Production of a Granulysin-Based, Tn-Targeted Cytolytic Immunotoxin Using Pulsed Electric Field Technology
Abstract
:1. Introduction
2. Results
2.1. Design of Plasmids for the Production of Recombinant SM3-Granulysin Immunotoxin
2.2. Production and Purification of the Recombinant Proteins in Pichia Pastoris. Obtention of the Intracellular Protein by Pulsed Electric Field Technology
2.3. Assays of Thermal Denaturation
2.4. Immunotoxin Binding to the Tn Antigen Expressed on the Cell Surface
2.5. In Vitro Cytotoxic Effect of GRNLY, SM3GRNLY and iSM3GRNLY
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Bacterial Strains, Plasmids, and Culture Conditions
4.3. Expression and Purification of Extracellular Recombinant Granulysin and SM3-Granulysin Immunotoxin in Pichia Pastoris
4.4. Expression and Purification of Intracellular Recombinant SM3-Granulysin Immunotoxin from Pichia Pastoris by Pulsed Electric Field Technology
4.5. Protein Stability Determinations
4.6. Flow Cytometry Analysis of Binding of SM3GRNLY or iSM3GRNLY to the Tn Antigen
4.7. Cytotoxicity Assays
4.8. Statistical Analysis
5. Patents
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
- Clayberger, C.; Krensky, A.M. Granulysin. Curr. Opin. Immunol. 2003, 15, 560–565. [Google Scholar] [CrossRef]
- Peña, S.V.; Hanson, D.A.; Carr, B.A.; Goralski, T.J.; Krensky, A.M. Processing, subcellular localization, and function of 519 (granulysin), a human late T cell activation molecule with homology to small, lytic, granule proteins. J. Immunol. 1997, 158, 2680–2688. [Google Scholar] [PubMed]
- Stenger, S.; Hanson, D.A.; Teitelbaum, R.; Dewan, P.; Niazi, K.R.; Froelich, C.J.; Ganz, T.; Thoma-Uszynski, S.; Melián, A.; Bogdan, C.; et al. An antimicrobial activity of cytolytic T cells mediated by granulysin. Science 1998, 282, 121–125. [Google Scholar] [CrossRef] [Green Version]
- Aporta, A.; Catalán, E.; Galán-Malo, P.; Ramírez-Labrada, A.; Pérez, M.; Azaceta, G.; Palomera, L.; Naval, J.; Marzo, I.; Pardo, J.; et al. Granulysin induces apoptotic cell death and cleavage of the autophagy regulator Atg5 in human hematological tumors. Biochem. Pharmacol. 2014, 87, 410–423. [Google Scholar] [CrossRef] [PubMed]
- Gamen, S.; Hanson, D.A.; Kaspar, A.; Naval, J.; Krensky, A.M.; Anel, A. Granulysin-induced apoptosis. I. Involvement of at least two distinct pathways. J. Immunol. 1998, 161, 1758–1764. [Google Scholar] [PubMed]
- Kaspar, A.A.; Okada, S.; Kumar, J.; Poulain, F.R.; Drouvalakis, K.A.; Kelekar, A.; Hanson, D.A.; Kluck, R.M.; Hitoshi, Y.; Johnson, D.E.; et al. A distinct pathway of cell-mediated apoptosis initiated by granulysin. J. Immunol. 2001, 167, 350–356. [Google Scholar] [CrossRef]
- Pardo, J.; Pérez-Galán, P.; Gamen, S.; Marzo, I.; Monleón, I.; Kaspar, A.A.; Susín, S.A.; Kroemer, G.; Krensky, A.M.; Naval, J.; et al. A role of the mitochondrial apoptosis-inducing factor (AIF) in granulysin-induced apoptosis. J. Immunol. 2001, 167, 1222–1229. [Google Scholar] [CrossRef] [Green Version]
- Al-Wasaby, S.; de Miguel, D.; Aporta, A.; Naval, J.; Conde, B.; Martínez-Lostao, L.; Anel, A. In vivo potential of recombinant granulysin against human tumors. OncoImmunology 2015, 4, e1036213. [Google Scholar] [CrossRef] [Green Version]
- Blanco-Toribio, A.; Lacadena, J.; Nuñez-Prado, N.; Álvarez-Cienfuegos, A.; Villate, M.; Compte, M.; Sanz, L.; Blanco, F.; Álvarez-Vallina, L. Efficient production of single-chain fragment variable-based N-terminal trimerbodies in Pichia pastoris. Microb. Cell. Factories 2014, 13, 116. [Google Scholar] [CrossRef] [Green Version]
- Ibáñez-Pérez, R.; Guerrero-Ochoa, P.; Al-Wasaby, S.; Navarro, R.; Tapia-Galisteo, A.; De Miguel, D.; Gonzalo, O.; Conde, B.; Martínez-Lostao, L.; Hurtado-Guerrero, R.; et al. Anti-tumoral potential of a human granulysinbased, CEA-targeted cytolytic immunotoxin. OncoImmunology 2019, 8, 1641392. [Google Scholar] [CrossRef] [Green Version]
- Ju, T.; Otto, V.; Cummings, R. The Tn antigen-structural simplicity and biological complexity. Angew. Chem. Int. Ed. Engl. 2011, 50, 1770–1791. [Google Scholar] [CrossRef] [PubMed]
- Martínez-Sáez, N.; Castro-López, J.; Valero-González, J.; Madariaga, D.; Compañón, I.; Somovilla, V.; Salvadó, M.; Asensio, J.; Jiménez-Barbero, J.; Avenoza, A.; et al. Deciphering the Non-Equivalence of Serine and Threonine O-Glycosylation Points: Implications for Molecular Recognition of the Tn Antigen by an anti-MUC1 Antibody. Angew. Chem. Int. Ed. Engl. 2015, 54, 9830–9834. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ahmad, M.; Hirz, M.; Pichler, H.; Schwab, H. Protein expression in Pichia pastoris: Recent achievements and perspectives for heterologous protein production. Appl. Microbiol. Biotechnol. 2014, 98, 5301–5317. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kotnik, T.; Frey, W.; Sack, M.; Meglič, S.; Peterka, M.; Miklavčič, D. Electroporation-based applications in biotechnology. Trends Biotechnol. 2015, 33, 480–488. [Google Scholar] [CrossRef]
- Martínez, J.; Delso, C.; Álvarez, I.; Raso, J. Pulsed Electric Field-assisted extraction of valuable compounds from microorganisms. Compr. Rev. Food Sci. Food Saf. 2020, 2020, 1–23. [Google Scholar] [CrossRef]
- Guo, Y.; Luan, G.; Shen, G.; Wu, L.; Jia, H.; Zhong, Y.; Li, R.; Li, G.; Shen, Y.; Sun, J.; et al. Production and characterization of recombinant 9 and 15 kDa granulysin by fed-batch fermentation in Pichia pastoris. Appl. Microbiol. Biotechnol. 2013, 97, 7669–7677. [Google Scholar] [CrossRef] [PubMed]
- Raso, J.; Condón, S.; Alvarez, I. Non-thermal processing: Pulsed electric field. In Encyclopedia of Food Microbiology, 2nd ed.; Elsevier/Academic Press: Cambridge, MA, USA, 2014; pp. 966–973. [Google Scholar]
- Posey, A.; Schwab, R.; Boesteanu, A.; Steentof, C.; Mandel, U.; Engels, B.; Stone, J.; Madsen, T.; Schreiber, K.; Haines, K.; et al. Engineered CAR T Cells Targeting the Cancer-Associated Tn-Glycoform of the Membrane Mucin MUC1 Control Adenocarcinoma. Immunity 2016, 44, 1444–1454. [Google Scholar] [CrossRef] [Green Version]
- Wilkie, S.; Picco, G.; Foster, J.; Davies, D.; Julien, S.; Cooper, L.; Arif, S.; Mather, S.; Taylor-Papadimitriou, J.; Burchell, J.; et al. Retargeting of human T cells to tumor-associated MUC1: The evolution of a chimeric antigen receptor. J. Immunol. 2008, 180, 4901–4909. [Google Scholar] [CrossRef] [Green Version]
- Yang, J.; Cai, H.; Liu, J.; Zeng, M.; Chen, J.; Cheng, Q.; Zhang, L. Controlling AOX1 promoter strength in Pichia pastoris by manipulating poly (dA:dT) tracts. Sci. Rep. 2018, 8, 1401. [Google Scholar] [CrossRef] [Green Version]
- Prabhu, A.; Veeranki, V. Metabolic engineering of Pichia pastoris GS115 for enhanced pentose phosphate pathway (PPP) flux toward recombinant human interferon gamma (hIFN-γ) production. Mol. Biol. Rep. 2018, 45, 961–972. [Google Scholar] [CrossRef]
- Meglič, S. Pulsed Electric Fields-Assisted Extraction of Molecules from Bacterial and Yeast Cells. In Handbook of Electroporation, 1st ed.; SpringerLink: Cham, Switzerland, 2017; pp. 2253–2270. [Google Scholar]
- Ganeva, V.; Galutzov, B.; Angelova, B.; Suckow, M. Human Ferritin Heavy Chain Expressed in Hansenula polymorpha. Appl. Biochem. Biotechnol. 2018, 184, 1286–1307. [Google Scholar] [CrossRef] [PubMed]
- Ganeva, V.; Stefanova, D.; Angelova, B.; Galutzov, B.; Velasco, I.; Arévalo-Rodríguez, M. Electroinduced release of recombinant β-galactosidase from Saccharomyces cerevisiae. J. Biotechnol. 2015, 211, 12–19. [Google Scholar] [CrossRef] [PubMed]
- Croce, M.; Colussi, A.; Price, M.; Segal-Eiras, A. Identification and characterization of different subpopulations in a human lung adenocarcinoma cell line (A549). Pathol. Oncol. Res. 1999, 5, 197–204. [Google Scholar] [CrossRef]
- Osinaga, E.; Pancino, G.; Porchet, N.; Berois, N.; De Cremoux, P.; Mistro, D.; Aubert, J.; Calvo, F.; Roseto, A. Analysis of a heterogeneous group of human breast carcinoma associated glycoproteins bearing the Tn determinant. Breast Cancer Res. Treat. 1994, 32, 139–152. [Google Scholar] [CrossRef] [PubMed]
- Kreitman, R.; Dearden, C.; Zinzani, P.; Delgado, J.; Karlin, L.; Robak, T.; Gladstone, D.; le Coutre, P.; Dietrich, S.; Gotic, M.; et al. Moxetumomab pasudotox in relapsed/refractory hairy cell leukemia. Leukemia 2018, 32, 1768–1777. [Google Scholar] [CrossRef]
- Mazor, R.; King, E.; Pastan, I. Strategies to Reduce the Immunogenicity of Recombinant Immunotoxins. Am. J. Pathol. 2018, 188, 1736–1743. [Google Scholar] [CrossRef]
- Mazor, R.; Onda, M.; Pastan, I. Immunogenicity of therapeutic recombinant immunotoxins. Immunol. Rev. 2016, 270, 152–164. [Google Scholar] [CrossRef] [Green Version]
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Guerrero-Ochoa, P.; Aguilar-Machado, D.; Ibáñez-Pérez, R.; Macías-León, J.; Hurtado-Guerrero, R.; Raso, J.; Anel, A. Production of a Granulysin-Based, Tn-Targeted Cytolytic Immunotoxin Using Pulsed Electric Field Technology. Int. J. Mol. Sci. 2020, 21, 6165. https://doi.org/10.3390/ijms21176165
Guerrero-Ochoa P, Aguilar-Machado D, Ibáñez-Pérez R, Macías-León J, Hurtado-Guerrero R, Raso J, Anel A. Production of a Granulysin-Based, Tn-Targeted Cytolytic Immunotoxin Using Pulsed Electric Field Technology. International Journal of Molecular Sciences. 2020; 21(17):6165. https://doi.org/10.3390/ijms21176165
Chicago/Turabian StyleGuerrero-Ochoa, Patricia, Diederich Aguilar-Machado, Raquel Ibáñez-Pérez, Javier Macías-León, Ramón Hurtado-Guerrero, Javier Raso, and Alberto Anel. 2020. "Production of a Granulysin-Based, Tn-Targeted Cytolytic Immunotoxin Using Pulsed Electric Field Technology" International Journal of Molecular Sciences 21, no. 17: 6165. https://doi.org/10.3390/ijms21176165