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Expression of Thermostable β-galactosidase ST0773 with E.coli

Author: ZhaoShengFeng
Tutor: GaoRenJun
School: Jilin University
Course: Biochemistry and Molecular Biology
Keywords: β-galactosidase thermostable expression
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 40
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Abstract


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.

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