DESIGN AND USE OF KAZAL-TYPE

摘要

Proteinase inhibitors KPI with the amino acid sequence (beginning with the N-terminus, written in single-letter amino acid code) X0-6 C (T) X2 (Y) X (P) (V) C G (S) (D) X2 T Y X-2-3C X (L) X5-12 G X C (KPI) where parentheses indicate that replacement with an amino acid of similar physical properties is possible and X marks positions in which any amino acid residue may occur. Preparation of recombinant proteinase inhibitors corresponding to the sequence shown above and of their derivatives containing C-terminal tail proteins or other moieties facilitating purification, and of glycosylated forms thereof. The examples include expression of KPII, KP12, KP13 and similar compounds that may be glycosylated: KPI1: VCTTEWDPVCGKDGKTYSNLCWLAEAGVGLDHEGEC KP12: EAVCTTEWDPVCGKDGKTYSNLCWLAEAGVGLDHEGEC KP13: EAVCTTEWDPVCGKDGKTYSNLCWLAEAGVGLDHEGECLEQKLISEEDLNSAVDHHHHHH The recombinant proteinase inhibitors are most effective on the bacterial and fungal proteinases. The presence of C-terminal tail and the glycosylation modify the activities in substrate-specific manner. Enhancement of the stability of biologically active peptides by their fusion to a KPI-type proteinase inhibitor. For example, fusion protein KPI1::CP, which consists of KPIl, coat protein of the potato Y virus, and a poly(His) tag, proved resistant to proteinase attack, could be used to generate antiserum to the PoI site of the viral coat protein, and assembled into rodshaped virus-like particles. The fusion of KPIl to beta-glucuronidase (GUS) rendered GUS resistant to proteinases without hampering its enzymatic activity. DNA templates encoding KPI-type proteinase inhibitors can be introduced into plants and other organisms to enhance their resistance to pathogens, herbivores, and other enemies.