哺乳動物細胞におけるヒトグランザイムの高収量およびコスト効率の高い発現システム
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
Gzmsは、CTLおよびNK細胞1の専門リソソームに局在高度に相同性セリンプロテアーゼのファミリーです。これらのキラー細胞の細胞傷害性顆粒はまた、排除2,3宛の標的細胞の認識にGzmsと同時にリリースされる膜破壊するタンパク質PFNとGNLYが含まれています。 （ – G、K、MおよびN GZMA）ヒトにおける5 Gzms（GZMA、B、H、KおよびM）、およびマウスでの10 Gzmsがあります。 GZMAとGZMBは最も豊富で広くヒトおよびマウス1で研究されています。しかし、最近の研究は、細胞死経路、ならびに健康と病気4の他、いわゆるオーファンGzmsによって媒介される追加の生物学的効果を検討し始めています。
PFN 5,6によって標的細胞に送達されたときGzmsの最もよく知られている機能は、GZMAおよびGZMBの、特に哺乳動物細胞におけるプログラム細胞死の誘導です。しかしながら、より最近の研究はまた、PFN 7,8によって独立細胞質ゾルデリバリーの、免疫調節および炎症に計り知れない影響を与えGzmsの細胞外効果を実証しました。 Gzmsの細胞質ゾルのエントリの後に効率的に殺された細胞のスペクトルはまた、最近、細菌9,10、さらには特定の寄生虫11に、哺乳動物細胞から広がりました。これらの最近の発見は、Gzmの研究者のための全く新しい分野を開きました。そのため、コスト効率、高収量哺乳動物発現系は、かなりのもの、将来の研究のための方法を容易にします。
天然ヒト、マウス、ラットGzmsが正常にCTLおよびNK細胞株12-14の顆粒画分から精製されています。しかし、我々の手で、このような精製技術の収量は0.1mg / L未満の細胞培養物（未発表の観察及び12）の範囲内です。また、パーソナルプラグインによる汚染のない単一Gzmのクロマトグラフィー分割R Gzmsおよび/ または顆粒中にも存在するタンパク質は、（未発表データと12,14）に挑戦されています。組換えGzms 16は、酵母、昆虫細胞は17とでさえ、このようなHEK 293 18,19などの哺乳動物細胞において、細菌15で製造しました。のみが哺乳動物発現系は、天然の細胞毒性タンパク質と同じ翻訳後修飾を有する組換え酵素を産生する可能性を負います。翻訳後修飾は、エンドサイトーシスによる特異的な取り込みおよび標的細胞20-22内のプロテアーゼの細胞内局在に関与しています。したがって、Gzm発現のためのプラスミド骨格としてpHLsec 23（ラドゥAricescuとイヴォンヌ·ジョーンズの寄贈、オックスフォード、英国の大学）を使用することにより、我々は、高収量のタンパク質産生のための簡単な、時間とコスト効率の高いシステムを構築しましたHEK293T細胞。 pHLsecは、ニワトリΒアクチンプロモーターとCMVエンハンサーを組み合わせました。一緒に、これらの要素はdemonstraテッド種々の細胞株24最強のプロモーター活性。また、プラスミドは、ウサギΒグロビンイントロン、最適化されたKozakおよび分泌シグナル、Lysの-の6xHisタグおよびポリAシグナルが含まれています。インサートは分泌シグナル、適切なN末端 ドメインを欠くタンパク質の最適な発現および分泌の効率を確保するのLys-の6xHisタグ（ 図1）との間で簡便にクローニングすることができます。 EK処理が分泌Gzmsを活性化（アクティブGzmsは、N末端で始まるようにGzmsの発現のために、我々は、エンテロキナーゼ（EK）のサイト（DDDDK）、続いて、ベクターが提供する分泌シグナルと内因性の分泌シグナル配列を置換しアミノ酸配列IIGG 25）。さらに、この方法のために有利なように、HEK293T細胞は、ダルベッコ改変イーグル培地（DMEM）で、低価格媒体中で急速に成長し、コスト効率の高いカルシウムリン酸トランスフェクション法に適しています。
Protocol
Representative Results
Discussion
GZMAとGZMBの古典と広範囲に研究の役割は、孔形成タンパク質PFN 1によるそれらのサイトゾル送達後、哺乳動物細胞におけるアポトーシスの誘導です。最近、Gzmsの細胞傷害性スペクトルは、細菌9,10、ならびに特定の寄生虫11に、哺乳動物細胞から大幅に広がりました。また、GZMAとGZMBの非古典的、細胞外の機能だけでなく、様々な孤児Gzmsの生物学的意義は依然として不?…
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 |
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