[Relationship between the structure--modified N- and C- termini and the biological activity of TNF alpha molecule]

In order to elucidate the relationship between N-, C-termini of TNF alpha and it's biological activity, a TNF alpha derivative 10 (TNF alpha D10) was prepared by changing amino acid at the N-terminus positions Ser(4), Ser(5), Asp(10) and C-terminus position Leu(157) to N-terminus Cys(4), Thr(5), Arg(10) and C-terminus Phe(157) with PCR site-directed mutagenesis. The results showed that the expression level of this mutein has not altered but its cytotoxic activity increased. This might result from trimer formation of TNF alpha D10 by changing N-terminal Ser(4) residue to Cys. HPLC showed that the molecular weight of TNF alpha D10 was 17kD, 35kD, 55kD respectively. In addition, we also found that the stability of TNF alpha D10 was less than that of TNF alpha when to be stored at -20 degrees C for two months. It might be caused by changing Leu(157) to Phe(157).