Production & purification of recombinant tumour necrosis factor-beta.
The effects of medium composition, temperature and tryptophan concentration on the growth and expression of a recombinant E. coli producing human tumour necrosis factor-beta (TNF-beta) were examined in shake flask cultures. We found that lower cultivation temperatures of 25 degrees C and 30 degrees C gave the best yield of soluble TNF-beta. A higher expression of total TNF-beta was obtained in defined medium. Fed-batch fermentations further confirmed that a lower mu was critical to obtaining high TNF-beta expression. This was shown to be due to the dilution effect at high mu, which affected the cell plasmid content. We found that we were unable to repress TNF-beta expression with tryptophan and TNF-beta was expressed in non-induced cultures. This has been attributed to the nature of the constructed clone, which is a low aporepressor producer, but carried a high copy number plasmid with a mutated rom gene. A rapid and improved method for the purification of TNF-beta has also been developed. The method utilised sequential steps of polyethyleneimine (PEI) and ammonium sulphate precipitation to remove most of the extraneous proteins and nucleic acids from the cell extracts. This was followed by DEAE chromatography. This procedure was found to be highly efficient and was used to purify large quantities of TNF-beta. Compared to an earlier protocol which did not include the PEI step, yields were higher and processing time was much shorter.
Mak
K W
KW
Department of Chemical Engineering, National University of Singapore.
Loh
K C
KC
Yap
M G
MG
D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics; D005285:Fermentation; D006801:Humans; D008233:Lymphotoxin-alpha / Q000096:biosynthesis* / Q000302:isolation & purification; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013696:Temperature; D014364:Tryptophan / Q000378:metabolism / Q000494:pharmacology
D003470:Culture Media; D008233:Lymphotoxin-alpha; D011994:Recombinant Proteins; D014364:Tryptophan