title: [Cloning, expression and bioinformatics analysis of cathepsin B of Echinococcus granulosus].
abstract: OBJECTIVE
  To clone and express cathepsin B gene of Echinococcus granulosus (EgCatB) and analyze EgCatB protein by using bioinformatics tools and online databases.
  
  
  METHODS
  The total RNA of E. granulosus was extracted and reversely transcribed into cDNA as the template sequence for PCR. The EgCatB gene was cloned by using the In-Fusion PCR cloning method and expressed by a wheat germ cell-free system, and then the recombinant protein was identified by Western blotting. The signal peptide, transmembrane helices and subcellular location of the EgCatB sequence were predicted by the online software SignalP 4.1, TMHMM sever v. 2.0 and TargetP 1.1 respectively. Subsequently, the homologue sequence and conserved sites were aligned by using BLASTP and GeneDoc software. Finally, the structures and the glycosylation modification site of the EgCatB encoding protein were analyzed and predicted in turn by ProtParam, SMART, Predictprotein, Swiss-model, NetOGlyc 4.0 and NetNGlyc 1.0 approaches.
  
  
  RESULTS
  The EgCatB gene was successfully amplified from cDNA of E. granulosus and expressed in the soluble fractions. The molecular weight of the expressed protein was estimated 35 kDa. The bioinformatics analysis revealed that EgCatB was a classical secreted protein containing a Pept_C1 domain. The homology analysis indicated that the amino acid sequence of EgCatB was highly conserved in the active enzyme sites. The protein structure prediction showed a catalytic active center was formed through Gln106, Cys112, His282 and Asn302. It was found that there were nine O-glycosylation sites in the EgCatB sequence, but no N-glycosylation sites.
  
  
  CONCLUSIONS
  The EgCatB gene is cloned and expressed successfully, and the recombinant protein is analyzed by bioinformatics approaches and structure predication. The study provides useful information for further functional study of the EgCatB protein.
authors: Ting
authors: T
authors: Zhang
authors: Li-Fang
authors: LF
authors: Jia
authors: Ying
authors: Y
authors: Chen
authors: Chuan
authors: C
authors: Ju
authors: Xiao-Jin
authors: XJ
authors: Mo
authors: Bin
authors: B
authors: Xu
authors: Shen-Bo
authors: SB
authors: Chen
authors: Jun-Hu
authors: JH
authors: Chen
authors: Wei
authors: W
authors: Hu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002401:Cathepsin B / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D019295:Computational Biology; D048209:Echinococcus granulosus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D015801:Helminth Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D015801:Helminth Proteins; D002401:Cathepsin B