解肝磷脂土地杆菌(Pedobacter heparinus)源唾液酸醛缩酶基因的异源表达及其活性研究

为发掘新型唾液酸醛缩酶,使其在大肠杆菌中得到高效表达,本研究首次从菌株Pedobacter heparinus中克隆疑似编码唾液酸醛缩酶的PhNeuLy3300基因片段,并构建可自主表达异源蛋白的组成型表达载体pRSF-EM5。在此基础上将克隆的目的基因分别导入该pRSF-EM5载体和常用的诱导型载体pET-30a,构建重组质粒pET30a-PhNeuLy3300和pRSF-EM5-PhNeuLy3300,经大肠杆菌表达系统异源表达目的蛋白,并进一步对两种重组蛋白酶进行电泳分析和活性研究。结果表明:编码唾液酸醛缩酶的基因片段全长为945 bp,共编码314个氨基酸残基;聚丙烯酰胺凝胶电泳显示两种重组蛋白表观分子量约为36.4 kDa,纯化后的pET30a-PhNeuLy3300重组蛋白浓度为3.29 mg/mL,证明经异源表达获得了两种重组目的蛋白酶;活性研究显示两种重组唾液酸醛缩酶均能在1 h内催化N-乙酰神经氨酸生成N-乙酰甘露糖胺,证明两种重组蛋白酶均具有较高的活性。

Heterologous Expression and Activity Study of a Novel Sialic Acid Aldolase Gene Derived from Pedobacter heparinus

In order to develop a new type of sialic acid aldolase and express it effectively in Escherichia coli, the gene PhNeuLy3300 putatively encoding sialic acid aldolase from Pedobacter heparinus was cloned. A constitutive pRSF-EM5 vector containing self-initiating promotors was constructed. The cloned sialic acid aldolase gene was transferred into the constitutive vector pRSF-ME5 and another commonly used inducible vector pET-30a to construct the recombinant plasmids pRSF-EM5-PhNeuLy3300 and pET30a-PhNeuLy3300, respectively. The constructs were heterologously expressed in E. coli, further electrophoretic analysis and activity studies were performed on the two recombinant proteases. The results showed that the gene fragment encoding sialic acid aldolase was 945 bp and encodes a total of 314 amino acid residues. Polyacrylamide gel electrophoresis showed that the apparent molecular weight of the two recombinant proteins was about 36.4 kDa. Bradford method measured that the concentration of purified pET30a-PhNeuLy3300 recombinant protein was 3.29 mg/mL, which proved that two recombinant proteases were obtained by heterologous expression. The activity studies showed that both recombinant sialic acid aldolases can catalyze N-Acetylneuraminic acid to produce the N-Acetylmannosamine within 1 h, which means that both recombinant proteases have high activity.