| Bone morohogenetic proteins, a group of proteins that was originally identified as bone inductive factors, have attracted much attention ever since their discovery. These polypeptide cytokines, except for BMP-1, comprise a sub-family of TGF-p superfamily due to their structural and functional similarities. Recent studies demonstrated that BMPs, except for BMP-1, play an important role not only in the embryonic bone differentiation and adult bone repair, but also in mesoderm induction/differentiation during embryo development, hematopoietic tissue development and nerves system development/repair. All these messages indicate that study of BMPs is both theoretically instructive and clinically promising.BMPs are distributed mainly in bone, however, extraction of BMPs from bone tissues have been proved complicated and less repetitive and low productivity (l-5mg/40kg wet bone). It's of great difficulty to generate single component BMP product enough for use. For clinical application, human-derived BMPs are the best choice. However, the scarcity of human bone tissues has made the application and mechanism research of BMPs rather limited.At least 20 hBMPs have been cloned, named hBMPl-15, CDMP-1, CDMP-2 hBMP-lB, 3B and 8B, respectively. hBMP-2,3, 4, 7 demonstrated higher content in human bone, while BMP-2,4,7 displayed higher osteogenic activity. The proprotein forms of BMPs comprise signal peptide in N-terminal, leading peptide in the middle and mature peptide. Mature BMPs can only beachieved after post-translational process of glycation, folding and excision of signal peptide and preprotein. No significant effect on the biological activities of hBMPs has been found in glycation, indicating the possibility of generating hBMPs in prokaryotic or low-grade eukaryotic expression systems.As a clinically promising bone inductive cytokine, BMP-2 has been successfully expressed in COS and CHO eukaryotic expression system, insect expression system and prokaryotic expression system as a biologically active protein product. However, problems are still present. Eukaryotic expression system generates low productivity and high cost; proteins expressed in insect expression system are difficult to purify; while prokaryotic expression systems generally express proteins in inclusion bodies, which needs additional renatruration process to obtain products with biological activities, and these processes, are always in poor efficiency, biologically instability and poor repeatability. Therefore, it's necessary to find a new express system that can produce bioactive mature rhBMP2 peptide with lower expense and simpler procedure. It will be very important for both basic research and clinical application.Yeast represents a protein expression system for it's unique biological characteristics as not only a microorganism but also a eukaryote. It has a protein folding system that is more sophisticate than E coli's, and can excrete correctly processed recombinant protein into culture medium. Yeast can also preliminarily glycates Asn of Asn-X-Ser/Thr motif. Compared with mammalian expression system, yeast culture has the advantages hi some aspects such as simple culture system and rapid proliferation.Pichia pastoris has been proved a successful high-efficiency yeast expression system characteristic of simple genetic manipulation, high level expression, and much similar to eukaryotic glycation pattern. With advanced high density culture techniques, Pichia pastoris culture can reach a density of 500 ODeoo/l, and a scale of 3000m3. Up to now, various exogenous proteins have been successfully expressed in Pichia pastoris system, indicating that Pichia pastoris is an expression system that can be used in both laboratory research and industrial production.In this research, we aim to use Pichia pastoris expression system to express human hBMP-2 mature peptides. Expression products are purified and biology activities are investigated for purpose of its clinical application.Using yeast expression system, we achieved the first... |
PDF Full Text Request