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Renaturation of reconstituted human bone protein-1 and making method of its preparation

A technology of osteogenic protein and preparation, which is applied in the preparation of osteogenic protein-1 preparation and recombinant human osteogenic protein-1 preparation, which can solve the problems of high cost, low output and limited use, and achieve the renaturation process easy-to-use effects

Inactive Publication Date: 2007-09-12
SHANDONG MEDICAL BIO TECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

OP-1 is a special protein that has been successfully expressed and refolded in eukaryotic CHO, but the yield is low and the cost is high
Although there are a large number of evaluation articles using the E. coli system to express human OP-1, they are all because of its daunting refolding (David CRueger, Biochemistry of bone morphogeneti proteins, in Bone Morphogenetic Proteins From Laboratory To Clinical Practice / Slobodan Vukicevic , KuberT.Sampath ed Basel.Bosdton.Berlin: Birkhauser.2002, 1-18) and its use is limited

Method used

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  • Renaturation of reconstituted human bone protein-1 and making method of its preparation
  • Renaturation of reconstituted human bone protein-1 and making method of its preparation
  • Renaturation of reconstituted human bone protein-1 and making method of its preparation

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Effect test

Embodiment 1

[0038] The rhOP-1 engineered bacteria are fermented, collected by centrifugation, lysed, and washed to obtain inclusion bodies. The inclusion bodies are denatured and dissolved by 8 mol / L urea, and then ion exchange (or / and hydrophobic, affinity and molecular exclusion) layer After analysis, collection and purification to a purity of over 95%, it was diluted to 200-600 μg / ml. Then put it into a conventionally treated dialysis bag (molecular weight cut-off of 10,000), in 6 mol / L of urea, 0.05 g / L of polyethylene glycol 4000, 0.4-0.6 mol / L of arginine, 20 mmol In the renaturation solution of phosphate buffer (pH8.0~9.5) in the renaturation solution of phosphate buffer (pH8.0~9.5), air oxidative renaturation at room temperature for 24~96 hours, and then undergo further ion exchange chromatography and size exclusion purification to make the dimer purity of the target protein reach 90 %above. Then 5 mol / L urea, 4 mol / L urea, 2 mol / L urea, and 1 mol / L urea were dialyzed into 20 mmo...

Embodiment 2

[0042] The rhOp-1 engineered bacteria were fermented, collected by centrifugation, lysed, and washed to obtain inclusion bodies, which were denatured and dissolved by 6 mol / L guanidine hydrochloride; ion exchange (or / and hydrophobic, affinity and molecular exclusion) After chromatographic collection and purification reach more than 95% of the purity, in the guanidine hydrochloride of 4 mol / liter, and the arginine of 0.2~0.5 mol / liter, the copper ion of 0.1 micromol / liter~0.1 mmol / liter (chloride copper or copper sulfate); 20 mmol / L phosphate refolding solution (pH8.0~9.0) was refolded at room temperature for 24 to 96 hours, and then further purified by ion exchange chromatography (or / and reversed phase, hydrophobic, Non-specific affinity and molecular exclusion) make the purity of the target protein reach more than 90%. The obtained sample is directly compounded with the carrier material (collagen, absorbent gelatin sponge, chitosan or coral powder, one of the synthetic polyme...

Embodiment 3

[0044] The inclusion bodies are denatured and dissolved with 8 mol / L urea, collected and purified by ion exchange chromatography to reach a purity of over 95%, and then diluted to 100-600 μg / ml. Then put it into a conventionally treated dialysis bag (molecular weight cut off is 10,000), and directly oxidize it in air at room temperature in the refolding solution of 6 mol / L urea and 20 mmol / L phosphate buffer (pH9.0~9.5). After renaturation for 24-96 hours, further ion-exchange chromatography and molecular exclusion purification are performed to make the dimer purity of the target protein reach more than 90%. Then 5 mol / L urea, 4 mol / L urea, 2 mol / L urea, and 1 mol / L urea were dialyzed into 20 mmol / L phosphate buffer solution, and freeze-dried to obtain rhOP-1 freeze-dried powder. Store at -20°C after ethylene oxide sterilization. Alternatively, the target protein whose dimer purity reaches more than 90% is directly compounded with a certain amount of absorbent gelatin sponge ...

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Abstract

The present invention discloses a recombinant human osteoplastic protein-1 renaturation and its preparation preparation method. It utilizes chromatography to obtain rh OP-1 monomer protein whose purity is above 95%. Said invention also provides the concrete steps of said preparation method, including: in 2M-6M urea or 1M-4M guanidine hydrochloride buffer solution making dialysis and renaturation, pH value of renaturation buffer solution is 8.0-10, renaturation temperature is room temperature or 4deg.C and renaturation time is 24-96 hr, then further making chromatographic purification, dialysis, sterilization, packaging and freeze-drying so as to obtain the renaturation preparation of recombinant human osteoplastic protein-1. Besides, said invention also provides the application method of said renaturation preparation of recombinant human osteoplastic protein-1.

Description

Technical field: [0001] The invention relates to a preparation method of osteogenic protein-1, in particular to a preparation method of recombinant human osteogenic protein-1 (rhOP-1). It uses genetic engineering means to express and mass-produce recombinant human osteogenic protein-1 mature peptide in Escherichia coli to refold recombinant human osteogenic protein-1 and prepare its preparations. After sterilization, the preparation prepared by the invention can be clinically used for repairing and orthopedic bone defects, and for maintaining and recovering alveolar bone in oral cavity tooth extraction wounds and the like. It belongs to the field of biotechnology and pharmaceuticals. Background technique: [0002] With the rapid development of genetic engineering technology, more and more people provide some protein and polypeptide products for clinical and industrial production through the heterologous expression of protein. So far, E.coli is still the preferred expressio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/14A61K38/18A61P19/00
Inventor 王世立韩金祥李俊玲车婧
Owner SHANDONG MEDICAL BIO TECH RES CENT
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