What is trypsin
Trypsin (C6H15O12P3) is a kind of protease. In vertebrates, it functions as a digestive enzyme. In the pancreas, it is synthesized as a precursor of the enzyme trypsinogen. It is secreted as a component of pancreatic juice and decomposed into activated trypsin under the restriction of enterokinase or trypsin. It is an endopeptidase that can cut the carboxyl side of lysine and arginine residues in the polypeptide chain. It not only functions as a digestive enzyme, but also limits the decomposition of precursors of other enzymes such as chymotrypsinogen, carboxypeptidase, and phospholipase, and has an activating effect. It is the most specific protease, and it has become an indispensable tool in determining the amino acid sequence of a protein.
Introduction of porcine trypsin
The relative molecular mass of porcine trypsinogen is about 24 000. According to the difference in isoelectric point, porcine trypsinogen can be divided into anionic (pl<6.8) and cationic (pl>6.8). Because the cationic type not only has a larger specific gravity, but also has better stability than the anionic type, the cationic porcine trypsinogen was selected for construction and expression. Porcine trypsinogen contains 12 cysteines, which can form 6 pairs of disulfide bonds (30-160, 48-64, 132-233, 139-206, 171-185, 196-220), which greatly Increased the difficulty of denaturation and renaturation of inclusion bodies in subsequent experiments.
Application of porcine trypsin
Porcine trypsin is a kind of trypsin, which can be used as an additive for the removal of surface adherent cells, the production of influenza virus vaccines, insulin and other proteins, the rapid hydrolysis of proteins, and the pretreatment of animal cells and tissues. There is a large demand for trypsin with high purity and strong activity. At present, a preparation process of recombinant porcine trypsinogen has been established, and the obtained recombinant porcine trypsin has good enzyme activity and does not contain other animal source pollution, which provides a certain experimental basis for industrialized research and production.
Characteristics of porcine trypsin
Porcine trypsin is unstable in nature and prone to autolysis. When constructing a recombinant porcine trypsinogen expression system, this autolysis feature makes it difficult for the eukaryotic expression system to obtain complete trypsinogen, and the activated product will affect the host. Cells can also be toxic, so consider choosing a prokaryotic expression system. In addition, the autolysis characteristics require that the activation conditions of porcine trypsinogen also need to be strictly controlled.
Preparation method of porcine trypsin
In the traditional preparation method, there are many separation and purification steps and a long time, which is easy to cause damage to the protein and cause inactivation. Resulting in low yield. Therefore, the preparation and production of porcine trypsin has gradually shifted from extraction from animal pancreas to recombinant expression production. The production of trypsin by constructing a recombinant porcine trypsinogen-derived Escherichia coli expression system can also avoid the risk of related pathogen contamination and the risk of carrying unknown viruses due to the animal origin of the donor.
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