Expression of biologically active anti-thrombosis protein lumbrokinase in edible sunflower seed kernel

Abstract

Lumbrokinase (LK) is an important fibrinolytic enzyme derived from earthworm. The capsules of the extracts of LK have been widely used. Unfortunately, the life cycle of earthworm is long, and extraction of lumbrokinases is generally a labor intensive and time-consuming activity. Also the extract is easily contaminated by multiple components. The successful expression of the recombinant LK provides a way to obtain single component with fibrinolytic activity. Meanwhile, it was reported that LK could be orally administered. Therefore, we have been attempting to produce recombinant LK using a safe and cost-effective production system. In this stduy, LK gene placed under the seed-specific promoter, napA, was expressed in the Helianthus annuus L. The SDS-PAGE and Western blot analysis confirmed the expression of recombinant LK (rLK). The yield of rLK was about 5.1 g/kg sunflower seeds as determined by ELISA. Fibrin plate assays revealed that the crude extraction of rLK from sunflower seed kernel contained a high level of fibrinolytic activity. Following oral administration of T1 generation of transgenic sunflower seed kernel, the prothrombin time (PT), the activated partial thromboplastin time (APTT), the thrombin time (TT) and the carrageenin-induced thrombosis were evaluated, using Balb/c mice as the thrombosis model. It was demonstrated that the oral treatment of mice with transgenic sunflower seed kernel had a significant anti-thrombotic effect. The finding provides a way for low-cost and seed kernel edible delivery of human therapeutic proteins.