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Method for producing recombinant human bone morphogenetic protein-2 mature peptide
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A morphogenetic protein and production method technology, applied in the field of protein purification, can solve problems such as the inability to realize efficient industrial production
Active Publication Date: 2014-07-16
HANGZHOU JIUYUAN GENE ENG
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[0012] In order to solve the technical defect that the production of recombinant human bone morphogenetic protein-2 (rhBMP-2) mature peptide cannot realize efficient industrial production in the prior art, the present invention provides a method different from the above-mentioned method, which is easy to operate, low in cost, suitable for Separation and purification method for large-scale scale-up production of high-purity and high-activity recombinant human bone morphogenetic protein-2 mature peptide
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example 1
[0037] Example 1 Solubilization of inclusion bodies
[0038] Dissolve the inclusion bodies in the lysate (7M guanidine hydrochloride, 50mM Tris-HCl, pH8.0, 1mM EDTA, 10mM DTT) at a ratio of 1 gram of inclusion body: 20mL of lysate, and stir at room temperature for more than 2 hours or at 4°C overnight. Then centrifuge at 12000rpm and 4°C for 20min, discard the precipitate and take the centrifugation supernatant. Protein concentration was determined by Bradford method.
[0039]Or solubilize by the following method. Dissolve inclusion bodies in lysate (8M urea, 50mM Tris-HCl, pH8.5, 1mM EDTA, 10mM DTT) at a ratio of 1 gram of inclusion body: 20mL of lysate, and stir at room temperature for more than 2 hours or overnight at 4°C . Then centrifuge at 12000rpm and 4°C for 20min, discard the precipitate and take the centrifugation supernatant. Protein concentration was determined by Bradford method.
example 2
[0040] Example 2 Renaturation of Inclusion Body
[0041] Refolding by dilution method. The refolding solution consists of: 100mM Tris-HCl, 0.5M L-Arg, 0.5M Urea, 1M NaCl, 2mM EDTA, pH8.5-9.5, 1mM oxidized glutathione, 5mM reduced glutathione. Slowly add the inclusion body enhancement solution into the refolding solution, control the final protein concentration to 0.05-0.3 mg / mL, and refold under a constant temperature environment of 4-20 degrees for 1-20 days.
example 3
[0042] The hydrophobic interaction chromatography of example 3 refolding solution
[0043] Firstly, hydrophobic interaction chromatography with low salt concentration is used, the chromatography filler is phenylsepharose Fast Flow, and the equilibrium solution is phe-A: 0.5M urea, 25mM CHES, 15mM Tris, pH8.5, 5% N, N-dimethylformamide , 1M NaCl. The eluent was phe-B: 0.5Murea, 25mM CHES, 15mM Tris, pH 8.5, 5% N,N-dimethylformamide. First, use the balance solution phe-A to balance, and the refolding solution to directly load the sample. After the sample is loaded, continue to wash with the balance buffer phe-A until the UV reaches the baseline, and then use the elution buffer phe-B to gradually reduce the salt concentration. Elution was performed and the main peak was collected.
[0044] In order to further improve the purity, the hydrophobic interaction chromatography with high salt concentration can continue to be used for collecting the main peak. The chromatographic fill...
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Abstract
The invention relates to a method for producing recombinant human bone morphogenetic protein-2 mature peptide. The method comprises the following steps: sampling a recombinant human bone morphogenetic protein-2 mature peptide solution with good renaturation into a balanced hydrophobic chromatography column, then performing a stepped-gradient elution which gradually reduces the salinity by an elution buffer solution, and collecting target peaks. In order to further enhance the protein purity of the target peaks, the purification method can be a multi-step hydrophobic interaction chromatography or be used by combining with the ion exchange chromatography. The method has the advantages of simple operation, lower cost, higher purification yield (more than 20%), higher purity (SDS-PAGE, HPLC and HPCE are greater than 97%) and the like, and is suitable for producing the recombinant human bone morphogenetic protein family.
Description
technical field [0001] The invention relates to the field of protein purification, in particular to a method for purifying and producing recombinant human bone morphogenetic protein-2 (rhBMP-2) mature peptide. Background technique [0002] In 1965, American doctor Urist (Urist MR. Bone: formation byautoinduction. Science, 1965, 150 (698): 893-899) found that the decalcified bone mesenchyme has ectopic osteogenesis, and the newly discovered ability to induce new The protein of bone formation is named bone morphogenetic protein (Bone Morphogenetic protein, BMP). Subsequently, many BMPs were discovered, except for BMP-1, they are all members of the TGF-beta superfamily, and they are very similar in gene or protein structure, or in biological function. Among them, the osteogenesis of BMP-2 is the most researched, and the industrialization research is also the most extensive. [0003] The full length of human BMP-2 is 396 amino acid residues, and the mature peptide with osteoge...
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