Fermentation And Expression Of Recombinant Trypsin In Escherichia Coli

Trypsin is an important member of the serine protease family. It is widely used in food production, pharmaceutical, clinical diagnosis, biochemical testing and so on. In the production of genetically engineered insulin, trypsin can be used to cut the pro insulin into active insulin. The trypsin mainly extracted from the pancreas of pigs, cattle. because of its animal source, There is a risk of unknown viruses and exogenous factors of pollution. And due to incomplete separation and purification, There are other enzymes（such as chymotrypsin） risk of substrate non-specific enzyme. The application of trypsin produced by this method in the production and analysis of insulin and other protein drugs is limited. It can produce high purity and high activity of trypsin by construct a strain of engineering bacteria to produce trypsinogen,which can meet the needs of research on protein and the production of pharmaceutical protein. E.coli expression system is the earliest and most widely used expression system in gene expression technology. The aim of this reserch is to study the expression of trypsin in Escherichia coli, and to provide a basis for its industrial production and application.In this experiment, we first optimize the codon preference of the trypsin gene sequence published in NCBI,then synthesis the gene fragment and Connected to p ET-22b（+）,Construction the vector named p ET22b-Trypsinogen. After enzyme digestion and sequencing, the vector was transferred into the host strain BL21 E.coli（DE3）, and the engineering bacteria expressing trypsin were obtained. The strain was named as p ET-pro TYP.Then to make sure the engineering bacteria can be stably passaged and expressed protein in the future, a research on the morphology and biochemical characteristics of engineering bacteria were made, and the engineering strain passage stability were investigated. The results showed that the recombinant trypsin engineering bacteria E. coli has a typical morphological and biochemical characteristics; after 50 generations later, plasmid stability exists in bacteria, and can express the target protein stably. the engineering bacteria can passaged stably.Subsequently, the experiments were conducted to induce the expression of engineering bacteria. The study found that the expression of trypsinogen was mainly in the form of inclusion bodies. In order to obtain a large number of inclusion bodies containing trypsinogen, the experiment was conducted to study the high density fermentation of 30 L, and the process and control of high density fermentation were simply explored. After the fermentation, the biomass of wet weight 102g/L was obtained by centrifugation. The results of SDS-PAGE electrophoresis analysis showed that the protein expression was high, and the expression level was 23.2%. The purpose of high density and high expression of cell fermentation was achieved.Finally, the trypsinogen were deformed and refolded, the activity of crude trypsin solution was obtained after digestion of the intestinal kinase. The crude enzyme was further purified and the purified trypsin enzyme activity was measured. Finally, the high activity and high purity of trypsin were processed by freeze drying. Finally, the complex condition of the inclusion body was determined: Protein concentration of 0.3 mg/m L, 100 m M glycine sodium hydroxide system, GSH/GSSG ratio of 1 mmol/L:0.3 mmol/L, complex buffer with p H 10. After this condition, the enzyme activity could be as high as 6200 units /m L. Refolded trypsinogen overnight after enterokinase digestion can be fully activated to get a crude enzyme solution. Crude enzyme solution was purified by ion exchange chromatography and hydrophobic interaction chromatography. finally got the purity of 95%, specific activity reached 10526 BAEE per mg of trypsin. The purification conditions after optimization:Hydrophobic chromatography conditions: buffer A-20 m M Tris-HCl+1.5 M（NH₄）₂SO₄,p H 8.0;buffer B-20 m M Tris-HCl,p H 8.0;0-100%B linear gradient 50CVIon exchange conditions: buffer A-20 m M Tris-HCl,p H8.0;buffer B-20 m M Tris-HCl+1M Na Cl,p H8.0;0-100%B linear gradient 30 CVThis experiment successfully constructed a strain of engineering bacteria for production of trypsin, the fermentation and purification of the target protein were studied, which provided a theoretical basis for industrial production of trypsin.