En High Yield og kostnadseffektive Expression System of Human Granzymes i pattedyrceller
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
De Gzms er en familie av høyt homologe serinproteaser lokalisert i spesialiserte lysosomene til CTL og NK-celler 1. De cytotoksiske granulater av disse killer celler inneholder også membranforstyrrende proteiner pFN og GNLY som er utgitt samtidig med Gzms på anerkjennelse av et mål celle bestemt for eliminering 2,3. Det er fem Gzms hos mennesker (GzmA, B, H, K og M), og 10 Gzms i mus (GzmA – G, K, M og N). GzmA og GzmB er den mest tallrike og omfattende studert hos mennesker og mus 1. Imidlertid har nyere studier begynt å undersøke celledødsveier samt flere biologiske effekter mediert av de andre, såkalte foreldreløse Gzms i helse og sykdom fire.
Den best kjente funksjon av Gzms, spesielt av GzmA og GzmB, er induksjon av programmert celledød i pattedyrceller når den leveres inn i målceller ved PFN 5,6. MenFlere nyere studier også vist effekten av de ekstracellulære Gzms med betydelig innflytelse på immunregulering og betennelse, uavhengig av cytosolisk levering av PFN 7,8. Spekteret av celler som er drept effektivt etter cytosolic oppføring av Gzms ble også nylig utvidet fra pattedyrceller til bakterier 9,10 og selv enkelte parasitter 11. Disse siste funn åpnet opp et helt nytt felt for Gzm forskere. Derfor vil en kostnadseffektiv, high-yield pattedyrekspresjonsvektor system vesentlig lette veien for de fremtidige studier.
Native menneske, mus og rotte Gzms har blitt renset fra granulatfraksjon av CTL og NK-cellelinjer 12-14. Men i våre hender utbyttet av slike renseteknikker er i området fra mindre enn 0,1 mg / l cellekultur (upublisert observasjon og 12). Videre er den kromatografiske oppløsning av et enkelt Gzm uten forurensning av other Gzms og / eller proteiner som også er til stede i granulene er utfordrende (upubliserte data og 12,14). Rekombinante Gzms ble produsert i bakterier, gjær 15 16, insektceller 17, og i og med i pattedyrceller som for eksempel HEK 293 18,19. Bare de pattedyrekspresjonssystemer bære potensial til å produsere rekombinante enzymer med posttranslational modifikasjoner som er identiske med det native cytotoksiske protein. Posttranslasjonelle modifikasjoner har vært implisert med det spesifikke opptak av endocytose og intracellulær lokalisering av protease innenfor målcellene 20-22. Derfor, ved hjelp pHLsec 23 (en slags gave av Radu Aricescu og Yvonne Jones, University of Oxford, UK) som plasmid ryggrad for Gzm uttrykk, vi etablert en enkel, tids- og kostnadseffektivt system for high-yield proteinproduksjon i HEK293T celler. pHLsec kombinerer en CMV forsterker sammen med en kylling Β-aktin promoter; sammen, disse elementene demonstrated den sterkeste promoter aktivitet i ulike cellelinjer 24. I tillegg inneholder plasmidet et kanin Β-globin intron, den optimaliserte Kozak og sekresjon signaler, et Lys-6xHis-tag og en poly-A-signalet. Innsatser kan klones hensiktsmessig mellom-sekresjonssignal, og Lys-6xHis-tag (figur 1) å sikre optimal ekspresjon og sekresjon effektivitet for proteiner som mangler egnede N-terminale domener. For ekspresjon av de Gzms, erstattet vi den endogene sekresjon-signalsekvensen med utskillelsen signal gitt av vektoren, etterfulgt av et enterokinase (EK) område (DDDDK), slik at EK behandling aktivert utskilte Gzms (aktive Gzms starte med den N-terminale aminosyresekvens IlgG 25). I tillegg til fordel for denne metoden, HEK293T celler vokser raskt i lavt priset medium, slik som Dulbeccos modifiserte Eagles medium (DMEM), og er godt egnet for kostnadseffektiv kalsiumfosfat transfeksjon metode.
Protocol
Representative Results
Discussion
Den klassiske og omfattende undersøkt rollen til GzmA og GzmB er induksjon av apoptose i pattedyrceller etter deres cytosoliske levering av pore-dannende protein PFN 1. Nylig ble den cytotoksiske spektrum for Gzms utvidet betydelig fra pattedyrceller til 9,10 bakterier, så vel som visse parasitter 11. Videre er de ikke-klassiske, ekstracellulære funksjoner av GzmA og GzmB samt den biologiske betydning av de forskjellige sjeldne Gzms er fortsatt uklar. Derfor, en robust tids- og kostna…
Disclosures
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 |
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