Identification of the heparin-binding domain of TNF-alpha and its use for efficient TNF-alpha purification by heparin-Sepharose affinity chromatography.
The N-terminus of the trimeric TNF-alpha molecule comprises two basic arginines within the short amino-acid sequence VRSSSR, which is here shown to be essential for binding of TNF-alpha to heparin-Sepharose. Mixed trimers containing full-length and DeltaN6-truncated subunits revealed a single VRSSSR sequence to be sufficient to achieve binding. On the basis of this newly identified heparin-binding domain, a new method for efficient purification of TNF-alpha is described. Affinity chromatography on heparin-Sepharose was introduced as a key step for highly purified TNF-alpha at a high yield. With minor modifications, this procedure can be used for TNF-alpha analogues that have full-length N-termini, as shown for the less toxic analogue LK-805.
Kenig
Maja
M
Lek Pharmaceuticals d.d., Verovskova 57, SI-1526 Ljubljana, Slovenia. maja.kenig@sandoz.com
Gaberc-Porekar
Vladka
V
Fonda
Irena
I
Menart
Viktor
V
D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D014409:Tumor Necrosis Factor-alpha / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D014409:Tumor Necrosis Factor-alpha; D006493:Heparin