A High Yield e Cost-efficient Sistema Espressione di granzimi umane in cellule di mammifero
Summary
We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.
Abstract
When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.
Introduction
I Gzms sono una famiglia di serina proteasi altamente omologhi localizzate nei lisosomi specializzati di CTL e cellule NK 1. I granuli citotossici di queste cellule killer contengono anche i-membrana interrompere proteine PFN e GNLY che vengono rilasciati contemporaneamente alle Gzms accreditato in una cella di destinazione destinata per l'eliminazione 2,3. Ci sono cinque Gzms nell'uomo (GzmA, B, H, K e M), e 10 Gzms nei topi (GzmA – G, K, M e N). GzmA e GzmB sono i più abbondanti e ampiamente studiati sugli esseri umani e nei topi 1. Tuttavia, studi più recenti hanno cominciato a studiare le vie di morte cellulare e gli effetti biologici supplementari mediati dalle altre, cosiddette Gzms orfani in materia di salute e di malattia 4.
La funzione più nota delle Gzms, in particolare di GzmA e GzmB, è l'induzione della morte cellulare programmata in cellule di mammifero quando espresso nelle cellule bersaglio da parte PFN 5,6. Tuttavia, Studi più recenti hanno anche dimostrato effetti extracellulari dei Gzms con profondo impatto sulla regolazione immunitaria e infiammazione, indipendentemente dalla consegna citosolica da PFN 7,8. Lo spettro di cellule che vengono uccisi in modo efficace dopo l'entrata citosolico dei Gzms è stato recentemente ampliato da cellule di mammifero a batteri 9,10 e anche alcuni parassiti 11. Queste scoperte recenti aperto un campo completamente nuovo per i ricercatori GZM. Pertanto, un alto rendimento del sistema di espressione mammifero economicamente efficiente faciliterà notevolmente la strada a tali studi futuri.
Umani, topi e ratti Gzms nativi sono stati purificati con successo dalla frazione granello di CTL e NK cellule linee 12-14. Tuttavia, nelle nostre mani il rendimento di tali tecniche di purificazione è nella gamma di cultura inferiore a 0,1 mg / L cella (osservazioni non pubblicate e 12). Inoltre, la risoluzione cromatografica di un singolo GZM senza contaminazione da other Gzms e / o proteine che sono presenti anche nei granuli è impegnativo (dati inediti e 12,14). Gzms ricombinanti sono state prodotte in batteri, lieviti 15 16, cellule di insetto 17 e in anche in cellule di mammifero, come HEK 293 18,19. Solo i sistemi di espressione di mammifero portano il potenziale di produrre enzimi ricombinanti con modificazioni post-identico alla proteina citotossica nativo. Modificazioni post-traduzionali sono stati implicati con l'assorbimento specifico per endocitosi e la localizzazione intracellulare della proteasi all'interno delle cellule bersaglio 20-22. Pertanto, utilizzando pHLsec 23 (un gentile dono di Radu Aricescu e Yvonne Jones, dell'Università di Oxford, Regno Unito) come spina dorsale plasmide per l'espressione GZM, abbiamo istituito un sistema semplice, tempo e conveniente per alto rendimento produzione di proteine in HEK293T cellule. pHLsec combina un esaltatore di CMV con un pollo Β-actina promotore; insieme, questi elementi dimoted il più forte promotore attività in varie linee cellulari 24. Inoltre, il plasmide contiene un coniglio Β-globina introne, segnali Kozak e secrezione ottimizzati, a-6xHis-tag Lys e un poli-A segnale. Gli inserti possono essere clonate convenientemente tra il segnale di secrezione e Lys-6xHis-tag (Figura 1) assicurando espressione ottimale e efficienza secrezione di proteine prive appropriate domini N-terminali. Per l'espressione delle Gzms, abbiamo sostituito la sequenza segnale di secrezione endogena con il segnale di secrezione fornito dal vettore seguito da un enterokinase (EK) sito (DDDDK) in modo che il trattamento EK attivato il Gzms secrete (Gzms attivi iniziare con N-terminale sequenza aminoacidica IIGG 25). Inoltre, a favore di questo metodo, le cellule HEK293T crescono rapidamente in basso prezzo medio, come Dulbecco Modified mezzo di Eagle (DMEM), e sono adatti per il calcio-fosfato metodo trasfezione conveniente.
Protocol
Representative Results
Discussion
Il ruolo classico e ampiamente studiato di GzmA e GzmB è l'induzione di apoptosi nelle cellule di mammifero dopo la loro consegna citosolico dal formano pori proteine PFN 1. Recentemente, lo spettro citotossica delle Gzms stato ampliato significativamente da cellule di mammifero ai batteri 9,10, così come a certi parassiti 11. Inoltre, i non classici, funzioni extracellulari di GzmA e GzmB nonché il significato biologico dei vari Gzms orfani sono ancora oscuri. Pertanto, l&#…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from the Novartis Foundation for Medical-Biological Research and from the Research Pool of the University of Fribourg (to MW). We thank Li Zhao, Zhan Xu, and Solange Kharoubi Hess for technical support, as well as Radu Aricescu and Yvonne Jones (Oxford University, UK) for providing the pHLsec plasmid, and Thomas Schürpf (Harvard Medical School) for helpful discussions.
Materials
| TRIzol Reagent | Invitrogen | 15596-026 | Total RNA isolation kit |
| SuperScript II Reverse Transcriptase | Invitrogen | 18064-014 | |
| Phusion High-Fidelity DNA Polymerase | NEB | M0530L | |
| EndoFree Plasmid Maxi Kit | QIAGEN | 12362 | |
| EX-CELL 293 Serum-Free Medium for HEK 293 Cells | Sigma | 14571C | |
| DMEM, high glucose, GlutaMAX Supplement, pyruvate | Gibco | 31966-021 | |
| SnakeSkin Dialysis Tubing, 10K MWCO | Thermo Scientific | 68100 | |
| Enterokinase from bovine intestine | Sigma | E4906 | recombinant, ≥20 units/mg protein |
| HisTrap Excel 5ml-column | GE Healthcare | 17-3712-06 | Nickel IMAC |
| HiTrap SP HP 5 ml-column | GE Healthcare | 17-1152-01 | S column |
| N-α-Cbz-L-lysine thiobenzylester (BLT) | Sigma | C3647 | GzmA substrate |
| Boc-Ala-Ala-Asp-S-Bzl (AAD) | MP Biomedicals | 2193608 _10mg | GzmB substrate |
| Suc-Ala-Ala-Pro-Leu-p-nitroanilide (AAPL) | Bachem | GzmM substrate | |
| 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) | Sigma | D8130 | Ellman`s reagent |
Referências
- Chowdhury, D., Lieberman, J. Death by a thousand cuts: granzyme pathways of programmed cell death. Annu Rev Immunol. 26, 389-420 (2008).
- Thiery, J., Lieberman, J. Perforin: a key pore-forming protein for immune control of viruses and cancer. Subcell Biochem. 80, 197-220 (2014).
- Krensky, A. M., Clayberger, C. Biology and clinical relevance of granulysin. Tissue Antigens. 73, 193-198 (2009).
- Bovenschen, N., Kummer, J. A. Orphan granzymes find a home. Immunol Rev. 235, 117-127 (2010).
- Lieberman, J. Granzyme A activates another way to die. Immunol Rev. 235, 93-104 (2010).
- Ewen, C. L., Kane, K. P., Bleackley, R. C. A quarter century of granzymes. Cell death and differentiation. 19, 28-35 (2012).
- Hiebert, P. R., Granville, D. J. Granzyme B in injury, inflammation, and repair. Trends Mol Med. 18, 732-741 (2012).
- Afonina, I. S., Cullen, S. P., Martin, S. J. Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme. B. Immunol Rev. 235, 105-116 (2010).
- Walch, M., et al. Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes. Cell. 157, 1309-1323 (2014).
- Lee, W. Y., et al. Invariant natural killer T cells act as an extravascular cytotoxic barrier for joint-invading Lyme Borrelia. Proc Natl Acad Sci U S A. , (2014).
- Kapelski, S., de Almeida, M., Fischer, R., Barth, S., Fendel, R. Antimalarial activity of granzyme B and its targeted delivery by a granzyme B-scFv fusion protein. Antimicrob Agents Chemother. , (2014).
- Thiery, J., Walch, M., Jensen, D. K., Martinvalet, D., Lieberman, J. Isolation of cytotoxic T cell and NK granules and purification of their effector proteins. Curr Protoc Cell Biol. Chapter 3 (Unit3 37), (2010).
- Shi, L., Yang, X., Froelich, C. J., Greenberg, A. H. Purification and use of granzyme B. Methods Enzymol. 322, 125-143 (2000).
- Masson, D., Tschopp, J. A family of serine esterases in lytic granules of cytolytic T lymphocytes. Cell. 49, 679-685 (1987).
- Lorentsen, R. H., Fynbo, C. H., Thogersen, H. C., Etzerodt, M., Holtet, T. L. Expression, refolding, and purification of recombinant human granzyme B. Protein Expr Purif. 39, 18-26 (2005).
- Sun, J., et al. Expression and purification of recombinant human granzyme B from Pichia pastoris. Biochem Biophys Res Commun. 261, 251-255 (1999).
- Xia, Z., et al. Expression and purification of enzymatically active recombinant granzyme B in a baculovirus system. Biochem Biophys Res Commun. 243, 384-389 (1998).
- Stahnke, B., et al. Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes. Mol Cancer Ther. 7, 2924-2932 (2008).
- Gehrmann, M., et al. A novel expression and purification system for the production of enzymatic and biologically active human granzyme. B. J Immunol Methods. 371, 8-17 (2011).
- Metkar, S. S., et al. Cytotoxic cell granule-mediated apoptosis: perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation. Immunity. 16, 417-428 (2002).
- Motyka, B., et al. Mannose 6-phosphate/insulin-like growth factor II receptor is a death receptor for granzyme B during cytotoxic T cell-induced apoptosis. Cell. 103, 491-500 (2000).
- Pinkoski, M. J., et al. Entry and trafficking of granzyme B in target cells during granzyme B-perforin-mediated apoptosis. Blood. 92, 1044-1054 (1998).
- Aricescu, A. R., Lu, W., Jones, E. Y. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr D Biol Crystallogr. 62, 1243-1250 (2006).
- Fukuchi, K., et al. Activity assays of nine heterogeneous promoters in neural and other cultured cells. In Vitro Cell Dev Biol Anim. 30 A, 300-305 (1994).
- Tran, T. V., Ellis, K. A., Kam, C. M., Hudig, D., Powers, J. C. Dipeptidyl peptidase I: importance of progranzyme activation sequences, other dipeptide sequences, and the N-terminal amino group of synthetic substrates for enzyme activity. Arch Biochem Biophys. 403, 160-170 (2002).
- Somanchi, S. S., Senyukov, V. V., Denman, C. J., Lee, D. A. Expansion, purification, and functional assessment of human peripheral blood NK cells. Journal of visualized experiments : JoVE. , (2011).
- Barry, M., et al. Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving Bid. Mol Cell Biol. 20, 3781-3794 (2000).
- Darmon, A. J., Nicholson, D. W., Bleackley, R. C. Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B. . Nature. 377, 446-448 (1995).
- Rajani, D. K., Walch, M., Martinvalet, D., Thomas, M. P., Lieberman, J. Alterations in RNA processing during immune-mediated programmed cell death. Proc Natl Acad Sci U S A. 109, 8688-8693 (2012).
- Domselaar, R., et al. Noncytotoxic inhibition of cytomegalovirus replication through NK cell protease granzyme M-mediated cleavage of viral phosphoprotein 71. J Immunol. 185, 7605-7613 (2010).
- Thiery, J., et al. Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis. Blood. 115, 1582-1593 (2010).
- Thiery, J., et al. Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells. Nat Immunol. 12, 770-777 (2011).
- Thomas, M. P., et al. Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins. J Immunol. 192, 5390-5397 (2014).
- Jacquemin, G., et al. Granzyme B-induced mitochondrial ROS are required for apoptosis. Cell death and differentiation. , (2014).
- Kummer, J. A., Kamp, A. M., Citarella, F., Horrevoets, A. J., Hack, C. E. Expression of human recombinant granzyme A zymogen and its activation by the cysteine proteinase cathepsin C. The Journal of biological chemistry. 271, 9281-9286 (1996).
- Hagn, M., Sutton, V. R., Trapani, J. A. A colorimetric assay that specifically measures Granzyme B proteolytic activity: hydrolysis of Boc-Ala-Ala-Asp-S-Bzl. Journal of visualized experiments : JoVE. , e52419 (2014).
- Huang, M. T., Gorman, C. M. Intervening sequences increase efficiency of RNA 3′ processing and accumulation of cytoplasmic RNA. Nucleic Acids Res. 18, 937-947 (1990).
- Luthman, H., Magnusson, G. High efficiency polyoma DNA transfection of chloroquine treated cells. Nucleic Acids Res. 11, 1295-1308 (1983).
- Magee, A. I., Grant, D. A., Hermon-Taylor, J., Offord, R. E. Specific one-stage method for assay of enterokinase activity by release of radiolabelled activation peptides from alpha-N-[3H]acetyl-trypsinogen and the effect of calcium ions on the enzyme activity. Biochem J. 197, 239-244 (1981).
