Role of polyethyleneimine in the purification of recombinant human tumour necrosis factor beta.
The chromatographic behaviour of recombinant human tumour necrosis factor beta (rhTNF-beta) (pI approximately 9.0) during cation-exchange chromatography at pH 7.5 is investigated. Without prior treatment of the Escherichia coli cell extract with polyethyleneimine (PEI), very little rhTNF-beta was bound to the column. However, upon addition of 5% PEI (100 microliters ml-1) to the cell lysate, rhTNF-beta was shown to bind to cation-exchange columns normally. TNF-beta was readily precipitated from the clarified cell extract by 20% ammonium sulphate, but ony ca. 25% of this precipitate could be re-solubilized for further purification. However, when 5% PEI was included in the solubilization buffer, the balance of the rhTNF-beta could be recovered. It is proposed that charge interaction between rhTNF-beta and nucleic acids in the cell extract is responsible for both of these anomalous phenomena, and that PEI (a cationic polyelectrolyte) was able to disrupt this interaction by displacing rhTNF-beta from the charge complex.
Loh
K C
KC
Bioprocessing Technology Centre, National University of Singapore, Singapore.
Yao
Z J
ZJ
Yap
M G
MG
Chung
M C
MC
D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D002417:Cattle; D011232:Chemical Precipitation; D002847:Chromatography, Agarose / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008233:Lymphotoxin-alpha / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011094:Polyethyleneimine / Q000737:chemistry*; D012313:RNA / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013950:Thymus Gland / Q000737:chemistry
D008233:Lymphotoxin-alpha; D011994:Recombinant Proteins; D012313:RNA; D011094:Polyethyleneimine; D004247:DNA; D000645:Ammonium Sulfate