Cloning And Functional Study Of Trypsin Gene From Euphausia Superba

Trypsin is one of the major proteolytic enzymes in the animal digestive tract,which belongs to the family of serine protease.At present,trypsin genes were cloned and identificated in invertebrate animal,such as crustaceans and insects.Antarctic krill is one of the single species of Southern Ocean and its with great biomass,belongs to Euphausiacea planktonic crustacean animal.In the Antarctic ecosystem,Antarctic krill is rich in biological resources,and have the special status of evolution.In this study,full-length cDNA of Antarctic krill trypsin gene was cloned using the technology of RACE,named as EsTrypsin-I.The full length of EsTrypsin-1 was of 864 bp with a 5 'untranslated region(UTR)of 14 bp,a 3' UTR of 49 bp,and an open reading frame of 801 bp encoding 266 amino acids residues.The predicted molecular weight is 28.23 kDa,isoelectric point is 4.20.The typical catalytic three conjoined structure His74-Asp125-Ser218,specific substrate binding site of Asp212,and 6 conserved cysteines were found in amino acid sequence of EsTrypsin-I.Homology analysis showed that,EsTrypsin-1 and crustacean animal(Penaeus vannamei and Penaeus japonicas)shared a high similarity.In the phylogenetic tree,EsTrypsin-1 and Penaeus vannamei and Penaeus japonicas cluster together.EsTrypsin-1 was Constructed recombinant expression vector and transformed into E.coli BL21(DE3)pLysS,and expressed by IPTG induction,the recombinant protein was isolated and purified,and its concentration was measured.The activity of recombinant protein was detected.The optimum catalyst temperature was about 40 ?,the optimum catalyst pH was about 8.The results show that EsTrypsin-1 belongs to the family members of trypsin,the recombinant EsTrypsin-1 protein with enzymatic activity.