人TFF2基因的原核表达与纯化

ZHUANG Yong-Hui; LI Si-Man; YU Guo-Yu; ZHANG Yong; XIANG Yang; ZOU Hao; LEE Wen-Hui

doi:10.3724/SP.J.1141.2012.02144

人TFF2基因的原核表达与纯化

doi: 10.3724/SP.J.1141.2012.02144

庄永辉^1,2,
李思熳^1,3,
余果宇^1,3,
张勇¹,
向阳¹,
邹浩⁴,
李文辉¹

1.
中国科学院和云南省动物模型与人类疾病机理重点实验室云南昆明 650223;
2.
云南省第二人民医院神经外科云南昆明 650021;
3.
昆明医学院生物化学教研室云南昆明 650500;
4.
昆明医学院第二附属医院肝胆外科云南昆明 650101

基金项目: “973”项目(2010CB529800); 国家基金委面上项目(81160302, 30870304); 中国科学院“西部之光”(Y102291081)

详细信息

中图分类号: Q786; Q251
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出版历程
- 收稿日期: 2011-12-14
- 修回日期: 2012-02-22
- 刊出日期: 2012-04-22

Bacterial expression and purification of biologically active human TFF2

1.
Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology,Kunming Yunnan 650223, China;
2.
Department of Neurosurgery, Yunnan No.2 People’s Hospital, Kunming Yunnan 650021, China;
3.
Department ofBiochemistry, Kunming Medical College, Kunming Yunnan 650500, China;
4.
Department of Hepatobiliary Surgery, the SecondAffiliated Hospital of Kunming Medical College, Kunming Yunnan 650101, China

Funds: This work was supported by grants from the National Basic Research Program of China (973 Program, 2010CB529800), the Chinese National Natural Science Foundation (81160302, 30870304), the “Western Light Project” from the Chinese Academy of Sciences (Y102291081), and the Science and Technology Department of Yunnan Province (2011C1139)

摘要

摘要: 人三叶因子2(hTFF2)具有促进细胞迁移和抑制细胞凋亡的活性,所以被认为是胃肠黏膜修复的启动者之一。因为从人组织中获得hTFF2比较困难,而且体外产生的重组hTFF2大都以融合蛋白的形式存在,所以该研究的目的是在体外产生不带任何融合蛋白的游离型hTFF2。hTFF2的开放阅读框被插入pET-32a(+)表达载体,然后在大肠杆菌中表达出带有硫氧还蛋白融合部分的hTFF2融合蛋白。进而利用融合蛋白的组氨酸标签使用镍亲和色谱柱以及反向高压色谱柱对目的蛋白进行纯化。23℃,FXa因子裂解纯度高达95%的融合蛋白以得到游离型hTFF2。在去除FXa因子和尚未被切开的融合蛋白后,获得的游离型hTFF2被SDS-PAGE和Western blotting所证实。重组游离型hTFF2的产量约为5mg/L,并且hTFF2能促进IEC-6细胞的迁移以及体外的伤口修复,而这些活性是依赖于ERK1/2的激活。同时,hTFF2也能抑制50μmol/L神经鞘氨醇所引起的HCT-116细胞的凋亡。总之,研究结果表明,在大肠杆菌中高产量地成功表达出具有生物学活性的游离型hTFF2,这为研究TFF2的分子机制,以及研制和开发TFF2的相关药物都提供很大的帮助。
- TFF2 /
- 表达 /
- 细胞迁移 /
- 抗凋亡 /
- 伤口修复
Abstract: Human trefoil factor 2 (hTFF2) is considered as one of the most important initiators of mucosal healing in the gastrointestinal tract by promoting cell migration and suppressing apoptosis. However, it is hard to obtain hTFF2 from human tissue and many recombinant hTFF2 produced in vitro exist as fusion proteins. The purpose of the present study was to produce native hTFF2 while maintaining its biological activities. The open reading frame of hTFF2 was inserted into a pET-32a(+) expression vector, and hTFF2-TRX fusion protein was successfully expressed in Escherichia coli and purified by Nickel-nitrilotriacetic acid affinity chromatography and reverse-phase HPLC steps. The recombinant fusion protein (purity>95%) was cleaved by Factor Xa at 23 ℃ to release hTFF2. After removal of Factor Xa and undigested fusion proteins, hTFF2 was purified and identified by SDS-PAGE and Western blotting. The yield of recombinant hTFF2 was about 5 mg/L. The recombinant hTFF2 could promote IEC-6 cells migration and in vitro wound healing via the activation of ERK1/2. Recombinant hTFF2 could also inhibit apoptosis of HCT-116 cells induced by 50 μmol/L ceramide.In summary, our results showed that the recombinant hTFF2 was expressed in E. coli and successfully purified after cleavage with the fusion partner with high yield while maintaining its biological activities. Recombinant hTFF2 might be useful for investigating the molecular mechanism of hTFF2 and development of hTFF2-related drugs.
- TFF2 /
- Expression /
- Cell migration /
- Anti-apoptosis /
- Wound healing

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参考文献(32)

[1]	A lison MR, Chinery R, Poulsom R, Ashwood P, Longcroft JM, Wright NA. 1995. Experimental ulceration leads to sequential expression of spasmolytic polypeptide, intestinal trefoil factor, epidermal growth factor and transforming growth factor alpha mRNAs in rat stomach[J]. J Pathol, 175(4): 405-414.
[2]	Babyatsky MW, deBeaumont M, Thim L, Podolsky DK. 1996. Oral trefoil peptides protect against ethanol-and indomethacin-induced gastric injury in rats[J]. Gastroenterology, 110(2): 489-497.
[3]	Bossenmeyer-Pourié C, Kannan R, Ribieras S, Wendling C, Stoll I, Thim L, Tomasetto C, Rio MC. 2002. The trefoil factor 1 participates in gastrointestinal cell differentiation by delaying G1S phase transition and reducing apoptosis[J]. J Cell Biol, 157(5): 761-770.
[4]	Chwieralski CE, Schnurra I, Thim L, Hoffmann W. 2004. Epidermal growth factor and trefoil factor family 2 synergistically trigger chemotaxis on BEAS-2B cells via different signaling cascades[J]. Am J Respir Cell Mol Biol, 31(5): 528-537.
[5]	Cook GA, Familari M, Thim L, Giraud AS. 1999. The trefoil peptides TFF2 and TFF3 are expressed in rat lymphoid tissues and participate in the immune response[J]. FEBS Lett, 456(1): 155-159.
[6]	Dignass A, Lynch-Devaney K, Kindon H, Thim L, Podolsky DK. 1994. Trefoil peptides promote epithelial migration through a transforming growth factor beta-independent pathway[J]. J Clin Invest, 94(1): 376-383.
[7]	Farrell JJ, Taupin D, Koh TJ, Chen D, Zhao CM, Podolsky DK, Wang TC. 2002. TFF2/SP-deficient mice show decreased gastric proliferation, increased acid secretion, and increased susceptibility to NSAID injury[J]. J Clin Invest, 109(2): 193-204.
[8]	Fox JG, Rogers AB, Whary MT, Ge ZM, Ohtani M, Jones EK, Wang TC. 2007. Accelerated progression of gastritis to dysplasia in the pyloric antrum of TFF2^-/- C57BL6 × Sv129 Helicobacter pylori-infected mice[J]. Am J Pathol, 171(5): 1520-1528.
[9]	Hanby AM, Poulsom R, Singh S, Elia G, Jeffery RE, Wright NA. 1993. Spasmolytic polypeptide is a major antral peptide: distribution of the trefoil peptides human spasmolytic polypeptide and pS2 in the stomach[J]. Gastroenterology, 105(4): 1110-1116.
[10]	Hoying JB, Williams SK. 1996. Effects of basic fibroblast growth factor on human microvessel endothelial cell migration on collagen I correlates inversely with adhesion and is cell density dependent[J]. J Cell Physiol, 168(2): 294-304.
[11]	Kinoshita K, Taupin DR, Itoh H, Podolsky DK. 2000. Distinct pathways of cell migration and antiapoptotic response to epithelial injury: structure-function analysis of human intestinal trefoil factor[J]. Mol Cell Biol, 20(13): 4680-4690.
[12]	Klemke RL, Cai S, Giannini AL, Gallagher PJ, de Lanerolle P, Cheresh DA. 1997. Regulation of cell motility by mitogen-activated protein kinase[J]. J Cell Biol, 137(2): 481-492.
[13]	Lalani EN, Williams R, Jayaram Y, Gilbert C, Chaudhary KS, Siu LS, Koumarianou A, Playford R, Stamp GW. 1999. Trefoil factor-2, human spasmolytic polypeptide, promotes branching morphogenesis in MCF-7 cells[J]. Lab Invest, 79(5): 537-546.
[14]	LaVallie ER, DiBlasio EA, Kovacic S, Grant KL, Schendel PF, McCoy JM. 1993. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm[J]. Nat Biotechnol, 11(2): 187-193.
[15]	Liu SB, He YY, Zhang Y, Lee WH, Qian JQ, Lai R, Jin Y. 2008. A novel non-lens βγ-crystallin and trefoil factor complex from amphibian skin and its functional implications[J]. PLoS One, 3(3): e1770.
[16]	Oertel M, Graness A, Thim L, Buhling F, Kalbacher H, Hoffmann W. 2001. Trefoil factor family-peptides promote migration of human bronchial epithelial cells: synergistic effect with epidermal growth factor[J]. Am J Respir Cell Mol Biol, 25(4): 418-424.
[17]	Perry JK, Kannan N, Grandison PM, Mitchell MD, Lobie PE. 2008. Are trefoil factors oncogenic?[J]. Trends Endocrinol Metab, 19(2): 74-81.
[18]	Poulsen SS, Thulesen J, Christensen L, Nex E, Thim L. 1999. Metabolism of oral trefoil factor 2 (TFF2) and the effect of oral and parenteral TFF2 on gastric and duodenal ulcer healing in the rat[J]. Gut, 45(4): 516-522.
[19]	Rosler S, Haase T, Claassen H, Schulze U, Schicht M, Riemann D, Brandt J, Wohlrab D, Müller-Hilke B, Goldring MB, Sel S, Varoga D, Garreis F, Paulsen FP. 2010. Trefoil factor 3 is induced during degenerative and inflammatory joint disease, activates matrix metalloproteinases, and enhances apoptosis of articular cartilage chondrocytes[J]. Arthritis Rheum, 62(3): 815-825.
[20]	Sands BE, Podolsky DK. 1996. The trefoil peptide family[J]. Annu Rev Physiol, 58(1): 253-273.
[21]	Siu LS, Romanska H, Abel PD, Baus-Loncar M, Kayademir T, Stamp GWH, Lalani el N. 2004. TFF2 (trefoil family factor2) inhibits apoptosis in breast and colorectal cancer cell lines[J]. Peptides, 25(5): 855-863.
[22]	Stewart EJ, Åslund F, Beckwith J. 1998. Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins[J]. EMBO J, 17(19): 5543-5550.
[23]	Storesund T, Hayashi K, Kolltveit KM, Bryne M, Schenck K. 2008. Salivary trefoil factor 3 enhances migration of oral keratinocytes[J]. Eur J Oral Sci, 116(2): 135-140.
[24]	Suemori S, Lynch-Devaney K, Podolsky DK. 1991. Identification and characterization of rat intestinal trefoil factor: tissue-and cell-specific member of the trefoil protein family[J]. Proc Natl Acad Sci USA, 88(24): 11017-11021.
[25]	Sun Y, Wu W, Wang L, Liang GP, Zhang Y, Lv SJ, Wang Z, Wang SL, Peng X. 2010. Overexpression of hTFF2 in the pET system and its in vitro pharmacological characterization[J]. Biomed Pharmacother, 64(5): 343-347.
[26]	Taupin D, Podolsky DK. 1999. Mitogen-activated protein kinase activation regulates intestinal epithelial differentiation[J]. Gastroenterology, 116(5): 1072-1080.
[27]	Taupin D, Podolsky DK. 2003. Trefoil factors: initiators of mucosal healing[J]. Nat Rev Mol Cell Biol, 4(9): 721-732.
[28]	Taupin DR, Kinoshita K, Podolsky DK. 2000. Intestinal trefoil factor confers colonic epithelial resistance to apoptosis[J]. Proc Natl Acad Sci USA, 97(2): 799-804.
[29]	Thim L. 1989. A new family of growth factor-like peptides. ’Trefoil’ disulphide loop structures as a common feature in breast cancer associated peptide (pS2), pancreatic spasmolytic polypeptide (PSP), and frog skin peptides (spasmolysins)[J]. FEBS Lett, 250(1): 85-90.
[30]	Thim L, Norris K, Norris F, Nielsen PF, Bj rn SE, Christensen M, Petersen J. 1993. Purification and characterization of the trefoil peptide human spasmolytic polypeptide (hSP) produced in yeast[J]. FEBS Lett, 318(3): 345-352.
[31]	Wong WM, Poulsom R, Wright NA. 1999. Trefoil peptides[J]. Gut, 44(6): 890-895.
[32]	Yu GY, Zhang Y, Xiang Y, Jiang P, Chen ZM, Lee W, Zhang Y. 2010. Cell migration-promoting and apoptosis-inhibiting activities of Bm-TFF2 require distinct structure basis[J]. Biochem Biophys Res Commun, 400(4): 724-728.

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人TFF2基因的原核表达与纯化

doi: 10.3724/SP.J.1141.2012.02144

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Bacterial expression and purification of biologically active human TFF2

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人TFF2基因的原核表达与纯化

doi: 10.3724/SP.J.1141.2012.02144

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Bacterial expression and purification of biologically active human TFF2

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