title: [Fusion construction, prokaryotic expression and structure characteristics prediction of bimolecular thrombopoietin].
abstract: OBJECTIVE
  Design, construction, expression in E coli and protein characteristics prediction of bimolecular thrombopoietin (T-T) with more stability, efficiency, and lower toxicity.
  
  
  METHODS
  The expression vectors of TPO and T-T, pET32 a(+)/TPO and pET32 a (+)/T-T, had been constructed by molecular cloning methods. Then, they were expressed in host bacterium. Their products were identified by Western blot. The protein characteristics, such as second structure, antigenicity, hydrophilicity, flexibility and isoelectric point, were predicted by DS Gene and Protscale software.
  
  
  RESULTS
  The expressing vectors pET32a(+)/TPO and T-T were constituted correctly and expressed in origami (DE3), and their expression efficiency were more than 40 percent of total protein. T-T was identified correctly by Western blot. DS Gene and Protscale software predict the protein characteristics of TPO sequences in T-T molecule were no change, there was high flexibility in the linker domain. But two amino acids in T-T molecule have been mutated, and an insert fragment with 34 amino acids following the linker had antigenicity, hydrophilicity, and beta-sheet structure.
  
  
  CONCLUSIONS
  We have constructed correctly and expressed T-T with high level in E Coli. Protein characteristics prediction of T-T accords with our design.
authors.affiliation: Department of Immunology, Affiliated Hospital of Academy of Military Medical Sciences, Laboratory of Immunoassay of National Center of Biomedical Analysis, Beijing 100039, China.
authors.forename: Si-Qi
authors.initials: SQ
authors.lastname: Guo
authors.forename: Yong-Zhi
authors.initials: YZ
authors.lastname: Xi
authors.forename: Zhi-Hong
authors.initials: ZH
authors.lastname: Yuan
authors.forename: Jian-Wu
authors.initials: JW
authors.lastname: Cui
authors.forename: Fei
authors.initials: F
authors.lastname: Liang
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D013926:Thrombopoietin / Q000235:genetics*
chemical_list: D011993:Recombinant Fusion Proteins; D013926:Thrombopoietin