微泡菌ALW1褐藻胶裂解酶AlgL14的表达及信息学分析

Expression and Bioinformatics Analysis of Alginate Lyase AlgL14 from Microbulbifer sp. ALW1

以微泡菌(Microbulbifer sp.) ALW1的基因组为模板,使用褐藻胶裂解酶AlgL14特异性引物进行PCR扩增,将扩增产物克隆至p MD18-T载体后进行测序,并对该基因编码的蛋白质序列进行生物信息学分析。将目的基因插入pET-28α(+)表达载体,转入Escherichia. coli BL21 (DE3)中进行诱导表达,并利用亲和层析进行重组蛋白纯化。结果显示,克隆基因的大小为1 350 bp,预测编码含有449个氨基酸残基的蛋白质。该蛋白质序列与其他菌株来源的褐藻胶裂解酶序列具有一定的相似性,预测克隆的目的基因编码褐藻胶裂解酶,归属于PL-14家族。褐藻胶裂解酶AlgL14的理论分子质量大小为48. 772 ku,理论等电点为6. 27。采用同源建模法建立菌株ALW1褐藻胶裂解酶AlgL14的三维结构,富含β-折叠。将目的基因在E. coli BL21 (DE3)中进行诱导表达,并纯化获得重组褐藻胶裂解酶。SDS-PAGE分析显示,表达的目的蛋白分子质量约为48. 8 ku。

The genomic DNA of Microbulbifer sp. ALW1 was used as the template for amplification of algi- hate lyase AlgL14 gene by using PCR with a pair of specific primers. The amplified products were cloned into pMD18-T vector and then sequenced. The deduced protein sequence of this gene was further analyzed by bioin- formatics. The target gene was inserted into pET-28α（ ＋ ） expression vector. The recombinant plasmid was transformed into Escherichia coli BL21 （DE3） and the target gene was induced to express. The recombinant alg- inate lyase AlgL14 was purified through affinity chromatography. The results showed that the cloned gene was 1 350 bp, encoding 449 amino acid residues. The target protein sequence shared certain identities with the alg-inate lyase sequences from other bacterial strains. So the cloned target gene was predicted to encode an alginate lyase belonging to PL-14 family. The theoretical molecular weight and pI of the alginate lyase AlgL14 were 48. 772 ku and 6.27, respectively. The three-dimensional structure of alginate lyase AlgL14 from ALW1 was constructed by homology modeling and presented β-strands rich structure. The target gene was transformed into Escherichia coli BL21 （DE3） and induced to express. Then the recombinant alginate lyase AlgL14 was purified. The molecular weight of the target protein was approximately 48.8 ku through SDS-PAGE analysis.