HETEROLOGOUS EXPRESSION AND PURIFICATION OF RECOMBINANT PROAPOPTOTIC HUMAN PROTEIN SMAC/DIABLO WITH EGFP AS FUSION PARTNER

Pál  SALAMON; Ildikó  MIKLÓSSY; Beáta  ALBERT; Mónika  KORODI; Katalin  NAGY; Ildikó  BAKOS; Szabolcs  LÁNYI; Csongor-Kálmán  ORBÁN

doi:10.24193/subbchem.2017.2.26

HETEROLOGOUS EXPRESSION AND PURIFICATION OF RECOMBINANT PROAPOPTOTIC HUMAN PROTEIN SMAC/DIABLO WITH EGFP AS FUSION PARTNER

Authors

Pál SALAMON Department of Bioengineering, Faculty of Economics, Socio-Human Sciences and Engineering, Sapientia Hungarian University of Transylvania, Miercurea Ciuc; Faculty of Pharmacy, University of Medicine and Pharmacy, Târgu Mureș, Romania. Email: salamonpal@uni.sapientia.ro. https://orcid.org/0000-0002-6803-1212
Ildikó MIKLÓSSY Department of Bioengineering, Faculty of Economics, Socio-Human Sciences and Engineering, Sapientia Hungarian University of Transylvania, Miercurea Ciuc, Romania. Email: miklossyildiko@uni.sapientia.ro. https://orcid.org/0000-0002-8037-7216
Beáta ALBERT Faculty of Applied Chemistry and Materials Science, Polytechnica University, Bucharest; Department of Bioengineering, Faculty of Economics, Socio-Human Sciences and Engineering, Sapientia Hungarian University of Transylvania, Miercurea Ciuc, Romania. Email: albertbeata@uni.sapientia.ro.
Mónika KORODI Department of Bioengineering, Faculty of Economics, Socio-Human Sciences and Engineering, Sapientia Hungarian University of Transylvania, Miercurea Ciuc, Romania. Corresponding author: orbancsongor@uni.sapientia.ro. https://orcid.org/0000-0002-9481-8422
Katalin NAGY Faculty of Applied Chemistry and Materials Science, Politehnica University, Bucharest, Romania. Corresponding author: orbancsongor@uni.sapientia.ro.
Ildikó BAKOS Faculty of Applied Chemistry and Materials Science, Politehnica University, Bucharest, Romania. Corresponding author: orbancsongor@uni.sapientia.ro.
Szabolcs LÁNYI Faculty of Sciences, University of Pécs, Hungary; Department of Bioengineering, Faculty of Economics, Socio-Human Sciences and Engineering, Sapientia Hungarian University of Transylvania, Miercurea Ciuc, Romania. Corresponding author: orbancsongor@uni.sapientia.ro.
Csongor-Kálmán ORBÁN Department of Bioengineering, Faculty of Economics, Socio-Human Sciences and Engineering, Sapientia Hungarian University of Transylvania, Miercurea Ciuc, Romania. Email: orbancsongor@uni.sapientia.ro. https://orcid.org/0000-0002-7509-748X

DOI:

https://doi.org/10.24193/subbchem.2017.2.26

Keywords:

SMAC/Diablo, apoptosis, heterologous expression, EGFP fusion proteins

Abstract

New proteins as molecular targets in development of therapies are discovered every day. However, study of their interactions with other proteins or binding partners in complex cellular environments has its limits. Therefore, high-yield production of these proteins in heterologous systems is a valid necessity, while obtaining these proteins linked to suitable fluorescent markers represents a step ahead in protein-protein interaction studies and cellular or subcellular localization. In this study, we present production of human SMAC/Diablo recombinant protein with EGFP as a fusion partner. High-yield expression of the fusion protein was carried out in E. coli Rosetta™(DE3)pLysS strain, and an acceptable purity of the protein was obtained after affinity chromatography purification and gel filtration. The obtained protein can be further used in protein-protein interaction studies, whereas our method represents a cost-effective and efficient production method for EGFP-fused proteins, applicable for a number of therapeutically important polypeptides.

References

G. Martinez-Ruiz, V. Maldonado, G. Ceballos-Cancino, J. Grajeda, J. Melendez-Zajgla, Journal of experimental & clinical cancer research, 2008, 27, 48.

A.M. Verhagen, P.G. Ekert, M. Pakusch, J. Silke, L. Connolly, G. Reid, D. Vaux, Cell, 2000, 102, 43.

C. Du, M. Fang, Y. Li, L. Li, X. Wang, Cell, 2000, 102, 33.

L. Ma, Y. Huang, Z. Song, S. Feng, X. Tian, W. Du, M. Wu, Nature, 2006, 13, 2079.

E. Mastrangelo, P. Vachette, F. Cossu, F. Malvezzi, M. Bolognesi, M. Milani, Biophysical Journal, 2015, 714.

R.Y. Tsien, Annu. Rev. Biochem, 1998, 67, 509.

M. Chalfie, Y. Tu, G. Euskirchen, W.W. Ward, D.C. Prasher, Science, 1994, 263, 802-805.

W. Denk, J.H. Strickler, W. W. Webb, Science, 1990, 248, 73.

R. Yuste, Nature, 2005, 902-904.

M.A. Ehrmann, C.H. Scheyhing, R.F. Vogel, Letters in Applied Microbiology, 2001, 32, 230.

R.M. Hoffman, Laboratory Investigation, 2015, 95, 432.

J. Sun, G.H. Kelemen, J.M. Fernandez-Abalos, M.J. Bibb, Microbiology, 1999, 145, 2221.

L.A. Kelley, S. Mezulis, C.M. Yates, M.N. Wass, M.J.E. Sternberg, Nature Protocols, 2015, 10, 845.

G. Wu, J. Chai, T.L. Suber, J.W. Wu, C. Du, X. Wang, Y. Shi, Nature, 2000, 408, 1008.

A. Royant, M. Noirclerc-Savoye, Journal of Structural Biology, 2011, 174, 385.

J. Glazyrina, E. Materne, T. Dreger, D. Storm, S. Junne, T. Adams, G. Greller, P. Neubauer, Microbial Cell Factories, 2010, 9, 42.

A. Sivashanmugam, V. Murray, C. Cui, Y. Zhang, J. Wang, Q. Li, Protein Science, 2009, 18(5), 936.

Z. Lai, R. Rahim, A. Ariff, R. Mohamad, Journal of Microbiology, Biotechnology and Food Sciences, 2016, 6(3), 905-910.

STUDIA UBB CHEMIA, Volume 62 (LXII), No. 2, Tome II, June 2017

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Published

2017-06-30

How to Cite

SALAMON, P. ., MIKLÓSSY, I. ., ALBERT, B. ., KORODI, M. ., NAGY, K. ., BAKOS, I. ., … ORBÁN, C.-K. . (2017). HETEROLOGOUS EXPRESSION AND PURIFICATION OF RECOMBINANT PROAPOPTOTIC HUMAN PROTEIN SMAC/DIABLO WITH EGFP AS FUSION PARTNER. Studia Universitatis Babeș-Bolyai Chemia, 62(2), 333–345. https://doi.org/10.24193/subbchem.2017.2.26