Expression Of Mature Peptide Gene Of Human Bone Morphogenic Protein-7 And Its Bioactivity Analysis

Bone morohogenetic proteins(BMPs), a group of multi-functional proteins that were identified as bone inductive factors, belong to the transforming growth factor beta (TGF-P) superfamily due to their structural and functional similarities. BMP-7, also known as osteogenic protein (OP-1), has attracted much attention ever since its discovery because of its great bone inductive function. Recent studies indicate that human BMP-7 can be generated sufficiently for basic research and clinical trials by genetic engineering techniques.BMP-7 is synthesized as a large precursor protein that is cleaved at the dibasic cleavage site (RXXR) to release the carboxy-terminal domain. Biologically active BMP-7 is a disulfide-linked homodimer, of the carboxy-terminal 139 amino acid residues that contains the characteristic seven conserved cysteine residues involved in the formation of the cysteine knot and the single interchain disulfide bond. The work mainly consisted of the following three parts:1. The gene encoding mature region of hBMP-7 was inserted into prokaryotic expression vector pMAL-p2x and transformed into competent E. coli ER2566. Then the hBMP7-MBP fusion protein was successfully produced which expressed secretly at high level in the periplasm. Fusion protein was purified to 80% by affinity purification and then was cleaved by Factor Xa. As a result, the interest protein hBMP-7 mature peptide monomer was obtained.2. Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, andposttranslational modification. So the aim was to use Pichia pastoris expression system to express biologically active human hBMP-7 mature peptides.The DNA sequence encoding the mature peptide of hBMP-7 was amplified by PCR and cloned into Pichia pastoris expression vector pPIC9K. The recombinant pPIC-hBMP7 was linearized and electroporated into Pichia pastoris SMD1168 strain. And the G418 hyper-resistant strains and Mut~+ transformants were screened. The expressing strain was expressed in soluble form by secreting into culture medium. Recombinant protein was of 8% total secreted protein and easily identified by Western Blot and ELISA with specific antibody binding activity.3. To increase the expression product, the gene of hBMP-7 mature peptide was modified but not changed amino acid, according to codon usage bias in Pichia pastoris expression system. Three low-usage ARG codons of gene fragment coding the mature peptide of hBMP-7 have been successfully converted into Pichia pastoris preferred ARG codons by Splicing overlap extension PCR for a high level expression of hBMP-7 mature peptide. The results showed that the production level of codon-optimized hBMP-7(2.5mg/L) had an improvement of 4-fold relative to that of non-codon-optimized hBMP-7(0.6mg/L). Due to glycosylation, the recombinant hBMP-7 mature peptide was produced as an 18kD under reducing conditions and as a approximately 36kD protein under non-reducing conditions in SDS-PAGE and was easily purified from culture supernatant by using Ni-NTA chromatography. Cytological assay demonstrated that rhBMP-7 mature peptide could differentiate fibroblasts cell to osteoblast.These results indicate that the rhBMP-7 mature peptide dipolymer obtained from the yeast expression system could be conductive to further study.