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Proceedings of the Society For... Oct 1959
Topics: Amylases; Animals; Feces; Glycoside Hydrolases; Intestines; Rats; Trypsin; beta-Fructofuranosidase
PubMed: 13802818
DOI: 10.3181/00379727-102-25174 -
Protein Expression and Purification Feb 2007The recently classified group III trypsins include members like Atlantic cod (Gadus morhua) trypsin Y as well as seven analogues from other cold-adapted fish species....
The recently classified group III trypsins include members like Atlantic cod (Gadus morhua) trypsin Y as well as seven analogues from other cold-adapted fish species. The eight group III trypsins have been characterized from their cDNAs and deduced amino acid sequences but none of the enzymes have been isolated from their native sources. This study describes the successful expression and purification of a recombinant HP-thioredoxin-trypsin Y fusion protein in the His-Patch ThioFusion Escherichia coli expression system and its purification by chromatographic methods. The recombinant form of trypsin Y was previously expressed in Pichia pastoris making it the first biochemically characterized group III trypsin. It has dual substrate specificity towards trypsin and chymotrypsin substrates and demonstrates an increasing activity at temperatures between 2 and 21 degrees C with a complete inactivation at 30 degrees C. The aim of the study was to facilitate further studies of recombinant trypsin Y by finding an expression system yielding higher amounts of the enzyme than possible in our hands in the P. pastoris system. Also, commercial production of trypsin Y will require an efficient and inexpensive expression system like the His-Patch ThioFusion E. coli expression system described here as the enzyme is produced in very low amounts in the Atlantic cod.
Topics: Adaptation, Physiological; Animals; Blotting, Western; Cold Temperature; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Gadus morhua; Recombinant Fusion Proteins; Substrate Specificity; Trypsin
PubMed: 16879980
DOI: 10.1016/j.pep.2006.06.008 -
Fish Physiology and Biochemistry Sep 2010Trypsin from the pyloric ceca of masu salmon (Oncorhynchus masou) cultured in fresh water was purified by a series of chromatographies including Sephacryl S-200,...
Trypsin from the pyloric ceca of masu salmon (Oncorhynchus masou) cultured in fresh water was purified by a series of chromatographies including Sephacryl S-200, Sephadex G-50 and diethylaminoethyl cellulose to obtain a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. The molecular mass of the purified trypsin was estimated to be approximately 24,000 Da by SDS-PAGE. The enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, and N ( alpha )-p-tosyl-L: -lysine chloromethyl ketone. Masu salmon trypsin was stabilized by calcium ion. The optimum pH of the masu salmon trypsin was around pH 8.5, and the trypsin was unstable below pH 5.0. The optimum temperature of the masu salmon trypsin was around 60 degrees C, and the trypsin was stable below 50 degrees C, like temperate-zone and tropical-zone fish trypsins. The N-terminal 20 amino acid sequence of the masu salmon trypsin was IVGGYECKAYSQPHQVSLNS, and its charged amino acid content was lower than those of trypsins from frigid-zone fish and similar to those of trypsins from temperate-zone and tropical-zone fish. In the phylogenetic tree, the masu salmon trypsin was classified into the group of the temperate-zone fish trypsin.
Topics: Amino Acid Sequence; Animals; Chromatography; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Intestine, Small; Japan; Molecular Sequence Data; Oncorhynchus; Phenylmethylsulfonyl Fluoride; Phylogeny; Sequence Analysis, Protein; Temperature; Trypsin; Trypsin Inhibitors
PubMed: 19680768
DOI: 10.1007/s10695-009-9336-4 -
Biochemical and Biophysical Research... Jun 2008Autoproteolytic stability is a crucial factor for the application of proteases in biotechnology. In contrast to vertebrate enzymes, trypsins from shrimp and crayfish are...
Autoproteolytic stability is a crucial factor for the application of proteases in biotechnology. In contrast to vertebrate enzymes, trypsins from shrimp and crayfish are known to be resistant against autolysis. We show by characterisation of a novel trypsin from the gastric fluid of the marine crab Cancer pagurus that this property might be assigned to the entire class of crustaceans. The isolated and cloned crab trypsin (C.p.TryIII) exhibits all characteristic properties of crustacean trypsins. However, its overall sequence identity to other trypsins of this systematic class is comparatively low. The high resistance against autoproteolysis was determined by mass spectrometry, which revealed a low susceptibility of the N-terminal domain towards autolysis. By homology modelling of the tertiary structure, the elevated stability was attributed to the distinctly different pattern of autolytic cleavage sites, which is conserved in all known crustacean trypsin sequences.
Topics: Amino Acid Sequence; Animals; Brachyura; Calcium; Enzyme Stability; Mass Spectrometry; Molecular Biology; Molecular Sequence Data; Peptide Hydrolases; Protein Conformation; Trypsin
PubMed: 18395521
DOI: 10.1016/j.bbrc.2008.03.128 -
Biotechnology Letters Jan 2008Two trypsin-like enzymes (TLEs) were purified from North Pacific krill (Euphausia pacifica) by ammonium sulfate precipitation, ion-exchange and gel-filtration...
Two trypsin-like enzymes (TLEs) were purified from North Pacific krill (Euphausia pacifica) by ammonium sulfate precipitation, ion-exchange and gel-filtration chromatography. The purified enzymes were identified as trypsins by LC-ESI-MS/MS analysis. The relative molecular mass of TLE I and TLE II were 33 and 32.3 kDa, respectively, with isoelectric points of 4.5 and 4.3, respectively. The TLEs showed excellent thermal stable in the crude extract and the purified TLEs were active over a wide pH (6.0-11.0) and temperature (10-70 degrees C) range. Compared with trypsins from other organisms, the purified TLEs had physiological efficiencies of 1.6-6.7-fold. The difference in Arg, Ile and Asp content might explain why E. pacifica TLEs have good thermal stability and physiological efficiency.
Topics: Amino Acid Sequence; Animals; Enzyme Activation; Enzyme Stability; Euphausiacea; Molecular Sequence Data; Trypsin
PubMed: 17987272
DOI: 10.1007/s10529-007-9511-6 -
Nature Aug 1961
Topics: Chemistry, Organic; Dimethyl Sulfoxide; Lipids; Trypsin; Water
PubMed: 13764239
DOI: 10.1038/191565a0 -
FEMS Yeast Research Jun 2005Trypsin is a highly valuable protease that has many industrial and biomedical applications. The growing demand for non-animal sources of the enzyme and for trypsins with...
Trypsin is a highly valuable protease that has many industrial and biomedical applications. The growing demand for non-animal sources of the enzyme and for trypsins with special properties has driven the interest to clone and express this protease in microorganisms. Reports about expression of recombinant trypsins show wide differences in the degree of success and are contained mainly in patent applications, which disregard the difficulties associated with the developments. Although the yeast Pichia pastoris appears to be the microbial host with the greatest potential for the production of trypsin, it has shown problems when expressing cold-adapted fish trypsins (CAFTs). CAFTs are considered of immense value for their comparative advantage over other trypsins in a number of food-processing and biotechnological applications. Thus, to investigate potential obstacles related to the production of CAFTs in P. pastoris, the cunner fish trypsin (CFT) was cloned in different Pichia expression vectors. The vectors were constructed targeting both internal and secreted expression and keeping the CFT native signal peptide. Western-blotting analysis confirmed the expression with evident differences for each construct, observing a major effect of the leader peptide sequence on the expression patterns. Immobilized nickel affinity chromatography yielded a partially purified recombinant CFT, which exhibited trypsin-specific activity after activation with bovine enterokinase.
Topics: Animals; Blotting, Western; Chromatography, Affinity; Cloning, Molecular; DNA; Enteropeptidase; Genetic Vectors; Perciformes; Pichia; Polymerase Chain Reaction; Recombinant Proteins; Temperature; Transformation, Genetic; Trypsin; Viscosity
PubMed: 15925313
DOI: 10.1016/j.femsyr.2005.02.007 -
Biochemistry. Biokhimiia Feb 2016Trypsins are key proteins important in animal protein digestion by breaking down the peptide bonds on the carboxyl side of lysine and arginine residues, hence it has...
Express Sequence Tag Analysis - Identification of Anseriformes Trypsin Genes from Full-Length cDNA Library of the Duck (Anas platyrhynchos) and Characterization of Their Structure and Function.
Trypsins are key proteins important in animal protein digestion by breaking down the peptide bonds on the carboxyl side of lysine and arginine residues, hence it has been used widely in various biotechnological processes. In the current study, a full-length cDNA library with capacity of 5·10(5) CFU/ml from the duck (Anas platyrhynchos) was constructed. Using express sequence tag (EST) sequencing, genes coding two trypsins were identified and two full-length trypsin cDNAs were then obtained by rapid-amplification of cDNA end (RACE)-PCR. Using Blast, they were classified into the trypsin I and II subfamilies, but both encoded a signal peptide, an activation peptide, and a 223-a.a. mature protein located in the C-terminus. The two deduced mature proteins were designated as trypsin-IAP and trypsin-IIAP, and their theoretical isoelectric points (pI) and molecular weights (MW) were 7.99/23466.4 Da and 4.65/24066.0 Da, respectively. Molecular characterizations of genes were further performed by detailed bioinformatics analysis. Phylogenetic analysis revealed that trypsin-IIAP has an evolution pattern distinct from trypsin-IAP, suggesting its evolutionary advantage. Then the duck trypsin-IIAP was expressed in an Escherichia coli system, and its kinetic parameters were measured. The three dimensional structures of trypsin-IAP and trypsin-IIAP were predicted by homology modeling, and the conserved residues required for functionality were identified. Two loops controlling the specificity of the trypsin and the substrate-binding pocket represented in the model are almost identical in primary sequences and backbone tertiary structures of the trypsin families.
Topics: Animals; Avian Proteins; Ducks; Expressed Sequence Tags; Gene Library; Models, Molecular; Phylogeny; Polymerase Chain Reaction; Sequence Alignment; Sequence Analysis, Protein; Structural Homology, Protein; Trypsin
PubMed: 27260395
DOI: 10.1134/S0006297916020097 -
Mutation Research Feb 1990When cells are trypsinized before irradiation a potentiation of X-ray damage may occur. This is known as the 'trypsin effect'. Potentiation of X-ray damage on cell...
When cells are trypsinized before irradiation a potentiation of X-ray damage may occur. This is known as the 'trypsin effect'. Potentiation of X-ray damage on cell killing was seen in V79 Chinese hamster cells but was marginal in Chinese hamster ovary (CHO K1) cells and not evident in murine Ehrlich ascites tumour (EAT) cells. Trypsinization did however increase the number of X-ray-induced chromosomal abnormalities in all 3 lines. To investigate the possibility that trypsin acts by digestion of proteins in chromatin, further experiments were performed to monitor DNA damage and repair. Induction of DNA breaks by X-rays was unaffected by trypsin but trypsinized EAT (suspension) cells repaired single-strand breaks (ssb) less rapidly than controls indicating an inhibitory effect of trypsin on ssb repair. However double-strand break (dsb) repair was unaffected by trypsin. It was also found that the EDTA solution in which the trypsin was dissolved also contributes to the inhibition of dsb repair. The results show that trypsinization can enhance X-ray-induced cell killing, chromosomal damage and DNA repair, the effect varying between cell lines.
Topics: Animals; Cell Cycle; Cell Line; Cell Survival; Chromatin; Chromosomes; DNA; DNA Repair; Filtration; Kinetics; Mammals; Trypsin; X-Rays
PubMed: 2300069
DOI: 10.1016/0027-5107(90)90078-i -
The Journal of Biological Chemistry Dec 1991Fusion of endosomes appears to be required at early steps of receptor-mediated endocytosis. These fusion events have been reconstituted using a cell-free assay and have...
Fusion of endosomes appears to be required at early steps of receptor-mediated endocytosis. These fusion events have been reconstituted using a cell-free assay and have been shown to require both cytosolic and membrane-associated proteins. We report here that trypsinization of endosomes completely inhibited fusion. Addition of untreated cytosol cannot restore fusion of trypsinized endosomes. However, fusion activity is restored by the addition of either untreated vesicles or a high salt extract containing peripheral membrane proteins (KE). KE contains both the membrane-associated factor(s) required for the reconstitution of fusion using trypsinized endosomes and the factors that are normally provided by the cytosol. The restorative activity of KE was sensitive to trypsin treatment or incubation at 100 degrees C, but was largely N-ethylmaleimide (NEM)-resistant. This and other criteria demonstrated that the trypsin-sensitive factor is distinct from N-ethylmaleimide-sensitive factor (NSF), an NEM-sensitive protein involved in vesicular fusion, and from other known factors that may participate in membrane fusion events. Preliminary fractionation studies indicate that the restorative activity of KE is associated with one or more high molecular weight proteins. The present study indicates that a novel trypsin-sensitive protein(s) is involved in endosome-endosome fusion. This factor is membrane-associated and is not found in an active form in cytosol as prepared.
Topics: Biological Transport; Cell Line; Chromatography; Cytosol; Endocytosis; Ethylmaleimide; Golgi Apparatus; Membrane Proteins; Microscopy, Electron; Organelles; Trypsin
PubMed: 1744137
DOI: No ID Found