Production Of Human Breast Cancer-associated Peptide And Pancreatic Spasmolytic Polypeptide In Escherichia Coli And Their Biological Activity

Background:Trefoil factor family(TFF),a new protein family,was discovered and named by Thim in 1989.TFF is composed of one structurally characteristic trefoil domain. A trefoil domain is defined as a sequence of 38 or 39 amino acid residues in which 6 cysteine residues are linked in the configuration of sequence 1-5,2-4,3-6,thus forming a characteristic three-leaved structure.The mammalian trefoil factor family contains three members:breast cancer-associated peptide(pS2 or TFF1),spasmolytic polypeptide(SP or TFF2),and intestinal trefoil factor(ITF or TFF3).Trefoil factors are expressed in several tissues of the body but most pronouncedly in the gastrointestinal(GI) tract,where the individual trefoil factor is expressed in a tissue-specific manner.Under physiological state, TFF1and TFF2 are expressed and synthesized in stomach,and TFF3 in small and large intestine.The trefoil factor is essential for protecting the epithelial layer of the gastrointestinal tract from damage and repairing epithelium after injury,and described as super protection factor of gastrointestinal tract.TFF has been attracted the researchers' attention for its potential pharmacological value.Three methods are generally available for producing TFF:peptides synthesis, preparation from natural sources and recombinant technology.Due to default tissue source and incompetence,peptides synthesis and preparation from natural sources could not be used to produce TFF on a large scale.Although gene recombination express is the best method of producing TFF,and every member of TFF has been expressed in Escherichia coli or yeast expression system.Due to the low output and great expensive,the industrialization product and clinical application of TFF are limited.It is necessary to search a new recombination expression method characteristic of high performance, shortcut and low expensive. Objective:To construct expression vector of rhTFF1 and rhTFF2,obtain rhTFF1 and rhTFF2 fusion protein with high production and high purity in Escherichia coli;to assay the stability of the rhTFF1 and rhTFF2 fusion protein in vitro simulated gastrointestinal environment and to explore their biological activity.Methods:1.Expression and purification of the rhTFF1 and rhTFF2 fusion protein in E.coli: hTFF1 and hTFF2 gene was obtained by RT-PCR,and then inserted into the MCS of the expression vector pET32a to construct the recombinant vector.After double enzyme digestion and gene sequencing,the recombinant vector was transformed into E.coli Origami B(DE3),and rhTFF1,rhTFF2 fusion protein was expressed by IPTG induction.The rhTFF1 and rhTFF2 fusion protein was purified by Ni-NTA affinity chromatography, determined by SDS-PAGE and Western blot analysis.2.Stability of the rhTFF1 and rhTFF2 fusion protein in simulated gastrointestinal environment in vitro.The rhTFF1 and rhTFF2 fusion protein were added to various concentration of pepsin or trypsin and digested at 37℃for 4 hours.Then the fusion protein of rhTFF1 and rhTFF2 were incubated in buffers with various pH values at 37℃for 5 hours.Results were analyzed by non-reducing tricine SDS-PAGE.3.To research the biological activity in vitro:①To observe the effect of different concentrations of the rhTFF1 and rhTFF2 fusion protein on proliferating activity of MKN-28 cell with MTT and CCK-8 assay after a 1d incubation.②To survey the migration in an in vitro injuryed model.Injury was established in confluent monolayer of MKN-28 cell,and injuried monolayers were cultured for 24h after adding culture medium and rhTFF1 and rhTFF2 fusion protein(30μg/ml).4.To research the biological activity in vivo:Mice were fed with anhydro-alcohol and inflicted with 30%total body surface area full thickness burns,and randomly divided into three groups:alcohol(A) or burned(B),treated with rhTFF1 or rhTFF2 fusion protein (F1 or F2),and normal control(C).Before and after injury,administered rhTFF1 or rhTFF2 fusion protein to mice group A and B.Injury index is calculated on 1,3,6,12 and 24h after alcohol iujury and 0.5,1,3 and 5d postburn.Results:1.As a result of RT-PCR,the 218bp and 356bp DNA fragment were obtained and the size of the amplification segment were consistent with the theoretical value(GenBank Accession No.NM003225 and No.NM005423).The recombinant vector was successfully constructed and the rhTFF1 and rhTFF2 fusion protein were expressed at a concentration of 169.6mg/L and 246.5mg/L.2.Western blot analysis showed that the rhTFF1 and rhTFF2 fusion protein had much good antigenicity and specificity.After purification,the purity was above 95%.3.The rhTFF1 fusion protein was robustly resistant to pepsin,even at a high enzyme/substrate(w/w) ratio of 1/1,more than 70%of the fusion protein of rhTFF1 existed. But it was decomposed at trypsin/substrate(w/w) ratio of 1/100.Different pH values ranging from 2.0 to 12.0 had little effect on the fusion protein of rhTFF1,and the percentage of residual fusion protein existed from 90%to 98%.4.The fusion protein of rhTFF2 was robustly resistant to trypsin and pepsin,even at a high enzyme/substrate(w/w) ratio at 1/1,more than half of rhTFF1 existed in fusion protein form.Different pH values ranging from 2.0 to 12.0 had little effect on the fusion protein of rhTFF2,and the percentage of residual fusion protein existed from 90%to 98%.5.The fusion protein of rhTFF1 and rhTFF2 could promote the generation and migration of MKN-28 cells when the valid concentration was 10μg/ml and 30μg/ml respectively.6.The fusion protein of rhTFF1 and rhTFF2 treatment(1mg/kg) significantly decreased the injury index and alleviated pathological damage in comparison with the control group.Conclusion:1.The rhTFF1 and rhTFF2 fusion protein were successfully produced in E.coli expression systems.The recombination protein have a good character in dissolubility. Compared with the calssic process,it has high yield and reducing the production cost.2.The rhTFF1 fusion protein possessed characteristics of pepsin resistance,acid and alkali resistance but lacked trypsin resistance.3.The rhTFF2 fusion protein possessed characteristics of protease resistance,acid and alkali resistance.4.In vitro study,the rhTFF1 and rhTFF2 fusion protein were demonstrated to have the ability to promote cell proliferation,and to enhance migration activity. 5.In vivo study,the rhTFF1 and rhTFF2 fusion protein enhanced the healing effect on acute gastric mucosal lesion in mice.