Trefoil Factor Family Research Articles

Trefoil factor family peptides in human saliva and cyclical cervical mucus. Method evaluation and results on healthy individuals

Trefoil peptides are 7-12 kDa molecules, se-creted by a variety of mucin-producing epithelial cells from different tissues and believed to be essential for protection and maintenance of gastrointestinal mucosa. Data on concentrations of trefoil peptides in secretions are limited. We validated in-house ELISA assays, developed for measurement of trefoil peptide concentrations (TFF1, TFF2 and TFF3) in serum, for use with saliva and cervical mucus. Saliva from healthy individuals (n=30), and cervical mucus as well as blood collected three times during the menstrual cycle from healthy women (n=18) were analyzed. Recovery of all trefoil peptides in the initial supernatants of saliva and (cervical mucus) were 86 and (92)% or more. Recovery of exogenously added trefoil peptides was 93 and (95)% or more. Western blotting showed that antibodies used in the TFF3-ELISA assay recognised one molecule of the same size as TFF3 in both saliva and cervical mucus. Median concentrations of TFF1, TFF2 and TFF3 in saliva and (cervical mucus) were 2.7 (2.7), 0.08 (0.58) and 14 (430) nmol/g protein, with a significant decrease in concentrations in cervical mucus after ovulation. Serum concentrations resembled previously measured values in blood donors and showed no cyclic change. Previously established ELISA assays can be employed for measurement of trefoil peptides in saliva and cervical mucus. TFF3 was the predominant trefoil peptide in both saliva and cervical mucus, and TFF3 in cervical mucus represents the highest concentration measured in a biological fluid to date.

Increased trefoil factor 3 levels in the serum of patients with three major histological subtypes of lung cancer

Lung cancer is the most common cause of cancer-related deaths in the world. The trefoil factor (TFF) family is composed of three thermostable, and protease-resistant proteins, named TFF1, TFF2 and TFF3. TFF protein levels have been found to be related to the development of various types of cancer. However, it is still unclear whether TFF proteins are differentially expressed in the serum of different histological subtypes of lung cancer compared to healthy individuals. In this study, we investigated the levels of TFF proteins in serum and lung tissues of 130 lung cancer patients (58 squamous cell lung carcinoma cases, 43 adenocarcinoma cases and 29 SCLC cases) and 60 healthy individuals. It was found that TFF1 and TFF2 have similar or slightly higher levels in these three subtypes of lung cancer compared to healthy individuals, while TFF3 levels were significantly higher in the examined lung cancer cases compared to healthy individuals. Immunoblot analyses of TFF1, TFF2 and TFF3 indicated that lung cancer tissues and lung cancer cell lines have a higher expression of the TFF3 protein, but not of TFF1 or TFF2 proteins, compared to tissues from healthy individuals or from the normal cell line. Quantitative RT-PCR analysis indicated higher levels of TFF3, but not TFF1 and TFF2, transcripts in lung cancer tissues or cell lines. These results show increased TFF3 levels in serum and lung tissues, suggesting that TFF3 may serve as a promising, easily detected biomarker of lung cancer.

The role of trefoil factor family in apparently healthy subjects administrated gastroprotective agents for the primary prevention of gastrointestinal injuries from low-dose acetylsalicylic acid: a preliminary study

It is well-known that acetylsalicylic acid induces gastrointestinal complication. Recently, trefoil factor family has been reported as a mucosal protective factor. We focused on trefoil factor family as one of defensive system for gastrointestinal injuries. The aim of this trial was to evaluate trefoil factor family levels in the serum of healthy subjects with low-dose acetylsalicylic acid. Low-dose acetylsalicylic acid with placebo or proton pump inhibitor or rebamipide were administered in 30 healthy subjects. Transnasal endoscopy was performed at 0, 24 h, 3 and 7 day. Changing of trefoil factor family (1,2,3) and numbers of gastric injuries were evaluated. The numbers of gastric injuries were significantly increased in the placebo group at 3 and 7 days. Injuries in the proton pump inhibitor group were not induced, in the rebamipide group were slightly induced. Trefoil factor family level in the placebo group were decreased in 3 and 7 days compared with prior to starting the trial. Trefoil factor family may have an important association with acetylsalicylic acid-induced gastrointestinal damage. Proton pump inhibitor and rebamipide prevented low-dose acetylsalicylic acid-induced gastrointestinal complications compared with the placebo group.

Expression of trefoil factor family-3 and β-catenin in different types of colorectal mucosal lesions

Expression of trefoil factor family-3 and β-catenin in different types of colorectal mucosal lesions

Validation of commercial assays for measurements of trefoil factor family peptides in serum

Trefoil peptides (TFF1, TFF2 and TFF3) are 7-12 kDa molecules, secreted by mucin-producing epithelial cells. Increased serum concentrations have been reported in a number of pathological conditions, which warrants the need for validated commercially available assays. We validated commercial assays for TFF1-3 and compared results obtained with our in-house assays, using serum from blood donors. Level of detection was: ≤ 0.008 nmol/L. Measuring ranges were: 0.032-0.51 (TFF1), 0.038-0.76 (TFF2) and 0.019-0.15 (TFF3) nmol/L. Imprecision (CV), judged from the measurement of serum pools in two levels, was below 9% (TFF2 and TFF3) but up to 18% (mean 0.41 nmol/L) for TFF1. No cross reactivity between the TFFs (concentrations > 100 nmol/L) was observed. The 95% non-parametric reference intervals were: <0.0032-0.53 (TFF1), 0.099-1.4 (TFF2) and 0.086-0.87 (TFF3) nmol/L. Comparing commercial to in-house assays (n=132), showed biases explained by differences in the calibrators (TFF1 and TFF2). A number of samples showed markedly different results. The commercial assays for TFF2 and TFF3 are acceptable for use on serum samples, while the TFF1 assay revealed a poor imprecision and a too narrow measuring range. Results obtained with the commercial and the in-house assays differed, partly because of differences in the calibrators employed.

Trefoil factor family peptides are increased in the saliva of children with mucositis

Mucositis is a painful ulcerative condition of the oral cavity and gastrointestinal tract, occurring in association with chemotherapy and radiotherapy regimes. Trefoil factor family peptides (TFF, trefoil peptides), present in saliva, contribute to epithelial restitution and repair and are therefore potentially important in the healing phase of mucositis. This study aimed to assess any changes in the levels of trefoil peptides in oncology patients with and without mucositis. Saliva was collected from healthy children, pretreatment oncology patients, neutropenic patients on treatment with no oral disease and mucositic patients. TFF1, 2 and 3 were quantified using ELISA. In healthy children TFF2 and 3 were positively correlated with age (r = 0.454, p = 0.01 for TFF2; r = 0.410, p = 0.05 for TFF3 Spearman rank correlation). TFF3 was higher in mucositis compared to all other groups. A linear regression prediction model indicated that TFF3, but not TFF1 and TFF2, was significantly different in mucositic and healthy controls, suggesting an altered pattern of trefoil peptide secretion (p = 0.021). This study is the first to focus on trefoil peptides in paediatric saliva. It shows the correlation between TFF2, TFF3 and age in healthy children. Paediatric mucositis disease occurs in the presence of increased concentrations and an altered pattern of trefoil peptides.

Decreased expression of TFF2 and gastric carcinogenesis

The trefoil factor family (TFF) of peptides, which are protease-resistant and have a strong affinity for mucins, play an important role in gastrointestinal mucosal protection and restitution. Prior studies have indicated that dysregulation of TFF2 was associated with cell migration, resistance to apoptosis, and gastric cancer invasion; however, the underlying mechanism associated with these actions remains unclear. Thus, to investigate the relationship between TFF2 and carcinogenesis in gastric cancer, TFF2 expression was analyzed by Western blot analysis in nine selected gastric cancer tissues and immunohistochemical staining was performed in paraffin-embedded samples from 157 gastric cancers. A reduced TFF2 expression was observed by Western blot analysis in the gastric cancer tissues. However, there was no significant difference in the TFF2 expression according to clinical and pathological parameters of the gastric cancers. To investigate the biological role of TFF2 in the development and progression of gastric cancer, a TFF2 expression plasmid was constructed for in vitro experiments of function. Introduction of TFF2 cDNA into a gastric cancer cell line did not affect tumor cell growth, cell migration or invasion. In conclusion, down-regulation of TFF2 in gastric cancer cells and restoration of TFF2 did not affect the tumorigenic potential of gastric cancer cells in vitro. The loss of TFF2 expression might be an early event of the multi-step process of gastrocarcinogenesis and may play a limited role in the mucosal protection of the normal gastric physiology.

TFF2 mRNA Transcript Expression Marks a Gland Progenitor Cell of the Gastric Oxyntic Mucosa

Gastric stem cells are located in the isthmus of the gastric glands and give rise to epithelial progenitors that undergo bipolar migration and differentiation into pit and oxyntic lineages. Although gastric mucus neck cells located below the isthmus express trefoil factor family 2 (TFF2) protein, TFF2 messenger RNA transcripts are concentrated in cells above the neck region in normal corpus mucosa, suggesting that TFF2 transcription is a marker of gastric progenitor cells. Using a BAC strategy, we generated a transgenic mouse with a tamoxifen-inducible Cre under the control of the TFF2 promoter (TFF2-BAC-Cre(ERT2)) and analyzed the lineage derivation from TFF2 mRNA transcript-expressing (TTE) cells. TTE cells were localized to the isthmus, above and distinct from TFF2 protein-expressing mucus neck cells. Lineage tracing revealed that these cells migrated toward the bottom of the gland within 20 days, giving rise to parietal, mucous neck, and chief cells, but not to enterochromaffin-like-cell. Surface mucus cells were not derived from TTE cells and the progeny of the TTE lineage did not survive beyond 200 days. TTE cells were localized in the isthmus adjacent to doublecortin CaM kinase-like-1(+) putative progenitor cells. Induction of spasmolytic polypeptide-expressing metaplasia with DMP-777-induced acute parietal cell loss revealed that this metaplastic phenotype might arise in part through transdifferentiation of chief cells as opposed to expansion of mucus neck or progenitor cells. TFF2 transcript-expressing cells are progenitors for mucus neck, parietal and zymogenic, but not for pit or enterochromaffin-like cell lineages in the oxyntic gastric mucosa.

Novel therapeutic strategies for acute lung injury induced by lung damaging agents: The potential role of growth factors as treatment options

The increasing threat from terrorism has brought attention to the possible use of toxic industrial compounds (TICs) and other lung-damaging agents as weapons against civilian populations. The way in which these agents could be used favours the development of generic countermeasures. Improved medical countermeasures would increase survivability and improve the quality of recovery of lung damaged casualties. It is evident that there is a dearth of therapeutic regimes available to treat those forms of lung damage that currently require intensive care management. It is quite possible that mass casualties from a terrorist incident or major industrial accident involving the release of large quantities of inhaled TICs would place a severe burden on already scarce intensive care facilities. The development of effective pharmacological approaches to assist the recovery of casualties suffering from acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) may improve the prognosis of such patients (which is currently poor) and would ideally be used as a means of preventing subjects from developing the pulmonary oedema characteristic of ALI/ARDS. Many promising candidate pharmacological treatments have been evaluated for the treatment of ALI/ARDS, but their clinical value is often debatable. Thus, despite improvements in ventilation strategies, pharmacological intervention for ALI/ARDS remains problematical. A new approach is clearly required for the treatment of patients with severely compromised lungs. Whilst the pathology of ALI/ARDS associated with exposure to a variety of agents is complex, numerous experimental studies suggest that generic therapeutic intervention directed at approaches that aim to upregulate repair of the damaged alveolar blood/air barrier of the lung may be of value, particularly with respect to chemical-induced injury. To this end, keratinocyte growth factor (KGF), epithelial growth factor (EGF) and basic fibroblast growth factor (bFGF) are emerging as the most important candidates. Hepatocyte growth factor (HGF) does not have epithelial specificity for lung tissue. However, the enhanced effects of combinations of growth factors, such as the synergistic effect of HGF upon vascular endothelial growth factor (VEGF)-mediated endothelial cell activity, and the combined effect of HGF and KGF in tissue repair should be investigated, particularly as the latter pair of growth factors are frequently implicated in processes associated with the repair of lung damage. Synergistic interactions also occur between trefoil factor family (TFF) peptides and growth factors such as EGF. TFF peptides are most likely to be of value as a short term therapeutic intervention strategy in stimulating epithelial spreading activities which allow damaged mucosal surfaces to be rapidly covered by epithelial cells.

Altered expression of a putative progenitor cell marker DCAMKL1 in the rat gastric mucosa in regeneration, metaplasia and dysplasia

BackgroundDoublecortin and calcium/calmodulin-dependent protein kinase-like-1 (DCAMKL1) is a candidate marker for progenitor cells in the gastrointestinal mucosa. Lineage cells in the gastric mucosa are derived from progenitor cells, but this process can be altered after injury. Therefore, we explored DCAMKL1 expression under pathological conditions.MethodsAn immunohistochemical analysis was performed in rat stomach with acute superficial injury, chronic ulcer, intestinal metaplasia and dysplasia.ResultsDCAMKL1 was exclusively expressed in immature quiescent cells in the isthmus of normal fundic glands, where putative progenitor cells are thought to reside. DCAMKL1-positive cells and proliferating cells shed into the lumen after superficial injury and re-appeared during the regenerative process, mainly in the superficial mucosa. In the marginal mucosa around the active ulcer, parietal and chief cells diminished, foveolar hyperplasia was evident, and trefoil factor family 2 (TFF2)/spasmolytic polypeptide-expressing metaplasia (SPEM) emerged at the gland base. DCAMKL1 cells re-emerged in the deep mucosa juxtaposed with SPEM and proliferating cells. In the healing ulcer, the TFF2 cell population expanded and seemed to redifferentiate to chief cells, while proliferating cells and DCAMKL1 cells appeared above and below the TFF2 cells to promote healing. SPEM appeared and PCNA cells increased in the intestinalized mucosa, and DCAMKL1 was expressed in the proximity of the PCNA cells in the deep mucosa. DCAMKL1, PCNA and TFF2 were expressed in different dysplastic cells lining dilated glands near SPEM.ConclusionThe ultrastructural appearance of DCAMKL1-positive cells and the expression patterns of DCAMKL1 in normal and pathological states indicate that the cells belong to a progenitor cell population. DCAMKL1 expression is closely associated with TFF2/SPEM cells after injury. DCAMKL1 cells repopulate close to proliferating, hyperplastic, metaplastic and dysplastic cells, and the progenitor zone shifts according to the pathological circumstances.

Human Intestinal TFF3 Forms Disulfide-Linked Heteromers with the Mucus-Associated FCGBP Protein and Is Released by Hydrogen Sulfide

TFF3 is a secretory peptide belonging to the trefoil factor family with a predicted size of 59 amino acid residues containing seven cysteine residues. It is predominantly expressed in intestinal goblet cells where it plays a key role in mucosal regeneration and repair processes. In the course of these studies, human colonic TFF3 was shown to exist mainly as a high molecular weight heteromer. Purification of this heteromer and characterization by LC-ESI-MS/MS analysis identified the IgG Fc binding protein (FCGBP) as the disulfide-linked partner protein of TFF3. FCGBP is a constituent of intestinal mucus secreted by goblet cells. Furthermore, low amounts of TFF3/monomer and only little TFF3/dimer were detected in human colonic extracts. Here, we show that these TFF3 forms can be released from the purified TFF3-FCGBP heteromer complex in vitro by reduction with hydrogen sulfide (H(2)S). Such a mechanism would be in line with the high H(2)S concentrations reported to occur in the lumen of the colon. Of special note, this points to intestinal mucus as a reservoir for a biologically active peptide. Also proteolytic processing of FCGBP was observed which is in line with multiple autocatalytic cleavages as proposed earlier by Johansson et al. (J. Proteome Res. 2009 , 8 , 3549 - 3557).

S1949 Serum Levels of Trefoil Factor Family Could Be Comprehensive Risk Markers for Gastric Cancer and Gastric Mucosal Damage

tissues was significantly higher than that in the adjacent normal tissue (73.3% vs 13.3%, P 0.05). 5. Multivariate analysis confirmed that the dual-high expression of SUMO-1 and P53 was an independent factor for predictors of progression risk. Conclusion: There was a high SUMO1 expression in colon cancer tissues. High expression of P53 in colon cancer may be related to SUMO Modification. SUMO1 may be useful as a novel target for therapy in colon cancer as well as a clinical indicator for tumor progression risk.

Abstract 2237: Expression of the trefoil protein TFF3 in human breast cancers

Abstract Expression of the trefoil protein TFF3 in human breast cancers The purpose of this study is to investigate expression of the trefoil factor family protein, TFF3 in a large series of human breast cancers. Trefoil factors are small secreted proteins that are expressed in mucus secreting epithelia. They are involved in mucosal protection in part by stimulation of cell motility. It has been proposed that the motogenic actions of trefoil proteins may facilitate tumor cell invasion and metastasis. Expression of TFF3 mRNA is regulated by estrogen in human breast cancer cell lines and strong association between estrogen receptor and TFF3 mRNA expression has been demonstrated in breast tumors by expression microarray analyses. A tissue microarray was constructed from 300 primary breast tumors and 76 metastatic deposits of breast tumors from patients who presented in the northern region of England between 2002 and 2003. TFF3 expression was evaluated by immunohistochemistry with an antibody raised against correctly-folded human TFF3 protein. Each sample was analyzed in duplicate. The intensity of the immunoreactions in 1,000 tumor cells was evaluated as: absent, weak, moderate or strong. The association of TFF3 expression with various clinico-pathological features and with estrogen receptor protein expression was tested statistically with SPSS software (p&amp;lt;0.01). Clear TFF3 expression is demonstrated in normal and malignant breast epithelial cells. There is no expression in stromal, endothelial or immune cells. Expression of TFF3 varied enormously between tumor samples. TFF3 expression is highest in mucinous and tubular breast adenocarcinomas. TFF3 is expressed at higher levels in lobular than in ductal breast cancers. TFF3 expression is associated strongly with estrogen receptor expression. There is a negative association between TFF3 expression and tumor grade. There is however a positive association between TFF3 expression and presence of vascular invasion and TFF3 expression is higher in metastatic breast tumor cells than in primary breast tumor cells. This is the first report of TFF3 protein expression in a large series of human breast tumors. The strong association between TFF3 and estrogen receptor suggests that TFF3 expression is dependent on estrogen in a significant proportion of breast tumors. The high expression of TFF3 in breast tumors with vascular invasion and in metastatic breast tumor cells supports the hypothesis that TFF3 predisposes towards breast cancer cell invasion. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2237.

IL-6/STAT3/TFF3 signaling regulates human biliary epithelial cell migration and wound healing in vitro

Interleukin-6 (IL-6), through activation of the signal transducer and activator of transcription 3 (STAT3) and trefoil factor family 3 (TFF3), has been implicated in the promotion of mouse biliary epithelial cell (BEC) proliferation and migration. However, it is still unclear whether the IL-6/STAT3/TFF3 signaling had similar effects on human BECs. Here, we showed that exposure of human BECs to recombinant IL-6 resulted in STAT3 phosphorylation and increased the expression of TFF3 at both mRNA and protein levels. Moreover, inhibition of STAT3 using RNA interference significantly abrogated IL-6-induced TFF3 expression. In an in-vitro wound healing model, IL-6 facilitated human BEC migration. This promotion of cell migration by IL-6 was blocked when STAT3 was knocked down. Interestingly, the addition of exogenous TFF3 could rescue the cell migration defects caused by STAT3 silencing. In conclusion, our data indicate that STAT3 plays a critical role in IL-6-induced TFF3 expression in human BECs and the IL-6/STAT3/TFF3 signaling is involved in human BEC migration and wound healing.

Copper binds the carboxy-terminus of trefoil protein 1 (TFF1), favoring its homodimerization and motogenic activity.

Trefoil protein 1 (TFF1) is a small secreted protein belonging to the trefoil factor family of proteins, that are present mainly in the gastrointestinal (GI) tract and play pivotal roles as motogenic factors in epithelial restitution, cell motility, and other incompletely characterized biological processes. We previously reported the up-regulation of TFF1 gene in copper deficient rats and the unexpected property of the peptide to selectively bind copper. Following the previous evidence, here we report the characterization of the copper binding site by fluorescence quenching spectroscopy and mass spectrometric analyses. We demonstrate that Cys58 and at least three Glu surrounding residues surrounding it, are essential to efficiently bind copper. Moreover, copper binding promotes the TFF1 homodimerization, thus increasing its motogenic activity in in vitro wound healing assays. Copper levels could then modulate the TFF1 functions in the GI tract, as well as its postulated role in cancer progression and invasion.

Trefoil Factor: from Laboratory to Clinic

Trefoil factor (TFF) family is a group of peptides with one or several trefoil factor domains in their structure, which are highly conserved in evolution, and are characterized by heat and enzymatic digestion resistance. The mammalian TFFs have three members (TFF1-3), and the gastrointestinal tract and the airway system are major organs of their expression and secretion. At certain physiological conditions, with a tissue-specific distribution, TFF plays an important role in mucosal protection and wound healing. But in the malignant tissues, TFF is widely expressed, correlated strongly with the genesis, metastasis and invasion of tumor cells. These phenomena indicated that TFF may be a possible common mediator of oncogenic responses to different stimuli. The biological functions of TFF involve complex regulatory processes. Single chain TFF may activate cell membrane receptors and induce specific signaling transduction. On the other hand, TFF can form a complex with other proteins to exert its biological effects. In clinical medicine, TFF is primarily applied as drugs in the mucosal protection, in the prevention and the treatment of mucosal damage-related diseases and as pathological biomarkers of tumors. At present the first hand actions and the molecular mechanisms related to TFFs are still the major challenges in TFF research. Furthermore, the discovery of the naturally occurring complex of TFF and crystallins is highly valuable to the understanding of the biological functions and action mechanisms of TFF.

ChemInform Abstract: Trefoil Factor Family (TFF) Peptides and Chemokine Receptors: A Promising Relationship.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Synthesis and localization of trefoil factor family (TFF) peptides in the human urinary tract and TFF2 excretion into the urine

Trefoil factor family (TFF) peptides promote regeneration and repair processes of mucous epithelia. They also probably play a key role in the remarkable regenerative capacity of the urinary tract epithelia. We have localized TFF1, TFF2, and TFF3 expression systematically in surgical specimens from the urinary tract by reverse transcription with the polymerase chain reaction, Western blot analysis, and immunohistochemistry. Urine samples from patients suffering from nephrolithiasis have been investigated and compared with those of healthy controls. TFF synthesis is detectable along the entire urinary tract epithelia. TFF3 synthesis is the most pronounced followed by TFF1, whereas TFF2 synthesis is occasionally detectable but only in trace amounts. In contrast, TFF2 is the predominant TFF peptide excreted into the urine, and significantly increased urinary TFF2 levels (together with occasionally raised TFF3 levels) have been observed in patients suffering from nephrolithiasis. Thus, we consider that TFF3 plays a major part in regeneration and restitution processes in urinary tract epithelia. TFF2 and probably also TFF3 are candidate biomarkers for nephrolithiasis and possibly other inflammatory conditions of the urinary tract.

Expression Analysis of Human Salivary Glands by Laser Microdissection: Differences Between Submandibular and Labial Glands

Both the major and minor salivary glands are the sources of saliva, a fluid vital for the maintenance of a healthy oral cavity. Here, the expression profiles of human submandibular (SMG) and labial glands (LG) were compared by RT-PCR analysis of laser microdissected mucous and serous cells, respectively. The focus was on trefoil factor family (TFF) genes, but also other genes encoding secretory proteins (mucins, lysozyme, amylase, statherin, and histatins) or aquaporin 5 were included. Immunofluorescence studies concerning TFF1-3, FCGBP, amylase, and lysozyme are also presented. It was shown that LGs clearly contain serous cells and that these cells differ in their expression profiles from serous SMG cells. Furthermore, all three TFF peptides, together with MUC5B, MUC7, MUC19, and FCGBP, were clearly detectable in mucous acini of both LGs and SMGs. In contrast, lysozyme was differentially expressed in LGs and SMGs. It can be expected that labial saliva may play a particularly important role for protecting the teeth.

Overexpression of hTFF2 in the pET system and its in vitro pharmacological characterization

The trefoil factor family 2 (TFF2), a member of the trefoil factor family, plays a critical role in the defense and repair of gastrointestinal mucosa. However, its widespread application is hampered by difficulties in large-scale production of the recombinant protein suitable for clinical use. The aim of the present study was to produce hTFF2 by Escherichia coli expression system and explore its in vitro pharmacological characterization. hTFF2 gene encoding mature peptide was obtained by RT-PCR, and then inserted into the expression vector pET32a to construct the recombinant pET32a-hTFF2. After confirmation by gene sequencing, pET32a-hTFF2 was transformed into E. coli Origami B(DE3), and TrxA-hTFF2 fusion protein was expressed by conventional IPTG induction in a shake flask and analyzed with SDS-PAGE and Western-blot. Subsequently, TrxA-hTFF2 was isolated by Ni-NTA affinity chromatography, and ultrafiltration. Finally, we tested the effect of hTFF2 on cell migration in an in vitro restitution model and cell proliferation by MTT assay. The data revealed that the recombinant vector pET32a-hTFF2 was constructed successfully. TrxA-hTFF2 fusion protein was expressed to 246.5 mg/L and its purity was above 95% after purification. SDS–PAGE and Western blot analyses showed that the fusion protein presented as a single band with a molecular weight of 32 kDa. In vitro model of wounding demonstrated that hTFF2 enhanced migration activity by three folds. MTT assay exhibited a statistically significant dose-dependent growth-enhanced effect. Collectively, the results suggest that the recombinant hTFF2 was expressed in E. coli with high production, purity and biological activity.