[Fed-batch fermentation of Escherichia coli that express fab fragment of anti-HBsAg].
To develop a fed-batch fementation process of E. coli TOP10 containing a recombinant plasmid pBAD/HBs Fab. Cells were grown in semi-defined medium at 37 degrees C, and the feed operation using glycerol as carbon source was performed when dissolved oxygen increased. When the target cell concentration reached to 64g/L, arabinose was added to a final concentration of 0.02%. Cells were grown for another 5h with the culture temperature decreased from 37 degrees C to 30 degrees C. In the whole process, cell growth was monitored by measuring OD600 of samples taken at 1/2h intervals and the dissolved oxygen was kept above 30%. After the fementation, E. coli pellets were collected for purification of Fab protein. The specificity of Fab protein was confirmed by Western blot, and binding activity to HBsAg was verified by Dot blot. Cell concentration we got is 96g wet bacteria per liter, the Fab protein is about 6% of total protein of the host, that is 80mg per liter. Stable fermentation parameters were obtained for fermentation to improve productivity of the Fab protein. The Fab protein was produced in the form of soluble biologically active protein, it's better than inclusion bodies from which biologically active protein can only be recovered by complicated and costly denaturation and refolding process.
An
Feng
F
Department of Forensic Pathology, Zhongshan Medical College, Sun Yat-sen University, Guangzhou 510080, China. anfenggz@hotmail.com
Chen
Yu-Chuan
YC
Fan
Lie-Ying
LY
Han
Huan-Xing
HX
D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation / Q000502:physiology*; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D013696:Temperature
D006514:Hepatitis B Surface Antigens; D007140:Immunoglobulin Fab Fragments