Expression, Renaturation And Purification Of RhBMP-2 In E.coli

In this article, recombinant plasmid pGEX-BMP-2 containing sequence of mature human bone morphorgenic protein (BMP-2) was constructed and transferred into E. coli. The main work included three parts: the expression of rhBMP-2 in different conditions, refolding of the aimed protein by dilution and dialysis, and refolding by hydroxyapatite (HA) chromatography.1. Expression.The author firstly studied the soluble expression of the aimed protein, both in fusion form with GST and non-fusion form. The result showed that , even in 19°C and with the inducing intensity of 0. 05mM and 0.1mM IPTG , the aimed protein was expressed as inclusion bodies and almost no soluble form was found. Then, we researched the influence of the concentration of IPTG on the expression of the aimed protein. We found that, when the concentration of IPTG ranged in 0.2~0.4mM, the expression yield reached the summit.2.Refolding by dialysis and dilution.This experiment chose the most commonly used procedures, step-dialyze and step-dilution to refold the inclusion bodies, adding low concentration urea to the refolding buffer as the solubilizationassistant. The refolding yield was very low in both procedures, below 5% in dilution and 1 % in dialyzation.3. Refolding by Hydraxyapatite chromatography.The author put forward a new refolding procedure, HA chromatography, based the well-known fact that native BMP-2 can bond with HA which is the main content of bone mineral materials. At first, we chose three different kinds of HA powder as the solid phase to study the bond ability of them to the aimed protein, and we found the non-sintering powder had the most strong affinity. During this course, we noted that, on the one hand, urea, the denature regent, could decrease the ability of HA to bond the aimed protein. On the other hand, without adding urea during refolding process, the denatured protein would aggregate in the column. Because of this, the urea should be added before and after loading the denatured protein, but its amount should be controlled. And the protein concentration in the loading buffer was optimized. In addition, we found that HA could bond glutathione, which will lead to a poor oxidizing ability of the refolding buffer if the on-column oxidization procedure was taken. To solve it, we put forward an after-column oxidization procedure, which got on very well with the refolding of denatured and reduced lysozyme. Then, we research the proper conditions for the after-column oxidization of rhBMP-2, including protein concentration, temperature, pH and ratio between reduced and oxidized glutathione. With all those optimized procedures, the refolding yield was enhanced to above 30% and simultaneously the purity of rhBMP-2 was about 90%. Finally, the bioactivity of the refolded and oxidized dimmer was tested by the induction of alkaline phosphate activity in C2C12 cells.