Expression of recombinant humanized anti-HBsAg Fab fragment in genetically engineered Pichia pastoris in high-density fermentation
- VernacularTitle:重组毕赤酵母菌高密度表达人源抗-HBs Fab抗体的研究
- Author:
Wenyin CHEN
;
Guirong RAO
;
Kuanyua SU
- Publication Type:Journal Article
- Keywords:
anti HBsAg Fab fragment;
high density fermentation;
Pichia pastoris;
affinity chromatography
- From:
Medical Journal of Chinese People's Liberation Army
2001;0(09):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To study the expression in high density fermentation of anti HBsAg Fab fragment in Pichia pastoris , and the purification and activity detection of expressed target protein. Method The high density fermentation of genetically engineered Pichia pastoris was proceeded in a 5L bioreactor using fed batch fermentation. The fermentation temperature was set at 28-30℃,the pH was 5 0-5 5, and the DO was kept over 20%. When the absorbance (OD 600 ) of the broth reached 400-450 (the first time of fermentation), 200-250 (the second time), and 300-350 (the third time), the induced phase was initiated, and the methanol concentration was 0 5%-1%. The fermentation ended after 96h's induction, the target protein was purified by affinity chromatography, and its activity was assessed by ELISA. Results It showed that the optimum initial cell density during methanol induced phase should be 300-350, which was good for control of the fermentation process and the expression of recombinant Fab. At the end of the fed batch phase, a yield of about 245mg/l of Fab was reached, and 98% purity could be reached as demonstrated by affinity chromatography. The results of ELISA showed that the supernatant of fermentation and the purified recombinant Fab could bind to HBsAg specifically. Conclusion The success of high density fermentation lays a sound foundation of mass production and clinical applications of recombinant humanized anti HBsAg Fab fragment
