Isolation Of Trypsin, CDNA Cloning And Sequence Analysis Of Pretrypsinogen From Pingxiang Red-transparent Crucian Carp (Carassius Auratus)

In this research, the trypsin of Carassius auratus var. Pingxiangnensis was purified by gel filtration and characterized; Pretrypsinogen gene was cloned, the deduced amino acids sequence and predicted protein structure were analysed. Main results are as follows:1,The isolation of trypsinWe got one kind of trypsin from Carassius auratus var. Pingxiangnensis by ammonium sulfate precipitation, dialysis and gel filtration through CHOM Sepharose4B-CL. the molecular weight was about 24 kDa by SDS-PAGE.2,The effect of activity of trypsin by pH and temperatureThe optimum temperature and pH of the trypsin for hydrolysis of BAEE (Nα-benzoyl-L-arginine ethyl ester) were 50℃and 7.53,The full length cDNA sequence of pretrypsinogen geneThe full length cDNA sequence was 866bp which cloned from Pingxiang red-transparent crucian carp, including a 5'untranslated region of 21bp and a 3' untranslated region of 107bp.4,Deduced amino acid sequence analysis from pretrypsinogenThe open reading frame of Carassius auratus var. Pingxiangnensis pretrypsinogen gene encoded 246 amino acid residues which composed by one signal peptide with 15 amino acid residues and one activation peptide with 8 amino acid residues. It contains the typical character structure of catalytic triad (His63, Asp107 and Ser200) and obligatory amino acid of Asp194; 6 conserved cystine bridges were founded by 12 cysteine residues.5,The similarity analysis of pretrypinogenCarassius auratus var. Pingxiangnensis and Spinibarbus sinensis were on the same embranchment deduced by phylogenetic tree, they have the highest similarity.6,The analysis of deduced amino acids sequence of pretrypsinogen and the protein structure predictionWith analyzing of deduced amino acids sequence of pretrypsinogen, the molecular weight was 26341.8Da, and pI was 6.48, In composition of amino acids, proportions of serine is relativity large, accounting for 13.82%, secondly, asparagine was 9.76%; It was predicted that the secondary structure of the protein contained four forms:α-helix, extended strand,β-turn and random coil.