The Approaching Of Refolding Of Human Bone Morphogenetic Protein-2 Mature Peptide (rhBMP-2m) In High Concentration

AIM: Bone morphogenetic protein-2(BMP-2) is one member of the bone morphogenetic protein (BMP) family. Up to date, more than 15 kinds of human BMP (hBMP) which could induce the situ and ectopic bone formation have been discovered, and the recombinant BMP-2 is the most powerful one for inducing of bone formation which could also coordinate with other members of BMPs. The activity pattern of the BMP-2 is homo- or hetero- dimmer, and the mature peptide of BMP-2 (BMP-2m) have 7 cysteines to form 3 intra-chain disulfide bonds and 1 inter-chain disulfide bond to form a activity dimmer.Escherichia coli (E. Coli) is the most commonly used recombinant protein expression systerm. Though the E. Coli does not have the eucaryotic post-translation modificaton and protein folding system, but it has several merits, such as the clear heredity background, safty, high-yield, simple manipulation, low cost, short manufacture time and so on, So it become the commonly used recombinant protein expression systerm and could not be replaced by some other expression system. The activity of the recombinant human bone morphogenetic protein-2 mature peptide (rhBMP-2m) prepared from eukaryotic cell is very well, but it could not generally used in clinic because of it's low--yield, and high cost. Our research team adopted nonfusion prokaryotic expression of rhBMP-2m to obtain the high expression quantity. Because the characteristics of the E. Coli system, the protein we got exists in inclusion bodies, and they do not have bioactivitivies. If we want to get the bioactive protein we have to refold the protein which exist in inclusion bodies. Since active BMP-m molecule is a dimmer with seven disulfide bonds, with strong hydrophobility, BMP is one of the most difficult renaturation protein and has no ideal refolding method for BMP today. In this paper, after the high density fermentation and purification of the rhBMP-2m, we investigate the refolding conditons of the rhBMP-2m and after that we detect the bioactivities of the refolding rhBMP-2m.METHODS:1. The high density fermentation of the rhBMP-2m: We used NBS 15L fermentation tank and the permanent dissolved oxygen (DO)-batch feeding cultivation to carry out the high density fermentation of the rhBMP-2m. During the fermentation process, pH 7.0 and 40%DO were kept all the time. The feedings were added 6 hours after the growth of the strain at 32℃, then 16h later the temperature was changed from 32℃to 42℃to induce the expression of the rhBMP-2m for 4h.2. The purification of the rhBMP-2m: STE, lysozyme, sodium deoxycholate and ultraphonic were used to split the harvest bateria after fermentation, then TritonX-100 and ultraphonic were used repeatly to wash the inclusion bodies for 4 times. After that positive-ion (SP-Sepharose FF) and negetive-ion (Q-Sepharose FF) exchange chromatography were employed in succession to further purify the rhBMP-2m.3. The refolding of the rhBMP-2m: The purified rhBMP-2m was refolded in different conditions, including dissimilar dialysis methods, changing the pH, temperature, oxidize-reduced pair and ion concentration. The refolding results were analyzed by non-reduction SDS-PAGE. Then, the refolding rhBMP-2m was passed through a 0.22μm micropore filter membrane for degermation, and the loss percentage was analyzed.4. The bioactivitivies of the refolded rhBMP-2m: According the method for detecting the activity of BMP established by Dr. Bang-Fu ZHU (National Patent: ZL 01 1 31813.9) First incubated the resuscitated C2C12 cells at 37℃for 48h, then inoculated the C2C12 cells at 24-hole-board, and made sure that each hole has 1×10~5 C2C12 cells. After that, added the unfolded rhBMP-2m and 3 different concentrations of refolded rhBMP-2m (0.2 mg/mL,0.02 mg/mL,0.002 mg/mL) to incubate with cells, and added 500μl culture medium to each hole, at last incubated the cells at 37℃for 24h to examine the fluorescence of cells for estimating the bioactivities of the refolded rhBMP-2m.RESULTS:1. The high density fermentation of the rhBMP-2m: At the end of the high density fermentation, OD600 values of the bacterial cultures were 60.5. The wet weight of the harvest bacteria in 4L fermentation broth was 386g. The percent of rhBMP-2m was accounted for 20% of the total bacterial protein.The inoculation's concentration, sterile environment, culture broth's ingredient, the feeding addition mode and speed, pH, DO, temperture and so on, all these fators influence the high density fermentation. So we have to keep eyes on these fantors during fermentation. The amount of target protein expressed in fermentation tank was always lower than in test tube. This phenomenon tell us that there are some other unknown impact factors which could effect the expression of the recombinant protein, so we should do more research work to solve these problem.2. The purification of the rhBMP-2m: After the high density fermentation the percent of rhBMP-2m was about 20% in the total protein of bacteria. The purity of the rhBMP-2m achieved to 75% after 4 times washings of the inclusion bodies, and the recovery rate is 51.19%. After purification by SP-Sepharose FF ion exchange chromatography, the purity of the rhBMP-2m reached up to 85%, and the total recovery rate was 32.23%. Succeeding Q-Sepharose FF ion exchange chromatography, the purity of the rhBMP-2m reached up to 95%, and the total recovery rate was 19.70%.From the washing and purification data we can see that more washing times and purify steps more pure of the interest protein and more loss of interest protein. In short, the protein's character, purity, recovery rate and cost should all be considered when purify one interest protein.3. The refolding of the rhBMP-2m: We investigated different factors which influence the refolding protein. We found the pH and the temperature affect the refolding significantly, while the concentration of ion and oxidize-reduced pair, and volume of dialysis refolding buffer have little effect. pH8.5 is benefit for refolding efficiency(30%) and pH4.8 is good for soluble of rhBMP-2m. The low temperature such as 4℃(30% refolding efficiency) was much better than 32℃(23% refolding efficiency). The refolding efficiency of high(10mg/ml) and low(0.1mg/ml) concentration of rhBMP-2m have no distinct difference. However, refolding rhBMP-2m at high concentration has the advantage in favor of the recovery of the renatured protein, reducing the lose of the protein, shortening the work time, lessening the work intension, and then will be propitious to using in industrial production. We also found that when the concentration of the rhBMP-2m was more than 6mg/ml it would precipitating out even under the changing pH condition. Considering the solubility and the recovery efficiency of rhBMP-2m, we devised the protocol which refolding rhBMP-2m at 6mg/ml by changing pH successively. The high pH is beneficial for refolding and low pH keeps the refolding rhBMP-2m soluble even after removing the deforming agent urea from the high concentration of the refolding protein. The quantity of the refolded rhBMP-2m dimmer had no difference between one-step(31.8%) and step-by-step dialysis(32.1%). The solubility of the refolded rhBMP-2m makes the degermation using 0.22μm micropore filter membrane possible, and the experiment has been proved it. The loss percentage was under 10%, and the dimmer percent of rhBMP-2m remained 30%. This renaturation protocol solve the difficult problem in refolding rhBMP at low protein concentration using previously, which leading severe losing of renatured rhBMP and difficult to degermation of the rhBMP before it applying in clinic.4. The bioactivitivies of the refolded rhBMP-2m: The bioactivies of the refolded rhBMP-2m was higher than unfolded rhBMP-2m. The fluorescence of 20μg/ml refolded rhBMP-2m of one-step and step-by-step dialysis of uera both are 20. This was 4 fold than the fluorescence of the unfolded which was 5. The bioactivies of the refolded rhBMP-2m was higher than unfolded rhBMP-2m in C2C12 cells (p<0.01), and there were no significant deviation between the two dialysis (p>0.05).In theoretic suppossing, the refolding efficiency of one-step dialysis of uera would be lower than step-by-step dialysis of uera, but either the non-reductive SDS-PAGE or the bioactivities analyzing results all suggested that there were no distinct difference between the two methods. It tells us that there are lots of unknown factors which influence the refolding process. That is why the refolding of recombinant protein is difficult.