Expression, Purification And Crystallization Of Recombinant Insulin-like Growth Factor Binding Protein-4 And Its Inhibitory Effect On Antler Cell Proliferation

Background and Objectives: Insulin-like growth factor binding protein-4 (IGFBP-4) is an important cytocine belonging to GH/IGF Somatotropic Axis. Nowadays more and more attentions were paid to IGFBP-4 for its wide range of growth-inhibitory and apoptosis-inducing actions on cells under biological and pathological conditions via IGF-dependent and IGF-independent mechanisms. It has been shown to take protective effect on common cancers such as lung cancer, prostate cancer, breast cancer, et al. Deer antlers are the only mammalian appendages that are cast and then fully regenerate each year. Therefore deer antler become a rare research model for regenerative medicine, which is a newly emerging field that holds exciting possibilities for the replacement of damaged or missing body parts in human beings. To study the relationship of function and structure of IGFBP-4 and the mechanism of deer antler regeneration, human IGFBP-4 cDNA was cloned and expressed in prokaryonic expression system, recombinant IGFBP-4 was purified, and experiments of its crystal growth and inhibitory effect to antler cells were conducted as well.Materials and Methods: The primers were designed and synthesized to amplify the human IGFBP-4 cDNA sequenc by PCR. The target gene was cloned into pGEX-6p-1 plasmid through BamHI and EcoRI restriction site which was designated pGEX-6p-1-BP4.The recombinant prokaryotic expression plasmid was confirmed by PCR amplification, restrictive enzymatic digestion and sequence analysis. Then the recombinant prokaryotic expression plasmid was transformed into E.coli. BL21(E3) cell. The stable transfectant, designated BL21-pGEX-6p-1-BP4, were selected by the addition of Ampilin to the growth medium, followed by a series of confirmation. Bl21-pGEX-6p-1-BP4 cells were cultured in LB medium till to OD600 value 0.6 to 0.8, then induced with IPTG to express recombinant IGFBP-4, expression was detected by SDS-PAGE electrophoresis.The recombinant IGFBP-4 existed in a resoluble GST fusion protein form, its purification was employed by affinity chromatography column, gel filtration chromatography and anion exchange column resourceQ. Crystals of recombinant IGFBP-4 were grown by the method of hanging-drop vapor-diffusion. Primary select solutions of crystal growing were provided with crystallization kits by XtalQuest Corporation.The biological activity of recombinant IGFBP-4 were examined by antler cell growing inhibit tests in vitro.Results and Conclusion:1.The recombinant prokaryotic expressing plasmid pGEX-6p-1-BP4 was constructed successfully;2.Stable transfectants BL21-pGEX-6p-1-BP4 were obtained and the IGFBP-4 was overexpressed successfully;3.The recombinant IGFBP-4 existed in a resoluble GST fusion protein form was purified by affinity chromatography column, gel filtration chromatography and anion exchange column resource Q.;4.Crystals of recombinant IGFBP-4were obtained by the method of hanging-drop vapor-diffusion;5.The growth-inhibitory and apoptosis-inducing effect of recombinant IGFBP-4 was shown in antler cells in vitro.