Skip to main content

Advertisement

SpringerLink
Log in

Recombinant Expression and Bioactivity Characterization of TAT-Fused Thymosin β10

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

Thymosin beta 10 (TB10) is one of the common members among beta-thymosins. Human TB10 is reported to play a role in anti-angiogenesis and inhibition of cell migration during the tumorigenesis or metastasis of some certain cancers. Thus, it would be a potent clinical agent. In the present study, the coding sequence of TB10 was optimized based on the codon preference of Escherichia coli and cloned to pET28a (+) by chemical synthesis and molecular cloning methods. The recombinant protein was highly expressed employing E. coli expressing system and purified by a simple step of Ni2+ affinity chromatography. The TEV proteinase recognition site was inserted in the His6-tag and the target protein for easy removal of the His6-tag. To improve the biological activity of TB10, the transactivator of transcription (TAT) short peptide, a transduction domain, was added to the N-terminus of TB10. About 14.3 mg of the recombinant TB10 proteins was obtained from 1 L bacterial culture. The functional analyses demonstrated that the recombinant TB10 proteins displayed the distinct inhibition on angiogenesis by chick embryo chorioallantoic membrane assay and endothelial cell migration by wound healing assay. The TAT-fused TB10 even had stronger effects, probably due to the better transduction into the cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

CAM:

Chick chorioallantoic membrane

E. coli :

Escherichia coli

EK:

Enterokinase

HUVEC:

Human umbilical vein endothelial cell

HIV:

Human immunodeficiency virus

Ras:

Rat sarcoma

RE:

Restriction endonuclease

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TAT:

Transactivator of transcription

TB4:

Thymosin β4

TB10:

Thymosin β10

TEV:

Tobacco etch virus protease

VEGF:

Vascular endothelial growth factor

References

  1. Sribenja S, Li M, Wongkham S et al (2009) Advances in thymosin β10 research: differential expression, molecular mechanisms, and clinical implications in cancer and other conditions. Cancer Invest 27:1016–1022. https://doi.org/10.3109/07357900902849640

    Article  CAS  PubMed  Google Scholar 

  2. Erickson-Viitanen S, Ruggieri S, Natalini P, Horecker BL (1983) Thymosin beta 10, a new analog of thymosin beta 4 in mammalian tissues. Arch Biochem Biophys 225:407–413

    Article  CAS  Google Scholar 

  3. Vassiliadou I, Leondiadis L, Ferderigos N et al (1999) Investigation of the epitopic structure of thymosin beta10 by epitope mapping experiments. Peptides 20:411–414

    Article  CAS  Google Scholar 

  4. Hannappel E (2007) beta-Thymosins. Ann NY Acad Sci 1112:21–37. https://doi.org/10.1196/annals.1415.018

    Article  CAS  PubMed  Google Scholar 

  5. Mannherz HG, Hannappel E (2009) The β-thymosins: intracellular and extracellular activities of a versatile actin binding protein family. Cell Motil Cytoskelet 66:839–851. https://doi.org/10.1002/cm.20371

    Article  CAS  Google Scholar 

  6. Tsitsiloni OE, Yialouris PP, Echner H et al (1992) Evidence for the extranuclear localization of thymosins in thymus. Experientia 48:398–402

    Article  CAS  Google Scholar 

  7. Yu FX, Lin SC, Morrison-Bogorad M et al (1993) Thymosin beta 10 and thymosin beta 4 are both actin monomer sequestering proteins. J Biol Chem 268:502–509

    CAS  PubMed  Google Scholar 

  8. Sribenja S, Wongkham S, Wongkham C et al (2013) Roles and mechanisms of β-thymosins in cell migration and cancer metastasis: an update. Cancer Invest 31:103–110. https://doi.org/10.3109/07357907.2012.756111

    Article  CAS  PubMed  Google Scholar 

  9. Zhang X, Ren D, Guo L et al (2017) Thymosin beta 10 is a key regulator of tumorigenesis and metastasis and a novel serum marker in breast cancer. Breast Cancer Res 19:15. https://doi.org/10.1186/s13058-016-0785-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zhang X-J, Su Y-R, Liu D et al (2014) Thymosin beta 10 correlates with lymph node metastases of papillary thyroid carcinoma. J Surg Res 192:487–493. https://doi.org/10.1016/j.jss.2014.05.066

    Article  CAS  PubMed  Google Scholar 

  11. Theunissen W, Fanni D, Nemolato S et al (2014) Thymosin beta 4 and thymosin beta 10 expression in hepatocellular carcinoma. Eur J Histochem 58:2242. https://doi.org/10.4081/ejh.2014.2242

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Santelli G, Califano D, Chiappetta G et al (1999) Thymosin beta-10 gene overexpression is a general event in human carcinogenesis. Am J Pathol 155:799–804

    Article  CAS  Google Scholar 

  13. Sribenja S, Sawanyawisuth K, Kraiklang R et al (2013) Suppression of thymosin β10 increases cell migration and metastasis of cholangiocarcinoma. BMC Cancer 13:430. https://doi.org/10.1186/1471-2407-13-430

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lee S-H, Zhang W, Choi J-J et al (2001) Overexpression of the thymosin β-10 gene in human ovarian cancer cells disrupts F-actin stress fiber and leads to apoptosis. Oncogene 20:6700–6706. https://doi.org/10.1038/sj.onc.1204683

    Article  CAS  PubMed  Google Scholar 

  15. Kim Y-C, Kim B-G, Lee J-H (2012) Thymosin β10 expression driven by the human TERT promoter induces ovarian cancer-specific apoptosis through ROS production. PLoS ONE 7:e35399. https://doi.org/10.1371/journal.pone.0035399

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lee S-H, Son MJ, Oh S-H et al (2005) Thymosin {beta}(10) inhibits angiogenesis and tumor growth by interfering with Ras function. Cancer Res 65:137–148

    CAS  PubMed  Google Scholar 

  17. Mu H, Ohashi R, Yang H et al (2006) Thymosin β10 inhibits cell migration and capillary-like tube formation of human coronary artery endothelial cells. Cell Motil Cytoskelet 63:222–230. https://doi.org/10.1002/cm.20117

    Article  CAS  Google Scholar 

  18. Zhang T, Li X, Yu W et al (2009) Overexpression of thymosin beta-10 inhibits VEGF mRNA expression, autocrine VEGF protein production, and tube formation in hypoxia-induced monkey choroid-retinal endothelial cells. Ophthalmic Res 41:36–43. https://doi.org/10.1159/000163220

    Article  CAS  PubMed  Google Scholar 

  19. Koutrafouri V, Leondiadis L, Avgoustakis K et al (2001) Effect of thymosin peptides on the chick chorioallantoic membrane angiogenesis model. Biochim Biophys Acta 1568:60–66

    Article  CAS  Google Scholar 

  20. Becker-Hapak M, McAllister SS, Dowdy SF (2001) TAT-mediated protein transduction into mammalian cells. Methods 24:247–256. https://doi.org/10.1006/meth.2001.1186

    Article  CAS  PubMed  Google Scholar 

  21. Nagahara H, Vocero-Akbani AM, Snyder EL et al (1998) Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat Med 4:1449–1452. https://doi.org/10.1038/4042

    Article  CAS  PubMed  Google Scholar 

  22. Fang L, Jia K-Z, Tang Y-L et al (2007) An improved strategy for high-level production of TEV protease in Escherichia coli and its purification and characterization. Protein Expr Purif 51:102–109. https://doi.org/10.1016/j.pep.2006.07.003

    Article  CAS  PubMed  Google Scholar 

  23. Grosjean H, Fiers W (1982) Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes. Gene 18:199–209

    Article  CAS  Google Scholar 

  24. Sun Q-M, Cao L, Fang L et al (2005) Expression, purification of human vasostatin120–180 in Escherichia coli, and its anti-angiogenic characterization. Protein Expr Purif 39:288–295. https://doi.org/10.1016/j.pep.2004.10.024

    Article  CAS  PubMed  Google Scholar 

  25. Pei G, Lan Y, Lu W et al (2018) The function of FAK/CCDC80/E-cadherin pathway in the regulation of B16F10 cell migration. Oncol Lett 16:4761–4767. https://doi.org/10.3892/ol.2018.9159

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Grada A, Otero-Vinas M, Prieto-Castrillo F et al (2017) Research techniques made simple: analysis of collective cell migration using the wound healing assay. J Invest Dermatol 137:e11–e16. https://doi.org/10.1016/j.jid.2016.11.020

    Article  CAS  PubMed  Google Scholar 

  27. Rho SB, Chun T, Lee S-H et al (2004) The interaction between E-tropomodulin and thymosin beta-10 rescues tumor cells from thymosin beta-10 mediated apoptosis by restoring actin architecture. FEBS Lett 557:57–63

    Article  CAS  Google Scholar 

  28. Li W, Song L, Wu S et al (2011) Expression, purification and characterization of a novel soluble human thymosin alpha1 concatemer exhibited a stronger stimulation on mice lymphocytes proliferation and higher anti-tumor activity. Int J Biol Sci 7:618–628

    Article  Google Scholar 

  29. Xue X-C, Yan Z, Li W-N et al (2013) Construction, expression, and characterization of thymosin alpha 1 tandem repeats in Escherichia coli. Biomed Res Int 2013:720285. https://doi.org/10.1155/2013/720285

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Wang F, Yu T, Zheng H, Lao X (2018) Thymosin Alpha1-Fc modulates the immune system and down-regulates the progression of melanoma and breast cancer with a prolonged half-life. Sci Rep 8:12351. https://doi.org/10.1038/s41598-018-30956-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Cha H-J, Jeong M-J, Kleinman HK (2003) Role of thymosin beta 4 in tumor metastasis and angiogenesis. J Natl Cancer Inst 95:1674–1680. https://doi.org/10.1093/jnci/djg100

    Article  CAS  PubMed  Google Scholar 

  32. Malinda KM, Sidhu GS, Banaudha KK et al (1998) Thymosin alpha 1 stimulates endothelial cell migration, angiogenesis, and wound healing. J Immunol 160:1001–1006

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors are grateful to Dr. Hua Zi-Chun for TEV and valuable suggestions. This work was supported by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, the Fujian Provincial Nature Science Foundation (2015J05052), the Chinese National Nature Sciences Foundation (31501232), the FAFU Science Fund for Distinguished Young Scholars (xjq201629), the FAFU Science grant for innovation (CXZX2018119), the Education and research projects for young teachers in Fujian provincial Education Hall (JT180137), and the science & technology innovation fund of Fujian Agriculture and Forestry University (CXZX2017512, KFA17583A).

Author information

Authors and Affiliations

  1. The Ministry of Education Key Laboratory of Biopesticide and Chemical Biology, School of Life Science, Fujian Agriculture and Forestry University, Fuzhou, China

    Kunzhi Jia & Ming Lin

  2. Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Ministry of Education/College of Crop Science, Fujian Agriculture and Forestry University, Jinshan, Fuzhou, 350002, China

    Defeng Kong & Qi Jia

Authors
  1. Kunzhi Jia
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ming Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Defeng Kong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qi Jia
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Qi Jia.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 1676 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jia, K., Lin, M., Kong, D. et al. Recombinant Expression and Bioactivity Characterization of TAT-Fused Thymosin β10. Protein J 38, 675–682 (2019). https://doi.org/10.1007/s10930-019-09855-2

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10930-019-09855-2

Keywords

Search

Navigation