嗜热β-半乳糖苷酶ST0773的表达探索
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摘要
β-半乳糖苷酶可以催化糖苷类化合物的合成，解决乳糖不耐受症以及作为酶类药物，对新生儿消化不良等方面具有重要的作用。本文采用生物信息学方法推测来自嗜热古细菌Sulfolobus Tokodaii strain 7基因组中的ORF0773为编码β-半乳糖苷酶的基因，构建了ST0773的大肠杆菌工程菌并对其可溶表达进行了初步的探索。首先培养古细菌并提取其基因组，设计引物并成功扩增出
ST0773基因，将其连接到pET-28b质粒上，转入E.coli BL21（DE3）中进行表达。然后分别在20℃、30℃和37℃条件下诱导目的蛋白表达，经SDS-PAGE检测，上清中目的蛋白表达量比较低，大部分以沉淀的形式存在。鉴于该酶氨基酸序列中含有12%的稀有密码子，我们将其转入E.coli BL21（DE3）CondonPlus-RIL宿主细胞中，进行不同温度和不同IPTG浓度的诱导表达，但是大部分目的蛋白仍然是以沉淀的形式存在，从沉淀蛋白电泳图来看，表达量有明显的提高。为克服包涵体的形成，构建了三种重组表达载体pET-15b-0773、pET-32a-0773和pHUE-0773，转入E.coli BL21（DE3）CondonPlus-RIL中诱导表达，发现目的蛋白绝大部分仍然是以沉淀的形式存在。结果表明，β-半乳糖苷酶ST0773在大肠杆菌中主要以包涵体形式表达，硫氧还蛋白融合表达和泛素融合表达也不能改善包涵体的形成。
Thermophilic archaea including sulfur-dependent archaea，reducing archaea andmethanogenic archaea live in hot springs，craters and other extreme environments.For the thermostable enzymes from thermophilic archaea have high temperaturetolerance，they have greater potential for industrial applications than normaltemperature enzyme. For this reason, thermostable enzymes are focused on day afterday.
β-galactosidases (EC.3.2.1.23) belong to glycoside hydrolases family.Accordingto amino acid sequence and protein fold，they are divided into 115families.Glycoside hydrolases play important roles in the degradation andtransformation of glycosides.β-galactosidases hydrolyze glycosidic bondβ-D-(1→4)and also have transglycosylation activity on galacto-oligosaccharide.They are widelydistributed in animals，plants，bacteria，fungi and yeast.
According to their activity，β-galactosidases are classified into GH-1、GH-2、GH-35 and GH-42.β-galactosidases from bacteria are mainly distributed in GH-2and GH-42，β-galactosidases from archaea are mainly distributed in GH-1 and GH-2.β-galactosidases from GH-2 and GH-42 have hydrolysis activity and hightransglycosylation activity.β-galactosidases play important roles in the production ofgalacto-oligosaccharide，medical，vegetables soften and mature，and environmentalengineering. Recent research work are focused on findring goodβ-galactosidase andhigh expressin ofβ-galactosidase.
Thermophilic archaea Sulfolobus tokodaii strain 7 which optimally growsgrows at 80℃with pH 2.5-3.0.Japanese scientist had sequenced its whole genomeby shot gun method in 2001.Here we reported the characterization of ST0773 proteinexpressed from the ST0773 gene from Sulfolobus tokodaii strain 7.With thebioinformatics methods，we found it contains 1152 base pairs and it encodes 483 amino acids and a stop codon.The putative molecular mass and the isoelectric pointof the ST0773 are 44674.1Da and pH 6.28.All the four cysteines in this enzyme donot form disulfide bond. Protein sequence analysis showed that this protein containsno signal peptide and no transmembrane region，but four potential glycosylationsites.
By the frequency of arginine codon usage and the ratio of (E+K)/(H+Q),wefound it belonged to a kind of thermophilic proteins.The observed structure domainand result from NCBI and Homologous Sequences Analysis indicated that theproduction of ST0773 belongs to glycoside hydrolases family 1 and contains of aconserved sequence IIVTENGIA.It showed 52% sequence homology withβ-galactosidase from Thermosphaera Aggregans.Structure obtained by homologymodeling showed that: ST0773 contained TIM barrel domain with the (β/α)8，theactive center residues (Glu-166,Glu-310 and Asn-247) were located in the corner ofβ-4,β-7 andβ-6.We obtained the gene ST0773 from the thermophiles SulfolobusTokodaii strain 7 with PCR，and then constructed the recombinant plasmid usingST0773 and pET-28b.The enzyme digested result,PCR result and sequencingshowed that we successfully constructed the recombinant plasmid.Then wetransfected the recombinant plasmid into the E.coli BL21 (DE3) host cells forprotein expression.The target protein could be overexpressed within E.coli under20℃、30℃、37℃for 6h，but most of the recombinant protein were expressed asprecipitation by SDS-PAGE analysis.And then we transfected the recombinantplasmid into E.coli BL21(DE3)-CondonPlus-RIL host cells for proteinexpression.The target protein could be overexpressed within E.coli under differenttemperatures and different concentration IPTG under 20℃,but most of therecombinant protein were also expressed as precipitation by SDS-PAGE analysis.Wethen constructed three expressed vectors(pET-15b-ST0773,pET-32a-ST0773,pHUEST0773)to improve the expression and realize soluble expression.Then wetransfected the recombinant plasmid into the E.coli BL21 (DE3) CodonPlus-RILhost cells for protein expression.The target protein could be overexpressed within E.coli under 20℃、30℃、37℃for 6h with 0.05mM IPTG，but most of the proteinwere also expressed as precipitation by SDS-PAGE analysis. But from expression ofprecipitation,fusion vector were more than non-fusion vector.Taking into account theconvenience of purification，we selected pHUE-ST0773 as expression vectorfinally，it lay the foundation for the next step enzymatic study.
ST0773基因，将其连接到pET-28b质粒上，转入E.coli BL21（DE3）中进行表达。然后分别在20℃、30℃和37℃条件下诱导目的蛋白表达，经SDS-PAGE检测，上清中目的蛋白表达量比较低，大部分以沉淀的形式存在。鉴于该酶氨基酸序列中含有12%的稀有密码子，我们将其转入E.coli BL21（DE3）CondonPlus-RIL宿主细胞中，进行不同温度和不同IPTG浓度的诱导表达，但是大部分目的蛋白仍然是以沉淀的形式存在，从沉淀蛋白电泳图来看，表达量有明显的提高。为克服包涵体的形成，构建了三种重组表达载体pET-15b-0773、pET-32a-0773和pHUE-0773，转入E.coli BL21（DE3）CondonPlus-RIL中诱导表达，发现目的蛋白绝大部分仍然是以沉淀的形式存在。结果表明，β-半乳糖苷酶ST0773在大肠杆菌中主要以包涵体形式表达，硫氧还蛋白融合表达和泛素融合表达也不能改善包涵体的形成。
Thermophilic archaea including sulfur-dependent archaea，reducing archaea andmethanogenic archaea live in hot springs，craters and other extreme environments.For the thermostable enzymes from thermophilic archaea have high temperaturetolerance，they have greater potential for industrial applications than normaltemperature enzyme. For this reason, thermostable enzymes are focused on day afterday.
β-galactosidases (EC.3.2.1.23) belong to glycoside hydrolases family.Accordingto amino acid sequence and protein fold，they are divided into 115families.Glycoside hydrolases play important roles in the degradation andtransformation of glycosides.β-galactosidases hydrolyze glycosidic bondβ-D-(1→4)and also have transglycosylation activity on galacto-oligosaccharide.They are widelydistributed in animals，plants，bacteria，fungi and yeast.
According to their activity，β-galactosidases are classified into GH-1、GH-2、GH-35 and GH-42.β-galactosidases from bacteria are mainly distributed in GH-2and GH-42，β-galactosidases from archaea are mainly distributed in GH-1 and GH-2.β-galactosidases from GH-2 and GH-42 have hydrolysis activity and hightransglycosylation activity.β-galactosidases play important roles in the production ofgalacto-oligosaccharide，medical，vegetables soften and mature，and environmentalengineering. Recent research work are focused on findring goodβ-galactosidase andhigh expressin ofβ-galactosidase.
Thermophilic archaea Sulfolobus tokodaii strain 7 which optimally growsgrows at 80℃with pH 2.5-3.0.Japanese scientist had sequenced its whole genomeby shot gun method in 2001.Here we reported the characterization of ST0773 proteinexpressed from the ST0773 gene from Sulfolobus tokodaii strain 7.With thebioinformatics methods，we found it contains 1152 base pairs and it encodes 483 amino acids and a stop codon.The putative molecular mass and the isoelectric pointof the ST0773 are 44674.1Da and pH 6.28.All the four cysteines in this enzyme donot form disulfide bond. Protein sequence analysis showed that this protein containsno signal peptide and no transmembrane region，but four potential glycosylationsites.
By the frequency of arginine codon usage and the ratio of (E+K)/(H+Q),wefound it belonged to a kind of thermophilic proteins.The observed structure domainand result from NCBI and Homologous Sequences Analysis indicated that theproduction of ST0773 belongs to glycoside hydrolases family 1 and contains of aconserved sequence IIVTENGIA.It showed 52% sequence homology withβ-galactosidase from Thermosphaera Aggregans.Structure obtained by homologymodeling showed that: ST0773 contained TIM barrel domain with the (β/α)8，theactive center residues (Glu-166,Glu-310 and Asn-247) were located in the corner ofβ-4,β-7 andβ-6.We obtained the gene ST0773 from the thermophiles SulfolobusTokodaii strain 7 with PCR，and then constructed the recombinant plasmid usingST0773 and pET-28b.The enzyme digested result,PCR result and sequencingshowed that we successfully constructed the recombinant plasmid.Then wetransfected the recombinant plasmid into the E.coli BL21 (DE3) host cells forprotein expression.The target protein could be overexpressed within E.coli under20℃、30℃、37℃for 6h，but most of the recombinant protein were expressed asprecipitation by SDS-PAGE analysis.And then we transfected the recombinantplasmid into E.coli BL21(DE3)-CondonPlus-RIL host cells for proteinexpression.The target protein could be overexpressed within E.coli under differenttemperatures and different concentration IPTG under 20℃,but most of therecombinant protein were also expressed as precipitation by SDS-PAGE analysis.Wethen constructed three expressed vectors(pET-15b-ST0773,pET-32a-ST0773,pHUEST0773)to improve the expression and realize soluble expression.Then wetransfected the recombinant plasmid into the E.coli BL21 (DE3) CodonPlus-RILhost cells for protein expression.The target protein could be overexpressed within E.coli under 20℃、30℃、37℃for 6h with 0.05mM IPTG，but most of the proteinwere also expressed as precipitation by SDS-PAGE analysis. But from expression ofprecipitation,fusion vector were more than non-fusion vector.Taking into account theconvenience of purification，we selected pHUE-ST0773 as expression vectorfinally，it lay the foundation for the next step enzymatic study.
引文
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