本研究將成熟之人類PDGF-B的cDNA，轉殖至大腸桿菌的蛋白質表現質體pEZZ18。此質體具有protein A signal sequence和ZZ domain。Protein A signal sequence可將表現之蛋白質送至培養基，有利於雙聚體之形成。ZZ domain與IgG sepharose具有很強之結合力，有利於純化。
利用不同培養基來培養大腸桿菌BL21，HB101及JM109等表現PDGF-BB的宿主以篩選最適合的組合，同時以Enzyme Linked ImmunoSorbent Assay (ELISA)偵測大腸桿菌異源表現之PDGF-B。以2xYT及M9做為培養基培養，而且以大腸桿菌BL21為宿主時，所表現之PDGF-B在胞外的濃度為最高；以SDS-polyacrylamide gel electrophresis (SDS-PAGE)及 Western blot證實經此表現系統所得到的PDGF-B可能具有雙硫鍵並且為雙聚體。 Platelet-derived growth factor (PDGF) is the major polypeptide growth factor of human serum and is a potent mitogen for cells of mesenchymal origin. It is stored in the ?granules of platelets, and is released into the serum during blood clotting. Biologically active PDGF consists of two inactive polypeptide chains, PDGF-A and PDGF-B, linked together by disulfide bonds. There are three isoforms, denoted as PDGF-AA, -AB, and -BB, which are homo- or heterodimers of related A and B polypeptide chains.
A cDNA fragment encoding the mature form of human PDGF-B chain was cloned into a plasmid pEZZ18 for heterogenous overexpression study. The plasmid pEZZ18 contains the protein A signal sequence and two synthetic ZZ domain. PDGF-B was expressed as a fusion protein with the ZZ peptide and secreted into aqueous culture medium.
In this study, E. coli strains BL21, HB101 and JM109 were cultured in the different media LB, 2xYT, super broth, TSY, and M9 medium. The production of PDGF-BB in growth media was monitored by Enzyme Linked ImmunoSorbent Assay (ELISA). The overexpressed PDGF-B was shown as dimers by SDS-polyacrylamide gel electrophresis (SDS-PAGE) and Western blotting analyses.
The highest PDGF-BB production was found using BL21 as host cultured in 2xYT and M9 medium.