title: Purification, characterization and cDNA cloning of a trypsin from the hepatopancreas of snakehead (Channa argus).
abstract: A trypsin was purified from the hepatopancreas of snakehead (Channa argus) by ammonium sulfate fractionation and a series of column chromatographies including DEAE-Sepharose, Sephacryl S-200 HR and Hi-Trap Capto-Q. The molecular mass of the purified trypsin was about 22 kDa, as estimated by SDS-PAGE. The optimum pH and temperature of the purified trypsin were 9.0 and 40°C, respectively. The trypsin was stable in the pH range of 7.5-9.5 and below 45°C. The enzymatic activity was strongly inhibited by serine proteinase inhibitors, such as MBTI, Pefabloc SC, PMSF, LBTI and benzamidine. Peptide mass fingerprinting (PMF) of the purified protein obtained 2 peptide fragments with 25 amino acid residues and were 100% identical to the trypsinogen from pufferfish (Takifugu rubripes). The activation energy (Ea) of this enzyme was 24.65 kJ·M(-1). Apparent K(m) was 1.02 μM and k(cat) was 148 S(-1) for fluorogenic substrate Boc-Phe-Ser-Arg-MCA. A trypsinogen gene encoding 247 amino acid residues was further cloned on the basis of the sequence obtained from PMF and the conserved site peptide of trypsinogen together with 5'-RACE and 3'-RACE. The deduced amino acid sequence contains a signal peptide of 15 residues and an activation peptide of 9 amino acid residues with a mature protein of 223 residues. The catalytic triad His-64, Asp-107, Ser-201 and 12 Cys residues which may form 6 disulfide bonds were conserved. Compared with the PMF data, only 2 amino acid residues difference were identified, suggesting the cloned trypsinogen is quite possibly the precursor of the purified trypsin.
authors.affiliation: College of Biological Engineering, The Key Laboratory of Science and Technology for Aquaculture and Food Safety, Jimei University, Xiamen, 361021, China.
authors.forename: Long-Zhen
authors.initials: LZ
authors.lastname: Zhou
authors.forename: Mi-Mi
authors.initials: MM
authors.lastname: Ruan
authors.forename: Qiu-Feng
authors.initials: QF
authors.lastname: Cai
authors.forename: Guang-Ming
authors.initials: GM
authors.lastname: Liu
authors.forename: Le-Chang
authors.initials: LC
authors.lastname: Sun
authors.forename: Wen-Jin
authors.initials: WJ
authors.lastname: Su
authors.forename: Min-Jie
authors.initials: MJ
authors.lastname: Cao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002845:Chromatography; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D005399:Fishes / Q000235:genetics*; D043143:Hepatopancreas / Q000187:drug effects / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007700:Kinetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D016415:Sequence Alignment; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D014357:Trypsin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D018076:DNA, Complementary; D015842:Serine Proteinase Inhibitors; D014357:Trypsin