Skip to main content

Advertisement

SpringerLink
Log in

Production of Bioactive Human PAX4 Protein from E. coli

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

Paired box 4 (PAX4) is a pivotal transcription factor involved in pancreatogenesis during embryogenesis, and in adults, it is key for β-cell proliferation and survival. Additionally, PAX4 also functions as a tumor suppressor protein in human melanomas. The present study demonstrates the production of bioactive recombinant human PAX4 transcription factor. At first, the inserts (PAX4 protein-coding sequence having tags at either ends) were cloned in an expression vector to give rise to pET28a(+)-HTN-PAX4 and pET28a(+)-PAX4-NTH genetic constructs, and these were then transformed into Escherichia coli (E. coli) for their expression. The HTN-PAX4 and PAX4-NTH fusion proteins produced were purified with a yield of ~ 3.15 mg and ~ 0.83 mg, respectively, from 1.2 L E. coli culture. Further, the secondary structure retention of the PAX4 fusion proteins and their potential to internalize the mammalian cell and its nucleus was demonstrated. The bioactivity of these fusion proteins was investigated using various assays (cell migration, cell proliferation and cell cycle assays), demonstrating it to function as a tumor suppressor protein. Thus, this macromolecule can prospectively help understand the function of human PAX4 in cellular processes, disease-specific investigations and direct cellular reprogramming.

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
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Dey C, Raina K, Haridhasapavalan KK, Thool M, Sundaravadivelu PK, Adhikari P, …, Thummer RP (2021) An overview of reprogramming approaches to derive integration-free induced pluripotent stem cells for prospective biomedical applications. Recent Adv iPSC Technol 231–287. https://doi.org/10.1016/B978-0-12-822231-7.00011-4

  2. Borgohain MP, Narayan G, Kumar HK, Dey C, Thummer RP (2018) Maximizing expression and yield of human recombinant proteins from bacterial cell factories for biomedical applications. Advances in Microbial Biotechnology, 1st edn. Apple academic press (447–486)

  3. Nagahara H, Vocero-Akbani AM, Snyder EL, Ho A, Latham DG, Lissy NA, …, Dowdy SF (1998) Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat Med 4(12):1449–1452. https://doi.org/10.1038/4042

    Article  CAS  PubMed  Google Scholar 

  4. Vargas N, Álvarez-Cubela S, Giraldo JA, Nieto M, Fort NM, Cechin S, …, Domínguez-Bendala J (2011) TAT-mediated transduction of MafA protein in Utero results in enhanced pancreatic insulin expression and changes in islet morphology. PLoS ONE 6(8). https://doi.org/10.1371/journal.pone.0022364

  5. Assenberg R, Wan PT, Geisse S, Mayr LM (2013) Advances in recombinant protein expression for use in pharmaceutical research. Curr Opin Struct Biol 23(3):393–402. https://doi.org/10.1016/j.sbi.2013.03.008

    Article  CAS  PubMed  Google Scholar 

  6. Sanchez-Garcia L, Martín L, Mangues R, Ferrer-Miralles N, Vázquez E, Villaverde A (2016) Recombinant pharmaceuticals from microbial cells: a 2015 update. Microb Cell Fact 15(1):1–7. https://doi.org/10.1186/s12934-016-0437-3

    Article  CAS  Google Scholar 

  7. Lorenzo PI, Juárez-Vicente F, Cobo-Vuilleumier N, Garcia-Dominguez M, Gauthier BR (2017) The diabetes-linked transcription factor PAX4: from gene to functional consequences. Genes 8(3):101. https://doi.org/10.3390/genes8030101

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Sosa-Pineda B (2004) The gene Pax4 is an essential regulator of pancreatic β-cell development. Mol Cells 18(3):289–294

    CAS  PubMed  Google Scholar 

  9. Brun T, Franklin I, St.-Onge L, Biason-Lauber A, Schoenle EJ, Wollheim CB, Gauthier BR (2004) The diabetes-linked transcription factor PAX4 promotes β-cell proliferation and survival in rat and human islets. J Cell Biol 167(6):1123–1135. https://doi.org/10.1083/jcb.200405148

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Sosa-pineda B, Chowdhury K, Torres M, Oliver G, Gruss P (1997) The Pax4 gene is essential for differentiation of insulin-producing β in the mammalian pancreas. Nature 386(6623):399–402. https://doi.org/10.1038/386399a0

    Article  CAS  PubMed  Google Scholar 

  11. Collombat P, Hecksher-Sørensen J, Broccoli V, Krull J, Ponte I, Mundiger T, Mansouri A (2005) The simultaneous loss of Arx and Pax4 genes promotes a somatostatin-producing cell fate specification at the expense of the α-and β-cell lineages in the mouse endocrine pancreas. Oxf Univ Press 2969–2980. https://doi.org/10.1242/dev.01870

  12. Napolitano T, Avolio F, Courtney M, Vieira A, Druelle N, Ben-Othman N, …, Collombat P (2015) Pax4 acts as a key player in pancreas development and plasticity. Seminars in Cell and Developmental Biology 44:107–114. https://doi.org/10.1016/j.semcdb.2015.08.013

    Article  CAS  PubMed  Google Scholar 

  13. Hata S, Hamada J-I, Maeda K, Murai T, Tada M, Furukawa H, …, Moriuchi T (2008) PAX4 has the potential to function as a tumor suppressor in human melanoma. Int J Oncol 33(5):1065–1071. https://doi.org/10.3892/ijo_00000095

    Article  CAS  PubMed  Google Scholar 

  14. Agrawal A, Narayan G, Gogoi R, Thummer RP (2021) Recent advances in the generation of β-cells from induced pluripotent stem cells as a potential cure for diabetes mellitus. Cell Biology and Translational Medicine, Volume 14: Stem Cells in Lineage Specific Differentiation and Disease, 1–27. https://doi.org/10.1007/5584_2021_653

  15. Narayan G, R, R. K., Thummer RP (2022) Direct reprogramming of somatic cells into induced β-cells: an overview (1–19). Springer International Publishing, Cham. https://doi.org/10.1007/5584_2022_756

    Book  Google Scholar 

  16. Thool M, Dey C, Bhattacharyya S, Sudhagar S, Thummer RP (2021) Generation of a recombinant stem cell – specific human SOX2 protein from Escherichia coli under native conditions. Mol Biotechnol 63:327–338. https://doi.org/10.1007/s12033-021-00305-y

    Article  CAS  PubMed  Google Scholar 

  17. Haridhasapavalan KK, Sundaravadivelu PK, Bhattacharyya S, Ranjan SH, Raina K, Thummer RP (2021) Generation of cell-permeant recombinant human transcription factor GATA4 from E. coli. Bioprocess Biosyst Eng 44(6):1131–1146. https://doi.org/10.1007/s00449-021-02516-8

    Article  CAS  PubMed  Google Scholar 

  18. Narayan G, Agrawal A, Joshi N, Gogoi R, Nagotu S, Thummer RP (2021) Protein production and purification of a codon-ptimized human NGN3 transcription factor from E. coli. Protein J 40(6):891–906. https://doi.org/10.1007/s10930-021-10020-x

    Article  CAS  PubMed  Google Scholar 

  19. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1–2):248–254. https://doi.org/10.1016/0003-2697(76)90527-3

    Article  CAS  PubMed  Google Scholar 

  20. Haridhasapavalan KK, Sundaravadivelu PK, Thummer RP (2020) Codon optimization, cloning, expression, purification, and secondary structure determination of human ETS2 transcription factor. Mol Biotechnol 62(10):485–494. https://doi.org/10.1007/s12033-020-00266-8

    Article  CAS  PubMed  Google Scholar 

  21. Narayan G, Kumar P, Agrawal A, Gogoi R, Nagotu S, Thummer RP (2021) Soluble expression, purification, and secondary structure determination of human PDX1 transcription factor. Protein Exp Purif 180(October 2020):105807. https://doi.org/10.1016/j.pep.2020.105807

    Article  CAS  Google Scholar 

  22. Dey C, Thool M, Bhattacharyya S, Sudhagar S, Thummer RP (2021) Generation of biologically active recombinant human OCT4 protein from E. coli. 3 Biotech 11(5):1–16. https://doi.org/10.1007/s13205-021-02758-z

    Article  Google Scholar 

  23. Wingfield PT (2015) Overview of the purification of recombinant proteins. Curr protocols protein Sci 80(1):1–6. https://doi.org/10.1002/0471140864.ps0601s80

    Article  Google Scholar 

  24. Ryan BJ, Henehan GT (2013) Overview of approaches to preventing and avoiding proteolysis during expression and purification of proteins. Curr protocols protein Sci 71(1):5–25. https://doi.org/10.1002/0471140864.ps0525s71

    Article  Google Scholar 

  25. Maertens, B., Spriestersbach, A., Von Groll, U., Roth, U., Kubicek, J., Gerrits, M., ? Schäfer, F. (2010). Gene optimization mechanisms: A multi-gene study reveals a high success rate of full-length human proteins expressed in Escherichia coli. Protein Science, 19(7), 1312–1326. https://doi.org/10.1002/pro.408

  26. Kurien BT, Scofield RH (2012) Extraction of proteins from gels: a brief review. Protein electrophoresis: methods and protocols 403–405. https://doi.org/10.1007/978-1-61779-821-4_33

  27. Smith, J. P., Ralbovsky, N. M., Lauro, M. L., Hoyt, E., Guetschow, E. D., Wang, F., ? others. (2022). Quantitation and speciation of residual protein within active pharmaceutical ingredients using image analysis with SDS-PAGE. Journal of Pharmaceutical and Biomedical Analysis, 207, 114393. https://doi.org/10.1016/j.jpba.2021.114393

  28. Kalousová A, Beneš V, Pačes J, Pačes V, Kozmik Z (1999) DNA binding and transactivating properties of the paired and homeobox protein pax4. Biochem Biophys Res Commun 259(3):510–518. https://doi.org/10.1006/bbrc.1999.0809

    Article  PubMed  Google Scholar 

  29. Lu J, Li G, Lan MS, Zhang S, Fan W, Wang H, Lu D (2007) Pax4 paired domain mediates direct protein transduction into mammalian cells. Endocrinology 148(11):5558–5565. https://doi.org/10.1210/en.2007-0636

    Article  CAS  PubMed  Google Scholar 

  30. Greenfield NJ (2006) Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 1(6):2876–2890. https://doi.org/10.1038/nprot.2006.202

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Bosnali M, Edenhofer F (2008) Generation of transducible versions of transcription factors Oct4 and Sox2, 389(7), 851–861. https://doi.org/10.1515/BC.2008.106

  32. Noguchi H, Xu G, Matsumoto S, Kaneto H, Kobayashi N, Bonner-Weir S, Hayashi S (2006) Induction of pancreatic stem/progenitor cells into insulin-producing cells by adenoviral-mediated gene transfer technology. Cell Transplant 15(10):929–938. https://doi.org/10.3727/000000006783981431

    Article  PubMed  Google Scholar 

  33. Grada A, Otero-Vinas M, Prieto-Castrillo F, Obagi Z, Falanga V (2017) Research Techniques made simple: analysis of collective cell Migration using the Wound Healing Assay. J Invest Dermatology 137(2):e11–e16. https://doi.org/10.1016/j.jid.2016.11.020

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The work was funded by the Ministry of Science and Technology, Government of India (Ref. No.: BT/COE/34/SP28408/2018) under NECBH outreach program hosted by IIT Guwahati sponsored by DBT. We also thank Prof. Siddhartha Sankar Ghosh, Department of Biosciences and Bioengineering and Centre for Nanotechnology, IIT Guwahati, for their assistance in flow cytometry.

Author information

Authors and Affiliations

  1. Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India

    Gloria Narayan, Akriti Agrawal & Rajkumar P Thummer

  2. Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India

    Plaboni Sen

  3. Organelle Biology and Cellular Ageing Lab, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India

    Shirisha Nagotu

Authors
  1. Gloria Narayan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Akriti Agrawal
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Plaboni Sen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shirisha Nagotu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rajkumar P Thummer
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

GN conceptualized and designed the study, performed the experiments, collected, assembled and analyzed the data, wrote the manuscript and approved the final draft of the manuscript; AA and PS performed the experiments, analyzed the data and approved the final draft of the manuscript; SN analyzed and interpreted the data and approved the final draft of the manuscript; and RPT conceptualized and designed the study, analyzed the data, supervised the experiments, wrote the manuscript, approved the final draft of the manuscript and financial support.

Corresponding author

Correspondence to Rajkumar P Thummer.

Ethics declarations

Competing Interests

The authors declare no competing interests.

Ethical Statement

Not applicable.

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.

Figure S1 Clonal selection to achieve maximal soluble expression.

Multiple transformed colonies harboring either HTN-PAX4 or PAX4-NTH constructs were screened to obtain maximal expression of full length PAX4 protein. (A) 12% SDS-PAGE analysis for multiple HTN-PAX4 and PAX-NTH clones (B) Representative western blots for data of (A). C (1–4), Clone (1–4); NT, HTN-PAX4; CT, PAX4-NTH; L, Lysate; S, Supernatant; kDa, kilodalton; α-His Ab, anti-Histidine antibody

Supplementary Material 2

Supplementary Material 3

Supplementary Material 4

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Narayan, G., Agrawal, A., Sen, P. et al. Production of Bioactive Human PAX4 Protein from E. coli. Protein J 42, 766–777 (2023). https://doi.org/10.1007/s10930-023-10143-3

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10930-023-10143-3

Keywords

Search

Navigation