Cloning And Heterologous Expression Of Thioredoxin And Cathepsin Genes From Euphausia Superba

The Euphausia superba has a circumpolar distribution with the highest concentrations in the Atlantic sector of the Southern Ocean. It is a key species of the Antarctic ecosystem and plays an important role both as feeder of algae, bacteria and micro-zooplancton and as a prey of vertebrates. Thioredoxin plays an important role in the maintenance of intracellular redox proteins, cathepsin is a kind of proteolytic enzymes mainly existed in the lysosomes, participate in many important physiological and pathological process which located in intracellular and extracellular.In this study, the full-length cDNA of three thioredoxin (EsTrx1, EsTrx2, EsTrx3) genes and part sequences of cathepsin (EsCat) genes from E. superba were cloned with the rapid amplification of cDNA end (RACE) technology. The coding regions of the EsTrx1, EsTrx2, EsTrx3 were inserted into pET-30a vector, and transformed into Escherichia coli BL21 (DE3) pLysS to achieve the recombinant expression, and detect the activity in vitro.Thioredoxins are small molecular protein evolutionary conserved in prokaryotic and eukaryotic, with the same "-Cys-Gly-Pro-Cys-" oxidation reduction activity center. Thioredoxins form protein two disulfide bond depended on the active site cysteine residues. The full-length cDNA of EsTrxl was of 621 bp, containing a 5’untranslated region (UTR) of 45 bp, a 3’UTR of 276 bp, and an open reading frame (ORF) of 300 bp encoding a polypeptide of 100 amino acids. The predicted molecular weight of EsTrxl was 11.08 kDa and the theoretical isoelectric point was 4.51. The similarity between EsTrxl and Trxl from Chinese Mitten Crab (Eriocheir sinensis) reached 68.6%. The three-dimensional structure of the EsTrx1 protein and Trx crystal structure (3ZZX(1.88 A)) from shrimp(Litopenaeus vannamei) had highly similarity (59.79%). The full-length cDNA of EsTrx2 was of 1276 bp, containing a 5’untranslated region (UTR) of 94 bp, a 3’UTR of 744 bp,and an open reading frame (ORF) of 438 bp encoding a polypeptide of 146 amino acids. The predicted molecular weight of the EsTrx2 was 16.05 kDa and the theoretical isoelectric point was 6.59. The similarity between EsTrx2 and Trx2 from Swimming crab (Portunus trituberculatus) reached 74%. The three-dimensional structure of the EsTrx2 protein and Trx crystal structure (1w4v(1.80 A))from human mitochondrial Trx had highly similarity (62.26%). The full-length cDNA of EsTrx3 was of 809 bp, containing a 5’untranslated region (UTR) of 123 bp, a 3’UTR of 311 bp, and an open reading frame (ORF) of 375 bp encoding a polypeptide of 125 amino acids. The predicted molecular weight of the EsTrx3 was 13.82 kDa and the theoretical isoelectric point was 4.88. The similarity between EsTrx3 and the Norway rat(Rattus norvegicus) reached 30.2%. The three-dimensional structure of the EsTrx3 protein and human Trx relative protein crystal structure (lwou(1.80 A)) had highly similarity (44.07%). The antioxidant activity of EsTrx2 showed raise with the increase of temperature from 4℃-37℃. The antioxidant activity of EsTrx3 also showed raise with the increase of temperature from 4℃-8℃, but it dropped with the increase at 25℃ and 37℃. These results together indicated that EsTrx2 and EsTrx3 could function as an important antioxidant. They have an important effect on maintaining redox state of the E. superba.The 3’UTR of 132 bp from EsCatC, a 3’UTR of 338 bp from EsCatC2,a 3’UTR of 481 bp and a part 5’UTR of 193 bp from EsCatD, a 3’UTR of 404 bp and a part 5’ UTR of 459 bp from EsCatL were cloned with RACE technology.