title: [Prokaryotic expression, purification and activity detection of the extracellular and intracellular domains of human IGF1R beta subunit].
abstract: OBJECTIVE
  To construct the prokaryotic expression vectors of extracellular domain (β-ED) and intracellular protein kinase domain (β-PKD) of insulin-like growth factor 1 receptor beta (IGF1R-β) subunit, purify the fusion proteins GST-IGF1R β-ED and GST-IGF1R β-PKD, and detect their activities.
  
  
  METHODS
  Human GST-IGF1R β-ED and GST-IGF1R β-PKD coding regions were amplified from human mammary cDNA library by PCR and cloned into the prokaryotic expression vector pGEX-KG. The fusion proteins GST-IGF1R β-ED and GST-IGF1R β-PKD were expressed in E.coli Rossate and purified by GST-Sepharose 4B beads. The expression of the fusion proteins were detected by Western blotting. The interactions of the proteins with mediator of epidermal growth factor receptor-2 (ERBB2)-driven cell motility (MEMO) protein were identified by GST pull-down assay.
  
  
  RESULTS
  GST-IGF1R β-ED and GST-IGF1R β-PKD recombinant plasmids were successfully cloned. Double enzyme digestion and sequencing confirmed that the inserted fragments were identical to the target ones. The fusion proteins were successfully induced in Rossate and Western blotting showed the expression as expected. GST pull-down assay revealed that GST-IGF1R β-PKD could interact with MEMO in vitro.
  
  
  CONCLUSIONS
  GST-IGF1R β-ED and GST-IGF1R β-PKD were successfully cloned and purified. In addition, GST-IGF1R β-PKD could interact with MEMO in vitro, which demonstrated the good activity of the purified proteins.
authors.affiliation: Institute of Biotechnology, Dalian Medical University, Dalian 116044; Beijing Institute of Biotechnology, Military Academy of Medical Sciences, Beijing 100850, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Guo
authors.affiliation: Second Artillery General Hospital, Beijing 100088, China.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Feng
authors.affiliation: Institute of Biotechnology, Dalian Medical University, Dalian 116044, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.affiliation: Department of Science and Technology, Military Academy of Medical Sciences, Beijing 100850, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Institute of Biotechnology, Dalian Medical University, Dalian 116044, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.affiliation: Institute of Biotechnology, Dalian Medical University, Dalian 116044, China.
authors.forename: Quanbo
authors.initials: Q
authors.lastname: Ji
authors.affiliation: Institute of Biotechnology, Dalian Medical University, Dalian 116044, China.
authors.forename: Xiaojie
authors.initials: X
authors.lastname: Xu
authors.affiliation: Institute of Biotechnology, Dalian Medical University, Dalian 116044, China.
authors.forename: Qinong
authors.initials: Q
authors.lastname: Ye
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D019159:Nonheme Iron Proteins / Q000235:genetics; D010957:Plasmids; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D017526:Receptor, IGF Type 1 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C527585:MEMO1 protein, human; D019159:Nonheme Iron Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D017526:Receptor, IGF Type 1