Fermentative production of insulin-like growth factor

• Main Authors: Chou, Shih-Min, 周世民
• Other Authors: Yuan-Chi Su, Jan-Hsiung Huang
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/07798308270809894579

碩士 === 國立臺灣大學 === 農業化學系 === 85 === In this study, GST-IGF-II fusion protein produced from GST(Glutathione S- transferase) gene fusion system was quantified bymeasuring GST activities. The relationship between CDNB assay anddensitometry was good. Hence, CDNB assay could be used as fastquantification method for these kinds of fusion protein. The growth and induction of E. coli BL21(DE3) pGEX/ IGF-II inHinton''s flask were better in mLB medium(CSL 10 mL/L, YE 5 g/L,NaCl 10 g/L) when compared to LB medium. When the scale was enlargedto 5 L fermenter and fed-batch was used to be high cell density culture,the cell density OD600 about 60 (dry cell weight 25.37 g/L) was achievedafter 11 hours. During the time, FM2 medium (starting medium :CSL 10 mL/L, YE 23.75 g/L, NaCl 10 g/L, tryptone 37.5 g/L，feedingsolution : 60% glucose) was used,and working volume was 2 L . Feedingstrategy was depended on glucose monitoring method. Because the limitationof dissolved oxygen, induction point was advanced to 7th hour. Celldensity OD600 about 40 (dry cell weight 19 g/L) was achieved after inducedwith IPTG for 9 hours. Finally, the IGF-II recovery from HCDC was sevenfoldhigher than that from Hinton''s flask. But there were lots of proteasedegradation fragments in HCDC IGF-II. The host-vector system of E. coli BL21(DE3) pGEX/IGF-II was very stable.After 100 generations, there were still above 80% host cells bearing plasmids.When induced with IPTG, 95% host cells had plasmids during the fermentationprocess in Hinton''s flask and in 5L fermenter. It was not easy to recovery IGF-II from inclusion bodies by simpletreatment. Therefore, we must avoid the inclusion bodies formation duringthe fermentation.