| Summary: | 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.
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