Purification production method for recombinant human bone morphogenetic protein-2

A morphogenetic protein and production method technology, applied in the field of protein purification, can solve the problems of low renaturation rate, unsuitable for industrial scale production, easy protein precipitation and low yield, etc.

Active Publication Date: 2015-03-25
YANTAI ZHENGHAI BIO TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The above production processes all have problems such as low renaturation rate and lo

Method used

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  • Purification production method for recombinant human bone morphogenetic protein-2
  • Purification production method for recombinant human bone morphogenetic protein-2
  • Purification production method for recombinant human bone morphogenetic protein-2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1, purification of recombinant human bone morphogenetic protein-2

[0059] 1. Dissolution of inclusion bodies

[0060] Inclusion body: the inclusion body containing the recombinant human bone morphogenetic protein-2 (rhBMP-2, sequence 1) was obtained through prokaryotic expression of Escherichia coli disclosed in the patent CN200510132792.9.

[0061] Inclusion body solution: 0.1mol / L Tris-HCl, 8mol / L guanidine hydrochloride, 10mmol / L DDT, pH9.0. Each concentration is the final concentration of the corresponding component in the solution.

[0062] Inclusion bodies were dissolved at a ratio of 1 gram of inclusion body: 30 mL of inclusion body lysate, shaken at 4°C and 150 r / min for 18 hours, centrifuged at 10,000 g for 10 min at high speed, and the supernatant was collected as inclusion body lysate.

[0063] 2. Purification before renaturation

[0064] Affinity chromatography and cation exchange chromatography were performed sequentially before protein renat...

Embodiment 2

[0109] The quality detection of embodiment 2, embodiment 1 gained protein product

[0110] 1. Detection of protein purity

[0111] Referring to Appendix IVC of the third part of the "Chinese Pharmacopoeia" in 2010, the purity of the protein stock solution obtained in Example 1 was tested by non-reducing SDS-polyacrylamide gel electrophoresis. The concentration of the separating gel was 15%, and the sample volume was not less than 10 μg (Coomassie Brilliant Blue R250 staining method).

[0112] The results showed that the purity of the rhBMP-2 dimer in the elution peak finally collected in Example 1 was 98.3% after scanning by the scanner. like Figure 7 shown.

[0113] 2. Calculation of protein yield

[0114] Referring to Appendix VIB of the third part of the "Chinese Pharmacopoeia" in 2010, the protein concentration of the protein stock solution obtained in Example 1 was measured by the lowrry method to be 0.75 mg / mL, and the rhBMP-2 stock solution could be obtained by purif...

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Abstract

The invention discloses a purification production method for recombinant human bone morphogenetic protein-2 (rhBMP-2 protein). The method comprises the following steps: 1) lysing an inclusion body containing rhBMP-2 protein, so as to obtain an inclusion body lysate; 2) successively performing affinity chromatography and cation exchange chromatography on the inclusion body lysate for separation purification, so as to obtain a denatured recombinant human bone morphogenetic protein-2 solution; 3) performing renaturation on the denatured rhBMP-2 protein, so as to obtain a renatured rhBMP-2 protein crude product; and 4) successively performing affinity chromatography and gel filtration chromatography for separation and purification, so as to obtain a renatured rhBMP-2 protein pure product. By using the provided method for purifying rhBMP-2 protein, the prepared product has the purity of 95% or more and the protein yield of 9.4%, and the endotoxin and DNA residues both accords with biological medicine quality requirements. The method is suitable for industrial large-scale production. Also the method provides reference for research and production of same-kind recombinant protein medicines.

Description

technical field [0001] The invention belongs to the field of protein purification and relates to a purification production method of recombinant human bone morphogenetic protein-2. Background technique [0002] Bone morphogenetic protein (BMP) is a hydrophobic protein secreted by bone matrix and belongs to the transforming growth factor (TGF-β) superfamily member. It was first proposed by Urist et al. in 1965, defined in 1972, and defined in 1982. An active protein isolated for the first time from demineralized bone matrix extracts. The BMP family includes BMP-1, 2, 4, 6, 7, 9, 12, 14, etc., among which BMP-2 can obviously induce the directional differentiation of undifferentiated fibroblasts into osteoblasts, and can regulate osteoblasts and osteoblasts. Chondrogenic differentiation, induction of ectopic bone formation and promotion of fracture healing functions. BMP-2 is the initiation factor of osteogenesis, which can accelerate bone reconstruction. It is the ideal targ...

Claims

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Application Information

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IPC IPC(8): C07K14/51C07K1/22C07K1/18C07K1/16C07K1/113
CPCC07K14/51
Inventor 张东刚李东升
Owner YANTAI ZHENGHAI BIO TECH
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