Cloning And Expression Of DNA Sequence Encoding Mature Peptide Of Human Bone Morphogenetic Protein 4

BMP-4 is a member of transforming growth factors 15(TGF-B) superfamily, which can induce cartilage and bone formation. It has great potential for medical therapeutic application in bone fracture and critical size bone defect. In this study, recombinant DNA technology was applied for producing recombinant human BMP-4 mature peptide.Genomic DNA was extracted from human hair root by cell lysis method. The DNA sequence encoding mature peptide of hBMP-4 was amplified by PCR and then inserted into pUCm-T vector. Then the encoding sequence was cloned into different prokaryotic expression vectors pBV220, pGEX-3X and pET42a. In additiong, the sequence was also cloned into eukaryotic expression vector pIC9.pBV220/hBMP-4 was transformed into E.coli DH5 a . After optimization of condition, the yield of non-fusion protein in E.coli DH5 a was 29.70mg/L medium, that mainly existed in inclusion bodies and accounted for 85.5% of the total protein in inclusion bodies, 17.4% of the total protein of E.coli DH5 a . With Western blotting, it was confirmed that the rhBMP-4 was produced by genetic engineering in prokaryotic cells. It was also proved in vitro that renatured rhBMP-4 had biological activity.Then pGEX-3X/hBMP-4 and pET42a/ hBMP-4 were transformed into DH5 a and BL21(DE3)pLysS. After optimization of condition, the yield of fusion protein in E.coli and E.coli BL21(DE3)pLysS was 5.85mg/L medium and 18.00mg/L medium. Fusion protein was purified by glutathione sepharose 4B affinity chromatography and cleaved by factor Xa. Then recombinant hBMP-4 with three additional amino acids at the N-terminal and human intact BMP-4 was obtained.With Western blotting, it was confirmed that the expressed products are hBMP-4. It was also proved in vitro that rhBMP-4 has biological activity. All of the results collected from these studies indicated:1. DNA extracted from hair root was a qualified template for PCR test.2. The DNA sequence encoding mature peptide of hBMP-4 has no difference between Chinese and White.3. Recombinant hBMP-4 expressed in non-fusion expression system has good biological activity after renaturation.4. Recombinant hBMP-4 expressed in fusion expression system cleaved by factor Xa has good biological activity.