Production of Human Breast Cancer-associated Peptide and Pancreatic Spasmolytic Polypeptide in Escherichia Coli and Their Biological Activity

Background: The 1989 Thim first discovered and named a new family of proteins, in view of its amino acid sequence for a special core sequence, during the six cysteine ??1-5,2-4 and 3-6 in the order , followed by a disulfide bond or two-phase, the formation of three cyclic structure, shaped like three leaves, it will be called the \peptide). The family includes: breast cancer-related peptide (breast cancer-associated peptide, pS2 or TFF1), pancreatic spasmolytic peptides (pancreatic spasmolyticpolypeptide, SP, or TFF2), and intestinal trefoil factor (intestinal trefoil factor, ITF or TFF3). The trefoil peptide is expressed primarily in the gastrointestinal tract, there is no or only a small amount of expression in other tissues. Physiological state TFF1 expression in the gastric mucosa; TFF2 expression in the stomach and pancreas; TFF3 expression in the intestine. Numerous studies show that the trefoil peptide has an important role in the protection of the gastrointestinal mucosa, digestive tract known as the \The 1982 Masiakowski first discovered and named the first member of the family of the trefoil peptide pS2 has more 25 years. The trefoil peptide a wide range of in-depth study included in the distribution in the body, gene mapping, recombinant expression, gene knockout, physical and chemical properties, antibody preparation, amino acid sequence analysis, spatial structure established as well as a large number of functional studies. The trefoil peptide clinical application of slow progress so far has not been at home and abroad trefoil peptide used in the clinical treatment reports, the key reason is that the trefoil peptide is very difficult to obtain. Trefoil peptide levels in the organization limited high tissue extraction cost, low-yield; peptide synthesis can not guarantee the correct spatial structure, synthetic product no biological activity. The use of genetic engineering methods is the best choice, either prokaryotic or eukaryotic expression system, each has some shortcomings and deficiencies, the recombinant expression and purification process is cumbersome, the production is not high, resulting in high production costs, limiting its industrialization production and clinical applications. In view of this, it is necessary to explore an efficient, fast, low-cost recombinant expression, the theoretical basis for the large-scale industrial production and clinical application of the trefoil peptide. Purpose: to build rhTFF1 and rhTFF2 fusion protein E. coli expression vector, recombinant expression in E. coli, to obtain high-purity recombinant fusion protein; fusion protein on the recombinant expression hTFF1 hTFF2 physical and chemical properties and biological activity. Method: 1.rhTFF1 and rhTFF2 Expression and purification of the fusion protein in Escherichia coli: hTFF1 hTFF2 of mature peptide cDNA sequence obtained by RT-PCR, respectively, cloned into plasmid pET32a to obtain a recombinant vector; transforming to a double digestion Origami B (DE3 ) induced expression, optimization of expression conditions for maximum expression yield; NI-NTA affinity chromatography, ultrafiltration purification of recombinant proteins. The identification of 2.rhTFF1 and rhTFF2: recombinant protein recombinant plasmid sequencing, SDS-PAGE and Western blot. Fusion proteins 3.rhTFF1 and rhTFF2 physical and chemical properties: the purified rhTFF1 rhTFF2 fusion protein were placed in simulated gastrointestinal liquid environment: 1 different concentrations of trypsin at 37 ° C for digestion 4h; (2) different concentrations of stomach 37 ° C digestion of the protease, 4h; ③ pH value of 2.0-12.0 buffer at 37 ° C were incubated for 5 h. Using non-reducing tricine SDS-PAGE analysis of the percentage residue rhTFF1 and rhTFF2 of the fusion protein to observe the stability of the fusion protein in simulated gastrointestinal environment rhTFF1 the rhTFF2. 4.rhTFF1 and rhTFF2 fusion protein in vitro biological activity of: (1) using the MTT assay and CCK-8 was observed rhTFF1 and rhTFF2 fusion protein on MKN-28 cells proliferation activity; (2) mechanical damage model MKN-28 cells was observed rhTFF1 cell migration rate rhTFF2. 5.rhTFF1 and rhTFF2 fusion protein in vivo biological activity: intragastric ethanol and 30% body surface area (Total BODY Surface Area TBSA) Ⅲ ° burn mouse model, to observe rhTFF1 and rhTFF2 fusion protein of the two injured mouse model of gastric mucosal injury and repair. Results: 1 to get hTFF1 and hTFF2 mature peptide cDNA sequence, the the Genbank database contains gene sequence is exactly the same, successfully constructed the reorganization of the target gene vector pET32a-hTFF1 the pET32a-hTFF2; E. coli Origami B (DE3) The expression product mainly in the cytoplasm of the bacterium, a small amount of inclusion body formation (less than 30%). The fusion protein recombinant hTFF1 and hTFF2 production of approximately 169.6mg / L and 246.5mg / L. 2.Western blot demonstrated the recombinant protein having a good antigenicity and specificity; purity of more than 95% of the recombinant protein by Ni-NTA affinity chromatography, ultrafiltration after centrifugation. Fusion protein 3.rhTFF1 in trypsin / protein ratio 1:100, i.e. hydrolysis, the degradation of form of rhTFF1 dimer gradually increased with the concentration of trypsin a dimer rhTFF1 ratio gradually decreased, to trypsin / protein ratio of 1:1, rhTFF1 dimer All degradation; rhTFF2 fusion protein until the trypsin / protein ratio of 1:1, the fusion protein 65.3% residual. rhTFF1 and rhTFF2 fusion protein pepsin / protein ratio of 1:1, after 4h the digestion rhTFF1 and rhTFF2 fusion protein residues, respectively, 73.7% and 50.7%. In a buffer of the pH value of 2.0-12.0, rhTFF1 and rhTFF2 of the fusion protein the amount of residue was 90% -98%, after a 37 ° C water bath for 5h. 4.rhTFF1 and rhTFF2 fusion proteins can promote MKN-28 cell proliferation and migration, the minimum effective concentration of 10μg/ml and 30μg/ml. 5 anhydrous ethanol gavage in mice and burned mice were given the 1mg/kg rhTFF1 or rhTFF2 of fusion protein orally, can significantly reduce the degree of damage to the gastric mucosa. Intragastric ethanol 6 hours heaviest damage control mice gastric mucosa, extensive bleeding, bleeding lesions showed a large area of ??the sheet or cord-like, rhTFF1 and rhTFF2 fusion protein therapy group injury significantly reduced erosion surface and bleeding point significantly less gastric mucosal injury index (36.66 ± 10.14,13.33 ± 2.42,24.83 ± 3.53, P <0.05 to 0.01), the pathological changes of gastric mucosa was significantly reduced. Burned mice gastric mucosal damage is the heaviest in the 24 hours after injury, the gastric mucosa of the control group visible bleeding points, or corn granular, rhTFF1 and rhTFF2 of fusion protein treatment group damage significantly reduced, bleeding significantly higher than the less gastric mucosal injury index (6.16 ± 1.95,2.00 ± 1.15,3.5 ± 0.95, P <0.05 to 0.01), respectively. Damage control group pathological changes of gastric mucosa erosion, hemorrhage, necrosis and ulceration of the main pathological changes of the treatment group was significantly reduced to congestion, edema mainly. Conclusion: 1.rhTFF1 and fusion protein rhTFF2 success in the expression of the E. coli system, the fusion protein soluble. Classic recombinant expression has the following advantages: (1) omitted the prokaryotic expression is complex due to the formation of inclusion bodies have too many complex process; (2) without the use of expensive enterokinase target protein; (3) without salting concentrate the target protein; (4) to maintain while reducing the expression of the biological activity of about 20% of the cost. 2.rhTFF1 fusion protein has good resistance to pepsin and pH stability, but poor resistance to trypsin. 3.rhTFF2 fusion protein has preferably the stomach, the trypsin-resistant and acid-base stability. 4. Vitro experiments show rhTFF1 the rhTFF2 fusion protein can promote the proliferation rate of in vitro cultured cells, accelerate the migration of cells to the damaged area. Vivo experiments showed, rhTFF1 and rhTFF2 fusion protein has a significant therapeutic effect on gastric mucosal damage after ethanol gavage and burn. In comparison, The fusion protein rhTFF1 the protective effect is better than rhTFF2 fusion protein.