EXPERIMENTAL AND THERAPEUTIC MEDICINE 19: 2149-2154, 2020 Extracellular production of recombinant sus scrofa trefoil factor 3 by Brevibacillus choshinensis HE-PING LI1*, CHUN-MEI XU1*, BING-YAN WEN1*, AN-QI LI2, GUANG-MING ZHA1, XIANG-YANG JIN1, YUN-ZE ZHAO1, LU-PING FENG1, YE-DONG CAO1, GUO-YU YANG1, YUE-YING WANG1 and KAI ZHONG1 1Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture, Henan Agricultural University, Zhengzhou, Henan 450002; 2Kansas International College, Zhengzhou Sias University, Xinzheng, Henan 451100, P.R. China Received June 27, 2019; Accepted December 12, 2019 DOI: 10.3892/etm.2020.8477 Abstract. Trefoil factor 3 (TFF3) is involved in cell adhe- TFF-peptides are small proteins secreted by a number of sion, motility and apoptosis, regulates mucosal immunity and mucosal epithelial cells (2). TFFs possess a common trefoil maintains the functional integrity of intestinal epithelia. The domain structural motif, forming characteristic disulfide bonds upregulation of TFF3 expression in the weaning rat intestine through six conserved cysteine residues, and are highly gastric attracted our interest. The present study hypothesized that acid and protease-resistant (2). TFFs help maintain mucosal TFF3 may serve a role in preventing diarrhea in weaning barrier integrity by protecting the gastrointestinal (GI) mucosa piglets, which is an important consideration in the pig farming against injury and improving repair following injury (3-5). industry. Previous recombinant TFF3 protein expression The pit cells in gastric mucosa primarily secret TFF1, whereas yields obtained from Escherichia coli were too low and the TFF2 is synthesized by the neck cells of the gastric mucosa bioactivity of the protein was poor. Hence, this expression and the Brunner's glands of the duodenal submucosa (6). The system was unsuitable for industrial applications. The present distribution of TFF1 and TFF2 is confined to the proximal study explored the production of recombinant sus scrofa TFF3 GI tract, whereas TFF3 is produced by the goblet cells and in a Brevibacillus choshinensis (B. choshinensis) expression secreted abundantly throughout the intestinal tract (3). TFF3 system, aiming to enhance the expression level of bioactive protects and repairs the gastrointestinal mucosa, maintains the protein. To achieve this, the sus scrofa TFF3-encoding gene tight junction barrier integrity and restores normal intestinal fragment was fused into an E. coli‑Brevibacillus shuttle vector permeability during inflammatory bowel disease pathogenesis pNCMO2. High levels of TFF3 (30 mg/l) were produced and (7). TFF3 is also present in the neural lobe of the porcine secreted into the B. choshinensis culture medium in soluble pituitary gland (8,9). form with a molecular mass of 13.6 kDa and high immunore- The GI tract acts as a barrier against bacteria and toxins activity in western blotting. Thus, Brevibacillus may be used to preventing digestive disorders. Weaning is an important produce useful mucosal factors for biochemical analyses and process during development of the pig (10). The structure and mucosal protection, and in industrial applications to produce function of the digestive system of a growing pig begins imme- novel inhibitors of diarrhea. diately to change when weaning begins (10). Weaning involves extensive exposure to novel antigens, improving the redistribu- Introduction tion of the microbial community in the small intestine (10). Piglets are susceptible to disease after weaning. When the GI The family of Trefoil factor (TFF)-peptides, previously known mucosal barrier function is impaired, the ecological balance as P-domain peptides, consists of 3 members (TFF1, 2 and 3) (1). among gut microbes is destroyed leading to enterogenous infection, resulting in diarrhea (11). Diarrheal piglets are of significant concern to the pig industry (10). Previous studies have revealed that TFFs can maintain the integrity of the intes- tinal epithelia in mice, heal wounds in humans and participate Correspondence to: Professor Yue-Ying Wang or Professor in cell adhesion, motility and apoptosis in vitro (12-17). TFFs Kai Zhong, Key Laboratory of Animal Biochemistry and Nutrition, are involved in mucosal immune modulation by regulating Ministry of Agriculture, Henan Agricultural University, 63 Nongye leukocyte recruitment and repairing tissue to ensure healthy Road, Zhengzhou, Henan 450002, P.R. China mucosal surfaces (18). In addition, the level of TFF3 expres- E-mail: [email protected] E-mail: [email protected] sion is increased in the intestine of weaning rats (19). Thus, the present study hypothesized that TFF3 may serve an important *Contributed equally role in preventing diarrhea in weaning piglets. In previous studies, TFF3 has been expressed in Key words: protein expression, trefoil factor 3, pNCMO2, Escherichia coli in an intracellular and soluble form (20-22). Brevibacillus choshinensis However, this method of production is not ideal because of the low yield and bioactivity of the produced TFF3 (20-22). 2150 LI et al: EXTRACELLULAR PRODUCTION OF RECOMBINANT Sus scrofa TREFOIL FACTOR 3 The present study developed a recombinant expression system as a template, and can quickly identify whether the colony for sus scrofa TFF3 fused with a 6xhis-tag in a strain of is a positive colony with the target gene. Plasmids extracted B. choshinensis. B. choshinensis is a Gram-positive bacterium, from the positive clones were sequenced by Takara Bio, Inc. which has the excellent advantage of secreting various extra- The primers were forward, 5'-GCg tcg acA TGG AGG CCA cellular proteins with high efficiency (23). The spore forming GGA TGT-3' and reverse, 5'-CGG ggt acc TTA GTG ATG TGA ability of B. choshinensis has been removed by genetic engi- TGG TGA TGG AAG GTG CAT TCT-3'; lower-case letters neering. In addition, this expression system is very powerful denote the enzyme restriction sites for SalI and KpnI and the for producing proteins with native structures, even when they underlined bases denote the nucleotide sequence encoding the contain disulfide bonds (23). pNCMO2 is a B. choshinensis‑E. 6xhis tag. To construct the sus scrofa TFF3 expression vector coli shuttle vector (24). The pNCMO2 vector includes the P2 pNCMO2-TFF3-6xhis, the fused TFF3-6xhis combined frag- promoter, which drives the transcription of cell wall protein ment from pMD19‑T‑TFF3‑6xhis was amplified by PCR. The but has no role in E. coli (24). The objective of the present following thermocycling conditions were used for the PCR: study was to explore the production of sus scrofa TFF3 by initial denaturation at 95˚C for 10 min; followed by 35 cycles B. choshinensis in vitro. of 95˚C for 30 sec, 56˚C for 30 sec and 72˚C for 30 sec; and a final extension 72˚C for 10 min. Digestion with SalI and Materials and methods KpnI followed by ligation at 16˚C overnight was used to clone the amplified fragment into the pNCMO2 vector to generate Bacteria, plasmids and media. B. choshinensis strain HB116 pNCMO2-TFF3-6xhis. DNA sequencing was performed to (Takara Bio, Inc.) was used in this study. E. coli DH5α confirm the cloned DNA sequence (Takara Bio, Inc.). competent cells (Sangon Biotech Co, Ltd.) were used for DNA manipulation. pNCMO2 (Takara Bio, Inc.) and pMD19-T B. choshinensis transfection. Competent cells of (Takara Bio, Inc.) were used as the vector and subcloning B. choshinensis HB116 were prepared by inoculating 1 ml plasmid, respectively. Milk-Tween (MT) medium containing bacterial solution in 100 ml MT medium (Shanghai Kemin 2% yeast extract, 10% glucose, 10% polypeptone, 5% meat Biotechnology Ltd.), which were then cultured at 37˚C extract, 0.01 % FeSO4·7H 2O, 0.001% ZnSO4·6H2O and 0.01% for 18 h. Bacteria were then collected by centrifugation at MnSO4·4H2O was used to culture strain HB116. E. coli DH5α 4,000 x g for 5 min at room temperature, and suspended in cells were cultured in Luria Broth medium (Oxoid; Thermo 50 mmol/l Tris-HCl (pH=8.5; Beijing Dingguo Changsheng Fisher Scientific, Inc.). NaOH was used to adjust the pH of all Biotechnology Co, Ltd.). Bacteria were then transfected with media to 7.0. Neomycin (20 µg/ml; Beijing Solarbio Science & vector DNA using electrophoretic transfer according to the Technology Co, Ltd.) was added to the media used to culture manufacturer's instructions (Takara Bio, Inc.). The empty bacteria containing pNCMO2 and derivatives. pNCMO2 vector was transfected as a negative control. A MicroPulser (Bio-Rad Laboratories, Inc.) was used with the RNA extraction and PCR. Total RNA from sus scrofa spleen Ec2 program. tissue (preserved in our laboratory) was isolated using a TRIzol® reagent kit (Takara Bio, Inc.) according to the manu- Protein expression of recombinant sus scrofa TFF3. B. choshi‑ facturer's instructions. cDNA synthesis was performed using nensis containing pNCMO2-TFF3-6xhis or pNCMO2 was a PrimeScript Reverse Transcriptase kit (Takara Bio, Inc.). cultured in liquid MT medium containing 20 mg/l neomycin Primers for the sus scrofa TFF3 gene were designed using at 30˚C for 60 h. Once the bacteria reached the logarithmic Primer v.5.0 software (Sangon Biotech. Co. Ltd.), according growth period at 37˚C, isopropyl β-D-1-thiogalactopyranoside to the gene sequence in the GenBank database (accession (final concentration 1 mmol/l) was added to the bacteria. no. NM_001243483). mRNA specific primers (Sangon Biotech Following induction, the collected bacteria were disintegrated Co, Ltd.) were: TFF3 forward, 5'-GCA TGG AGG CCA GGA by ultrasound (JY88-II Ultrasonic Cell Disruptor; Bio-Equip) TGT-3' and reverse, 5'-CGG TTA GAA GGT GCA TTC T-3'. The and lysed in PBS (pH=7.4). The bacterial precipitate and super- PCR program to amplify the TFF3 gene from cDNA was 95˚C natant were subsequently used in SDS-PAGE. Supernatants and for 5 min, followed by 35 cycles of 95˚C for 30 sec, 55˚C for cells were separated by centrifugation at 12,000 x g for 20 min 20 sec and 72˚C for 30 sec with a final extension step at 72˚C at 4˚C.