vector to construct pUCm-T-F238. The product was sequenced. The GenBank accession number was DQ202401. Lumbrokinase F238 comprised 738bp and included an open reading frame that encoded a polypeptide of 245 amino acid residues, containing a signal peptide of 7 amino acid residues and a mature peptide of 238 amino acid residues. Both nucleotide and amino acid sequences homologies were 99% after the sequence was compared with Lumbricus rubellus F-III-2. There were two base pair mutations, which subsequently caused two amino acid mutations. The characteristics and structure of F238 was analysed and predicted with biology softwares and databases. The pl of F238 was 4.61. It had eleven Cysteines, which formed three disulfide bonds. Its secondary structure mainly consisted of beta-sheet. Lumbrokinase F238 had serine active center. It was a protease in trypsin family of serine protease superfamily. Lumbrokinase gene F238-m without signal peptide sequence was obtained by PCR using pUCm-T-F238 as template. The expression vector pPIC9-F238-m was constructed by inserting gene F238-m into yeast expression and secretion plasmid pPIC9. Plasmid pPIC9-F238-m was linearized with BgIII and then transformed into Pichia pastoris strain GS115 cell by electroporation method. Phenotypes of transformants were screened in MM and MD plates to ensure the integration of lumbrokinase gene F238-m into yeast chromosome DNA. Methanol was added to a final concentration of 0.5% for the expression of recombination protein every 24h to maintain induction. The result of SDS-PAGE showed that the molecular weight of the expression product was about 28 kDa, in correspondence with the theoretical molecular weight. After the induction of expression, the fibrinolytic activity of the supernatant was measured using artificial fibrin plates. Then the engineering strain of high activity was obtained, and the fibrinolytic activity was up to 100 U/mL.