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Chimie

生物哺乳动物细胞光交联图谱和 Bioorthogonal 化学中化学探针的遗传受体优化

Published: April 09, 2018
doi:
10.3791/57069
, , ,
1Institute of Biochemistry, Faculty of Life Sciences,University of Leipzig

Summary

本文提出了一种简便的荧光分析方法, 用于评价将非规范氨基酸 (ncAAs) 纳入哺乳动物细胞表达的蛋白质中的氨基酰 tRNA 合成酶/tRNA 对的效率。介绍了 ncAAs 在 G 蛋白耦合受体 (受体) 研究中的应用, 包括结合位点的光交联映射和活细胞的 bioorthogonal GPCR 标记。

Abstract

非规范氨基酸 (ncAAs) 的基因融合通过琥珀停止密码子抑制是一个强大的技术, 安装人工探针和反应基团直接在蛋白质的活细胞。每个 ncAA 都由一个专用的正交抑制-tRNA/氨基酰 tRNA 合成酶 (AARS) 对导入宿主有机体。不同 ncAAs 的并网效率有很大差异, 在某些情况下是不理想的。通过操作 AARS 或 tRNA 可以提高正交对。然而, tRNA 或 AARS 的定向进化使用大型图书馆和死/活选择方法是不可行的哺乳动物细胞。在此基础上, 提出了一种简便、稳健的荧光检测方法, 用于评价哺乳动物细胞中正交对的有效性。该化验允许在合理的时间内进行适量的 AARS/tRNA 变种的筛查。利用这一方法生成新的 tRNAs, 显著提高 pyrrolysine 正交系统的效率, 以及 ncAAs 在 G 蛋白耦合受体 (受体) 的研究中的应用, 这是 ncAA 的挑战对象。诱变。首先, 系统地将一张照片交联的 ncAA 在一个受体的胞外表面, 在完整的受体的结合部位直接映射到活细胞中。其次, 通过将最后一代 ncAAs 加入 GPCR, 证明了用荧光染料进行超快无催化剂受体标记, 利用 bioorthogonal 应变促进逆翅果桤木 cycloaddition (SPIEDAC) 在活细胞上的作用。由于 ncAAs 可以被广泛地应用于任何蛋白质, 它的大小, 该方法是普遍感兴趣的一些应用。此外, ncAA 公司不需要任何特殊的设备, 很容易在标准生物化学实验室进行。

Introduction

化学探针在蛋白质中的遗传结合是一种有效的方法, 可以直接在活细胞的本机语境中研究蛋白质功能的结构和动态方面。如今, 数以百计的非规范氨基酸 (ncAAs) 配备了最不同的化学基团, 可以通过生物合成1,2,3,4,专门纳入蛋白质。在他们之间, 你发现光敏 ncAAs 例如相片交联剂 5, 相片笼6, 7, 8, 9 和相片转换氨基酸10,11, 含氨基酸的烯烃和炔烃无催化剂 bioorthogonal 化学2,12,13,14,15,16 、17、携带丹磺酰18、香豆素9、19、prodan20、21显影的氨基酸以及配备其他生物物理探针的氨基酸作为以及与后平移修改1,2,3,4,22,23,24,25。

ncaa 的遗传编码是由一个专门的氨基酰 tRNA 合成酶 (AARS) 配对的同源抑制-tRNA, 其中结合了 ncaa 的反应琥珀停止密码在定期核糖体合成。ncAARS/tRNA 对被设计成在宿主有机体中是正交的,即不与内生对进行交叉交谈。该技术在原核和真核宿主中都有很好的建立, 并且很容易适用于哺乳动物细胞。对 ncAA 纳入哺乳动物细胞是基于三主要正交系统: tyrosyl 系统, 结合 TyrRS 从大肠杆菌26与 tyrosyl 琥珀色抑制器从 B. 嗜热脂肪 27(EcTyrRS/Bst山药对),大肠杆菌leucyl 系统 (EcLeuRS/tRNA列在CUA对)6,18,28和古细菌 pyrrolysyl 系统 (PylRS/tRNAPyl对)3, 藉以 tRNAPyl是自然琥珀抑。一般来说, 每个 ncAA 都被一个专业的 ncAARS 认可。根据 ncaa 的结构, ncAARS 是通过 TyrRS、LeuRS 或 PylRS 的定向进化获得的, 尽管一些合成酶系可以接受不止一个 ncaa。

将正交对通过简单地使用质粒载体导入细胞中。最常见和有效的质粒是 bicistronic 和编码的合成酶和 tRNA 形成正交对29。第二种质粒编码的兴趣的蛋白质, 轴承一个琥珀密码子在指定的修改地点被联合转染。ncAA 只是添加到细胞生长培养基中。然而, 不同的专业群体经常使用不同的质粒结构, 即使是同一个 ncAA 的合并。构造在基因的排列不同在载体, 合成酶的类型, 密码子使用在合成酶基因, 促进者用法, tRNA 的变异和 tRNA 表达盒的数量。另外, 由于不同合成酶系的催化效率、tRNA 的质量和其他因素的不同, 不同 ncAAs 的结合效率可能会有很大的差异,如30。因此, 在手边有一个快速而可靠的方法来评估正交对的效率, 无论是选择最适合的系统为理想的应用和执行一些优化步骤, 以提高整体蛋白表达收益 率。

我们已经建立了一个简单和稳健的荧光检测方法来评估正交对29 (图 1) 的效率。在该检测中, 细胞与质粒编码的正交对, 连同 bicistronic 报告质粒编码两个绿色荧光蛋白轴承一个琥珀停止密码子在允许的位置 (EGFP标记) 和mCherry 基因。全细胞裂解物的红色和绿色荧光在一个96井板上的平板阅读器上以不同的通道读出。绿色荧光的强度与琥珀色抑制的效率直接相关, 而红荧光强度则直接估计出所测样品的大小和转染效率。关于基于荧光辅助细胞分类的类似化验, 请阅读31、32, 该化验结果立即全面评估了整个细胞的蛋白质表达, 这更代表通常的实验条件, 并提供一个更容易的数据获取和处理标准软件。总的来说, 该方法的主要优点是可以同时分析大量样品中的介质。利用这一方法, 我们筛选了一个合理设计的抑制 tRNAs 库, 以提高 Pyl 正交系统30的效率。这项工作描述的实验协议, 以执行这一试验, 并显示其应用的例子, 包括优化的正交对组合的照片交联的叠氮基苯丙氨酸 (Azi) 和比较采用不同氨基酸的效率 (图 2)。

在过去的几年中, ncAA 的工具已经被证明是非常强大的研究 G 蛋白耦合受体的结构和功能方面 (受体)33,34,35,36,37,38. 在人类中, 受体形成了一个大家庭的膜受体 (800 名成员), 并代表治疗药物的主要目标。受体的直接结构表征仍然具有挑战性, 对其进行研究需要补充生物化学方法。我们率先使用的照片交联 ncAAs 地图 GPCR 表面和发现配体绑定口袋34。利用我们的优化系统 Azi 合并, 我们系统地纳入 Azi 整个 juxtamembrane 领域的 GPCR 直接在活体哺乳动物细胞。在紫外线照射下, Azi 形成一种高度反应的 nitrene 物种, 共价键捕获相邻分子。当配体添加到系统中时, Azi 作为近距离探针来揭示受体的位置接近束缚配体。这样, 在 B 类 GPCR 内促肾上腺皮质激素释放因子受体类型 1 (CRF1R) 33 上首次公布了神经肽激素 Urocortin I (Ucn1) 的结合模式。最近, 我们在同一受体38上公开了不同的激动剂和拮抗器的结合模式。其他受体39、40、41、42的其他肽和小分子配体的 orthosteric 和构结合点也采用了类似的方法。本文介绍了在 GPCR 表面光交联测绘实验室中应用的实验协议。该方法相对快速, 简单, 不需要任何特殊设备, 因此适用于标准生物化学实验室。重要的是, 该方法不仅提供了一个有价值的工具, 不仅可以识别3D 结构数据稀缺的配体结合点, 而且还可以补充现有的体外数据, 并从完全后 translationally 修改后的受体中得到信息。活细胞的生理环境。

新近开发的新型 ncAAs 轴承, 适用于超快无催化剂 bioorthogonal 化学的侧链化学基团, 已开辟了将最后一代显影安装到蛋白质中的超级分辨率成像的可能性。直接在活单元格2,43上。 此类化学锚包括 SCOK14中的紧张 cyclooctyne, 双环 [6.1. 0] 壬炔BCNK12, 17, 和跨 cyclooctenes 在 TCO *K13, 15, 17 等 ncAAs窝藏冰片16,17,44或 cyclopropene45,46基团。bioorthogonal 化学的笨重 ncAAs 由 PylRS 的变体组成, 通常表示为 PylRSAF (指示m Y271A Y349F 中的突变 barkeri 和 PylRS), 以及其他即席进化 ncAARSs 17, 44. bioorthogonal 锚与四嗪类试剂47 通过逆电子需求翅果-桤木 cycloaddition 的反应, 在几分钟43, 48 内给出高标号率。然而, 这一强大的方法, 以标签受体是一个挑战, 由于整体效率低的正交 ncAA 公司合并系统。利用我们的增强 Pyl 系统, 我们最近展示了这种氨基酸的高产量纳入受体和超快 GPCR 标记在活体哺乳动物细胞表面30。标记的受体仍然功能, 因为他们的生理内化后激活受体与激动剂。本文介绍了将 bioorthogonal 锚定为受体的实验协议, 以及下面的标记步骤。用小亮显影装备受体是通过先进显微技术研究活细胞 GPCR 结构动力学的第一个基本步骤。

Protocol

1. 基于荧光的筛选效率 (图 1) 维持 Dulbecco 修饰鹰培养基中的 HEK293 细胞 (DMEM; 高葡萄糖, 4 毫米谷氨酰胺, 丙酮酸) 补充 10% (v/v) 胎牛血清 (血清), 100 U/毫升青霉素和100µg/毫升链霉素在37°c, 95% 湿度和 5% CO2。 在转染前的前一天将细胞播种。 分离细胞为5分钟在37°c 在0.05% 胰蛋白酶或 PBS 补充与0.5 毫米 EDTA。使用1毫升胰蛋白酶/EDTA 的10厘米菜。用10卷的…

Representative Results

荧光检测的轮廓在图 1中描述。这项化验在三宗申请中使用。首先, 对 Pyl 正交对中的赖氨酸 (中行) 的一些 tRNA 变体进行筛选。赖氨酸 (中行) 是一种与 Pyl 类似的氨基酸 sterically。由于 Pyl 在商业上不可用, 赖氨酸 (中行) 通常被用作 PylRS 的标准基板。筛选的 tRNAs 基于 tRNAPyl。每个 tRNA 变种在回路和茎的单基或基对的突变承担合理设计, 以提高 tR…

Discussion

该协议描述了一个简单而可靠的方法, 以评估正交对的效率, 将 ncAAs 纳入哺乳动物细胞表达的蛋白质。这种方法的主要优点是, 在广泛使用的基于资产管制系统的测试中, 它允许同时准备和测量大量的样本, 并提供了使用普通软件可以轻松分析的数据。在哺乳动物细胞中进行正交对分析的中等吞吐量方法的有效性对于新的正交对的发展和现有的改进是非常重要的。事实上, 不可能通过生成大型随机库…

Divulgations

The authors have nothing to disclose.

Acknowledgements

这项工作是由德意志 Forschungsgemeinschaft (DFG) 根据赠款 CO822/2-1 (艾美诺特计划) 和 CO822/3-1 到中

Materials

Chemicals
Acryamide/Bisacrylamide 30% (37,5:1) Carl Roth 3029.1
Ammonium persulfate (APS) Carl Roth 9592.2
p-Azidophenylalanine (Azi) Bachem F-3075.0001
Boric acid Sigma Aldrich B6768
Bromphenolblue Sigma-Aldrich B0126-25G
Bovine serum albumine (BSA) Carl Roth 8076.2
Carbobenzyloxy-L-lysine (Lys(Z)) NovaBiochem 8540430100
Cyclooctyne-L-lysine (SCOK) Sichem SC-8000
DMEM Life Technologies 41966052
DMSO Carl Roth A994.2
DTT Carl Roth 6908.1
enhanced chemiluminescence reagent (ECL)  home-made 10 mg/l luminol in 0.1 M Tris-HCl pH 8.6 ; 1100 mg/l  p-coumaric acid in DMSO ; 30 % H2O2 (1,000 : 100 : 0.3) [Quelle Laborjounal]
EDTA Carl Roth 8043.1
EGTA Carl Roth 3054.1
endo-bicyclo[6.1.0]nonyne-L-lysine (BCNK) Sichem SC-8014
FBS Thermo Fisher (Gibco) 10270106
FluoroBrite DMEM Thermo Fisher (Gibco) A1896701
Glycerol Carl Roth 7533.1
Glycin Carl Roth 3908.3
HEPES Carl Roth 9105.3
Hoechst 33342 Sigma Aldrich B2261
KCl Carl Roth 6781.3
Lipofectamine 2000  Thermo Fisher 11668019
Luminol Applichem A2185,0005
Methanol Carl Roth 0082.3
MgCl2 Carl Roth 2189.2
NaCl Carl Roth HN00.2
Na-Lactate Sigma-Aldrich 71718-10G
NaOH Grüssing 121551000
PBS Sigma-Aldrich P5493-1L
p-Coumaric acid Sigma-Aldrich C9008-1G
poly-D-lysine hydrobromide Corning 354210
PEI Polysciences 23966
Penicillin/Streptomycin Thermo Fisher (Gibco) 11548876 (15140-122)
PMSF Carl Roth 6367.1
PNGase F NEB P0704L
Protease Inhibitor Roche 11873580001
PVDF membrane Immobilon-P Millipore IPVH00010
Skim Milk Powder  Sigma 70166
Sodium dodecyl sulfate (SDS) Carl Roth CN30.2
Tetrazine-Cy3 Jena Bioscience CLK-014-05
Tetramethylethylenediamine (TEMED) Carl Roth 2367.3
trans-Cyclooctene-L-lysine (TCO*K) Sichem SC-8008
TRIS Sigma-Aldrich T1503
Triton X-100 Carl Roth 3051.4
Trypsin 2.5% Thermo Fisher (Gibco) 15090046
Tween 20 Carl Roth 9127.2
Wasserstoffperoxid (30%) Merck 1.07210.0250
Cell lines
HEK293 cells German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) ACC-305
HEK293T cells German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) ACC-635
Equipment
Crosslinker Bio-Link 365 nm Bio-Budget Technologies GmbH 40-BLX-E365 5 x 8 Watt tubes
Plate Reader BMG LABTECH FLUOstar Omega  BMG LABTECH
Plasmids
Plasmid E2AziRS The huminized gene for E2AziRS was synthesized by Geneart (Life Technologies) Plasmid containing 4 tandem copies of the suppressor tRNA Bst-Yam driven by the human U6 promoter and one copy of a humanized gene for the enhanced variant of the Azi-tRNA synthetase (EAziRS) driven by a PGK promoter
POI-TAG mutant plasmids Plasmid encoding the POI driven by the CMV promoter, C-terminally fused to the FLAG-tag, bearing a TAG codon at the desired position 
CRF1R-95TAG-EGFP Cloned in the MCS of pcDNA3.1
HA-PTH1R-79TAG-CFP Cloned in the MCS of pcDNA3.1
Arrestin3-FLAG Synthesized by Genart (Life Technologies) Cloned in the MCS of pcDNA3.1
Antibodies
Anti-FLAG-HRP M2 antibody conjugate  Sigma-Aldrich A8592  monoclonal, produced in mouse clone M2
Goat-anti-rabbit-HRP antibody Santa Cruz sc-2004
Rabbit-anti-CRF antibody home-made PBL #rC69 polyclonal [Turnbull]
Rabbit-anti-Ucn1 antibody home-made PBL #5779 polyclonal [Turnbull]
DOWNLOAD MATERIALS LIST

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Tags

GPCRPhoto-crosslinkingBioorthogonal ChemistryNon-canonical Amino AcidsAmber Codon SuppressionFluorescence AssayMammalian CellsProtein-protein InteractionsLigand BindingLive Cell Imaging
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Serfling, R., Seidel, L., Böttke, T., Coin, I. Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells. J. Vis. Exp. (134), e57069, doi:10.3791/57069 (2018).
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