Expression of cholera toxin B-lumbrokinase fusion protein in Pichia pastoris--the use of transmucosal carriers in the delivery of therapeutic proteins to protect rats against thrombosis.

Cholera toxin B-subunit (CTB) has been widely used to facilitate antigen delivery by serving as an effective mucosal carrier molecule for the induction of oral tolerance. However, whether CTB can be used as a transmucosal carrier in the delivery of not only vaccines but also therapeutic proteins has not been widely studied. Thus, we investigate here the concept of receptor-mediated oral delivery of lumbrokinase (LK) proteins which is an important fibrinolytic enzyme derived from earthworm. CTB and LK, separated by a furin cleavage site, was expressed via Pichia pastoris. The activity and proper folding of recombinant protein in yeast were confirmed by Western blot analysis, fibrin plate assays, and G(M1)-ganglioside ELISA. Following oral administration of recombinant protein, the thrombosis model of rats and mice revealed that the oral treatment of rCTB-LK has a more significant anti-thrombotic effect on animals compared with rLK. It is possible to conclude that CTB can successfully enhance its fusion protein LK to be absorbed. The use of CTB as a transmucosal carrier in the delivery of not only vaccines but also therapeutic proteins was supported.