title: [Cloning, expression and bioinformatics analysis of pyruvate dehydrogenase of Echinococcus granulosus].
abstract: OBJECTIVE
  To clone and express Echinococcus granulosus pyruvate dehydrogenase (EgPDH) gene and analyze EgPDH protein with bioinformatics tools and online database.
  
  
  METHODS
  The total RNAs of E. granulosus was extracted and reversely transcribed into cDNA. The EgPDH gene was cloned into pET28b to construct the recombinant vector and expressed in E. coli BL21 (DE3) system subsequently. The signal peptide, transmembrane helices and subcellular location in EgPDH sequence were analyzed by the online software SignalP4.1, TMHMM sever v.2.0 and TargetP1.1, respectively. Subsequently, the structure of EgPDH was predicted by SMART. Finally, the homologue sequence and conserved sites were aligned by using BLASTP and GeneDoc among the homologous sequences of EgPDH. Based on the alignment of PDH sequence, an evolutionary tree of E. granulosus and other species were constructed by the neighbor joining method of MEGA6 software.
  
  
  RESULTS
  The EgPDH gene was successfully amplified from cDNA of E. granulosus and expressed in the soluble fractions. The bioinformatics analysis revealed that EgPDH was a classical secreted protein and contained transketolase domain. The homology analysis revealed that the amino acid sequence of EgPDH was highly conserved in catalytic sites Glu57, Leu72, Ile86 and Phe114. The phylogenetic tree analysis of PDH proteins showed the closest relationship between E. granulosus and E. multilocularis.
  
  
  CONCLUSIONS
  An EgPDH gene is cloned and expressed successfully, and the recombinant protein is analyzed by the bioinformatics approaches and structure predication. The study provides useful information for further functional study of the EgPDH protein.
authors.forename: Ying
authors.initials: Y
authors.lastname: Chen
authors.forename: Wu-liji
authors.initials: WL
authors.lastname: Ao
authors.forename: Ting
authors.initials: T
authors.lastname: Zhang
authors.forename: Qun-feng
authors.initials: QF
authors.lastname: Wu
authors.forename: Zhi-sheng
authors.initials: ZS
authors.lastname: Dang
authors.forename: Jun-hu
authors.initials: JH
authors.lastname: Chen
authors.forename: Wei
authors.initials: W
authors.lastname: Hu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D019295:Computational Biology; D048209:Echinococcus granulosus / Q000201:enzymology*; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data
chemical_list: D007658:Ketone Oxidoreductases; C053164:pyruvate dehydrogenase (NADP+)