New eGFP Mutant with Intact C- and N-Termini and Affinity for Ni 2+

A. G. Tarabarova; Тарабарова А. Г.; M. S. Yurkova; Юркова М. С.; A. N. Fedorov; Федоров А. Н.

doi:10.31857/S0555109923060193

New eGFP Mutant with Intact C- and N-Termini and Affinity for Ni²⁺

Authors: Tarabarova A.G.¹, Yurkova M.S.¹, Fedorov A.N.¹
Affiliations:
1. Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences
Issue: Vol 59, No 6 (2023)
Pages: 614-621
Section: Articles
URL: https://journals.rcsi.science/0555-1099/article/view/232499
DOI: https://doi.org/10.31857/S0555109923060193
EDN: https://elibrary.ru/CXYUQZ
ID: 232499

Cite item

Full Text

Open Access
Restricted Access

Access granted
Restricted Access

Subscription Access

Abstract
About the authors
References
Supplementary files
Statistics

Abstract

The green fluorescent protein GFP has long been used in research practice as a molecular tool. It is often used as a fusion partner. To create fusion constructs, target molecules are attached to the N- or C-terminus of GFP. On the other hand, the N- or C-termini of GFP required to create fusion constructs are also used to attach affinity tags that is greatly facilitating purification. Simultaneous introduction of affinity tag and GFP to both or the same end of GFP can create steric hindrances both in the process of biosynthetic folding of the construct and in its affinity purification. This work is devoted to the production of GFP with a His-tag introduced into the polypeptide chain. This work resulted in eGFP157_7H protein with an embedded His-tag and free N- and C-termini to create fusion proteins. The added His-tag will allow purification of the construct with GFP by metal-chelated affinity chromatography under native conditions. The resulting eGFP157_7H variant retained the original fluorescent properties completely similar to those of wild-type eGFP.

Keywords

GFP, internal His-tag, IMAC, protein purification in native condition

References

Heger T., Stock C., Laursen M. J., Habeck M., Dieudonné T., Nissen P. In: Methods in Molecular Biology. Advanced Methods in Structural Biology. / Ed. Â. Sousa, L. Passarinha. N.Y.: Springer US, 2023. P. 171–186.
Le Bail A., Schulmeister S., Perroud P.-F., Ntefidou M., Rensing S.A., Kost B. // Front. Plant Sci. 2019. V. 10. P. 456.
Xiang X., Li C., Chen X., Dou H., Li Y., Zhang X., Luo Y., Xiang X. // Methods in Mol. Biol. 2019. P. 255–269. https://doi.org/10.1007/978-1-4939-9170-9_16
Nakamura S., Fu N., Kondo K., Wakabayashi K.-I., Hisabori T., Sugiura K. // J. Biol. Chem. 2021. V. 296. P. 100134. https://doi.org/10.1074/jbc.RA120.016847
Barnard E., Timson D.J. In: Methods in Molecular Biology. Molecular and Cell Biology Methods for Fungi. / Ed. A. Sharon. Totowa. N.J.: Humana Press, 2010. V. 638. P. 303–317. https://doi.org/10.1007/978-1-60761-611-5_23
Pedelacq J.-D., Waldo G.S., Cabantous S. // Methods Mol. Biol. 2019. V. 2025. P. 423–437.
Alam S.R., Mahadevan M.S., Periasamy A. // Current Protocols. 2023. V. 3. P. e689. https://doi.org/10.1002/cpz1.689
Nilsson J., Ståhl S., Lundeberg J., Uhlén M., Nygren P.-åke // Protein Exp. Purif. 1997. V. 11. P. 1–16.
Booth W.T., Schlachter C.R., Pote S., Ussin N., Mank N.J., Klapper V. et al. // ACS Omega. 2018. V. 3. P. 760–768.
Hochuli E. // J. Chromatogr. 1988. V. 444. P. 293–302.
Knecht S., Ricklin D., Eberle A. N., Ernst B. // J. Mol. Recognit. 2009. V. 22. P. 270–279. https://doi.org/10.1002/jmr.941
Chung Y.H., Volckaert B.A., Steinmetz N.F. // Bioconjugate Chem. 2023. V. 34. P. 269–278.
Hu Y.-C., Tsai C.-T., Chung Y.-C., Lu J.-T., Hsu J.T.-A. // Enzyme and Microb. Technol. 2003. V. 33. P. 445–452.
Jiang C., Wechuck J.B., Goins W.F., Krisky D.M., Wolfe D., Ataai M.M., Glorioso J.C. // J. Virology. 2004. V. 78. № 17. P. 8994–9006. https://doi.org/10.1128/JVI.78.17.8994-9006.2004
Biswal J.K., Bisht P., Subramaniam S., Ranjan R., Sharma G.K., Pattnaik B. // Biologicals. 2015. V. 43. C. 390–398.
Ye K., Jin S., Ataai M.M., Schultz J.S., Ibeh J. // J. Virology. 2004. V. 78. P. 9820–9827.
Opitz L., Hohlweg J., Reichl U., Wolff M.W. // J. Virol. Methods. 2009. V. 161. P. 312–316.
Cheeks M.C., Kamal N., Sorrell A., Darling D., Farzaneh F., Slater N.K.H. // J. Chromatogr. A. 2009. V. 1216. P. 2705–2711.
Fan J. Xiao P., Kong D., Liu X., Meng L., An T. et al. // Vaccines. 2022. V. 10. P. 170. https://doi.org/10.3390/vaccines10020170
Paul D.M., Beuron F., Sessions R.B., Brancaccio A., Bigotti M.G. // Sci Rep. V. 6. P. 20696. https://doi.org/10.1038/srep20696
Edelheit O., Hanukoglu A., Hanukoglu I. // BMC Biotechnol. 2009. V. 9. P. 61. https://doi.org/10.1186/1472-6750-9-61
Miles A.J., Ramalli S.G., Wallace B.A. // Protein Sci. 2022. V. 31. P. 37–46.
Drew E.D., Janes R.W. // Nucleic Acids Res. 2020. V. 48. P. W17–W24. https://doi.org/10.1093/nar/gkaa296
Mamontova A.V., Shakhov A.M., Lukyanov K.A., Bogdanov A.M // Biomolecules. 2020. V. 10. № 11. P. 1547. https://doi.org/10.3390/biom10111547
Arpino J.A.J., Reddington S.C., Halliwell L.M., Rizkallah P.J., Jones D.D. // Structure. 2014. V. 22. P. 889–898.
Chiang C.-F., Okou D.T., Griffin T.B., Verret C.R., Williams M.N.V. // Arch. Biochem. Biophys. 2001. V. 394. P. 229–235.
Leibly D.J., Arbing A.M., Pashkov I., DeVore N., Waldo G.S., Terwilliger TC., Yeates T.O. // Structure. 2015. V. 23. P. 1754–1768.
Williams D.E., Williams D.E., Dolgopolova E.A., Pellechia P.J., Palukoshka A., Wilson T.J. et al. // J. Am. Chem. Soc. 2015. V. 137. P. 2223–2226.
Zimmer M. // Chem. Rev. 2002. V. 102. P. 759–782.
Hovmöller S., Zhou T., Ohlson T. // Acta Cryst D. 2002. V. 58. P. 768–776.
Pettersen E.F., Goddard T.D., Huang C.C., Couch G.S., Greenblatt D.M., Meng E.C., Ferrin T.E. // J. Comput. Chem. 2004. V. 25. P. 1605–1612.