[Prokaryotic expression, purification and activity detection of the extracellular and intracellular domains of human IGF1R beta subunit].

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.

Conclusion: 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.