earticle

영어

Enterokinase is a heterodimeric serine protease consisting of 82~140kDa heavy chain and 35-62kDa light chain. The light chain is a catalytic subunit of enterokinase, which consists of a chymotrypsin-like serine protease. Because of high specificity for the amino-acid sequence
(Asp4)Lys, enterokinase has a great potential as a fusion protein cleavage reagent. Recombinant HEKL was produced in transgenic rice cell suspension culture with the rice amylase 3D promoter. RAmy3D promoter is highly expressed by sugar starvation. The expression of recombinant HEKL as mRNA and protein in transgenic rice cell was detected by Northern and Western blot analyses, recombinant HEKL was observed about 43 kDa in Western blot analysis. The activity of recombinant HEKL was determined by cleavage of the fusion protein EGFP-hTNFa as substrate, which contained a enterokinase cleavage site (DDDDK) between two moieties. Under optimal conditions the hTNFa product with expected size of 17kDa was well detected on Western blot analysis. These results demonstrate the use of HEKL derived from plant cell suspension culture as proteases (This Study was supported by a grant from the Ministry of Knowledge Economy).