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A cyclic chromatography renaturation method for recombinant human bone morphogenetic protein-2

A morphogenetic protein and circulating layer technology, which is applied in the fields of bone morphogenetic factors, peptide preparation, animal/human proteins, etc. problems, to achieve the effect of being suitable for industrial scale production, reducing protein precipitation, and small processing volume

Active Publication Date: 2018-07-20
YANTAI ZHENGHAI BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current renaturation process has problems such as easy protein precipitation, low renaturation rate, cumbersome process route, long production cycle, large consumption of renaturation solution and high cost, and is not suitable for industrial scale production.

Method used

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  • A cyclic chromatography renaturation method for recombinant human bone morphogenetic protein-2
  • A cyclic chromatography renaturation method for recombinant human bone morphogenetic protein-2
  • A cyclic chromatography renaturation method for recombinant human bone morphogenetic protein-2

Examples

Experimental program
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Effect test

Embodiment 1

[0074] Example 1. Refolding of recombinant human bone morphogenetic protein-2rhBMP-2 inclusion bodies by affinity chromatography

[0075] 1. Denaturation of rhBMP-2 inclusion bodies

[0076] Inclusion body solution: 0.1mol / L Tris-HCl, 8mol / L urea, 0.1mol / L DTT, 5mmol / L EDTA; pH8.5. Each concentration is the final concentration of the corresponding component in the solution, and the solvent is water.

[0077] Denaturation: Dissolve inclusion bodies at a ratio of 1 gram of recombinant human bone morphogenetic protein-2rhBMP-2 inclusion body: 10 mL of inclusion body solution, shake at 25°C and 100 r / min for 6 hours, centrifuge at 12,000 g for 30 minutes at high speed, and collect the supernatant The solution is denatured rhBMP-2 protein solution.

[0078] 2. Refolding of rhBMP-2 by affinity chromatography after denaturation

[0079] 1. Pre-purification

[0080] In order to improve the purity of the protein and reduce the content of impurities such as nucleic acid, the denatur...

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Abstract

The invention discloses a recombinant human bone morphogenetic protein-2 recycling chromatography renaturation method. The method comprises 1, carrying out denaturation on a human bone morphogenetic protein-2 inclusion body to obtain a denatured protein, and carrying out affinity chromatography renaturation on the denatured protein, and collecting the eluent to obtain the recombinant human bone morphogenetic protein-2 dimer subjected to renaturation. The human bone morphogenetic protein-2 has an amino acid sequence shown in the formula 2 in the sequence table. An experiment proves that the method realizes rhBMP-2 protein renaturation, has the advantages of high protein concentration, less reagent consumption and renaturation rate of 70% or more, is suitable for industrial large-scale production and provides reference for research and development of the same type of a recombinant protein drug.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the field of gene recombinant protein medicine, and relates to a cyclic chromatography renaturation method for 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 decalcified bone matrix extract. 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 osteogen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/51C07K1/22C07K1/113C07K1/18
CPCC07K14/51
Inventor 彭继学张东刚屈长宏刘启省杨建军
Owner YANTAI ZHENGHAI BIO TECH
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