[结构修饰的肿瘤坏死因子α分子N端和C端与生物活性之间的关系]

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

作者信息

Chang J, Li X, He X, Lu Q, Yu H, Cai W, Li C, Zhao S

机构信息

State Key Laboratory of Genetic Engineering, Fudan University, Shanghai.

出版信息

Yi Chuan Xue Bao. 1995;22(5):329-35.

PMID:8679272

Abstract

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).

摘要

为了阐明肿瘤坏死因子α（TNFα）的N端、C端与其生物学活性之间的关系，通过PCR定点诱变将N端位置的丝氨酸（Ser）4、丝氨酸5、天冬氨酸（Asp）10以及C端位置的亮氨酸（Leu）157替换为N端的半胱氨酸（Cys）4、苏氨酸（Thr）5、精氨酸（Arg）10以及C端的苯丙氨酸（Phe）157，制备了一种TNFα衍生物10（TNFα D10）。结果表明，该突变体的表达水平未改变，但其细胞毒性活性增强。这可能是由于将TNFα D10的N端丝氨酸4残基替换为半胱氨酸后形成了三聚体。高效液相色谱（HPLC）显示，TNFα D10的分子量分别为17kD、35kD、55kD。此外，我们还发现，当在-20℃储存两个月时，TNFα D10的稳定性低于TNFα。这可能是由于将亮氨酸157替换为苯丙氨酸157所致。