JoVE Journal > Immunology and Infection
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Immunologia e infezioni

在大肠杆菌中生成特异的乙酰蛋白的简易协议

Published: December 09, 2017
doi:
10.3791/57061
, , , ,
1Department of Chemistry and Biochemistry,University of Arkansas, 2Cell and Molecular Biology Program,University of Arkansas, 3Department of Biological Sciences,University of Arkansas, 4Fayetteville High School

Summary

基因编码扩展是研究广泛的生物过程, 包括蛋白质乙酰化的有力工具。在这里, 我们演示了一个轻便的协议, 利用这种技术, 在特定的地点, 在大肠杆菌细胞生成均匀乙酰蛋白。

Abstract

在蛋白质的特定位置发生的后平移修饰被证明在多种细胞过程中发挥重要作用。其中, 可逆赖氨酸乙酰化是最广泛分布在生活的所有领域之一。尽管已经进行了大量的质谱 acetylome 研究, 但对这些假定的乙酰化靶点的进一步定性是有限的。一个可能的原因是, 它是很难产生纯乙酰蛋白质在理想的位置, 大多数经典的生物化学方法。为了克服这一挑战, 基因编码扩展技术已被应用于使用一对工程 pyrrolysyl tRNA 合成酶变种, 其同源 tRNA 从Methanosarcinaceae物种, 以指导 cotranslational 公司成立acetyllysine 在特定的地点的蛋白质的利益。在首次应用于组蛋白乙酰化的研究中, 这种方法促进了对各种蛋白质的乙酰化研究。在这项工作中, 我们展示了一个简便的协议, 以生产现场特定的乙酰蛋白, 使用模型细菌大肠埃希菌作为宿主。以苹果酸脱氢酶为例。

Introduction

转录后的修饰 (PTMs) 的蛋白质发生后, 翻译过程, 并产生了共价键增加功能基团的氨基酸残留, 在几乎所有的生物过程中扮演重要的角色, 包括基因转录, 应激反应, 细胞分化和新陈代谢1,2,3。到目前为止, 已确定了大约400独特的 PTMs4。基因组和蛋白质的复杂性在很大程度上被蛋白质 PTMs, 因为它们调节蛋白质活性和定位, 并影响与其他分子的相互作用, 如蛋白质、核酸、血脂和因子5。

在过去二十年中, 蛋白质乙酰化一直处于 PTMs 研究的前沿6、7、8、9、10、11、12。在组蛋白中首次发现的赖氨酸乙酰化超过50年前的13,14, 已被仔细检查, 并已知存在于超过80转录因子、调节器和各种蛋白质15中, 16,17。对蛋白质乙酰化的研究不仅为我们提供了对其调节机制的更深的了解, 而且也为一些因功能失调而引起的疾病进行了指导治疗18,19,20,21,22,23. 据认为, 赖氨酸乙酰化只发生在真核生物, 但最近的研究表明, 蛋白质乙酰化也发挥关键作用的细菌生理学, 包括趋化性, 耐酸, 活化, 稳定致病性岛屿及其他毒力相关蛋白24,25,26,27,28,29。

一种常用的方法来生化的特点赖氨酸乙酰化是使用定点诱变。谷氨酰胺被用作 acetyllysine 的模拟, 因为它的大小和极性相似。精氨酸被用作非乙酰赖氨酸的模拟, 因为它在生理条件下保留了正电荷, 但不能乙酰。但是, 这两种模拟都不是真正的 isosteres, 并且并不总能产生预期的结果30。最严谨的方法是在特定的赖氨酸残留物中生成均匀的乙酰蛋白, 这对于大多数经典方法来说是困难或不可能的, 因为在自然界中, 赖氨酸乙酰化的低化学计量性7,11。这一挑战已被解开的遗传代码扩展战略, 采用了工程 pyrrolysyl-tRNA 合成酶变种从Methanosarcinaceae物种的收费 tRNAPyl与 acetyllysine, 利用主机用于抑制 mRNA 中的 UAG 停止密码子的平移机械, 并指示在目标蛋白31的设计位置中合并 acetyllysine。最近, 我们优化了这个系统, 改进了 EF Tu 绑定 tRNA32和升级后的 acetyllysyl-tRNA 合成酶33。此外, 我们在苹果酸脱氢酶34和氨-tRNA 合成酶35的乙酰化研究中应用了这种增强的并网系统。本文以苹果酸脱氢酶 (MDH) 为例, 研究了从分子克隆到生物化学鉴定的纯乙酰蛋白的生成方法, 并以此作为演示的例证。

Protocol

1. 靶基因的定点诱变 注意: MDH 在pCDF-1向量中以CloDF13原点和20到 4034的副本号表示在 T7 启动子下。 介绍了琥珀色停止密码在该基因的位置140的引物 (向前底漆: GGTGTTTATGAC标记AACAAACTGTTCGGCG 和反向底漆: GGCTTTTTTCAGCACTTCAGCAGCAATTGC), 按照指示的网站定向诱变试剂盒。 放大含有野生型苹果脱水基因的模板质粒, 通过聚合酶链反应 …

Representative Results

乙酰 MDH 蛋白的产量为每 1 l 培养15毫克, 而野生型 MDH 为31毫克, 每 1 l 培养。用 SDS-页分析纯化蛋白, 如图 1所示。使用野生类型的 MDH 作为正控制34。从细胞中纯化的 acetyllysine (AcK) 合并系统和突变的mdh基因, 但在生长培养基中没有 AcK, 被用作阴性对照。用 acetyllysine 抗体检测纯化蛋白的赖氨酸乙酰化, 如图 2</stron…

Discussion

非氨基酸 (ncAAs) 的基因合并是基于对一个被分配的密码子的镇压, 主要琥珀色的中止密码子 UAG36,37,38,39, 由 ncAA 充电 tRNA包含相应的子。众所周知, UAG 密码子是公认的释放 factor-1 (RF1) 的细菌, 它也可以抑制附近同源转运从主机的标准氨基酸 (cAAs), 如赖氨酸和酪氨酸40,<sup class="xr…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了国立卫生研究院 (AI119813) 的支持, 这是来自阿肯色州大学的起步, 也是来自阿肯色州生物科学研究所的奖项。

Materials

Bradford protein assay Bio-Rad 5000006 Protein concentration
4x Laemmli Sample Buffer Bio-Rad 1610747 SDS sample buffer
Coomassie G-250 Stain Bio-Rad 1610786 SDS-PAGE gel staining
4-20% SDS-PAGE ready gel Bio-Rad 4561093 Protein determination
Ac-K-100 (HRP Conjugate) Cell Signaling 6952 Antibody
IPTG CHEM-IMPEX 194 Expression inducer
Nε-Acetyl-L-lysine CHEM-IMPEX 5364 Noncanonical amino acid
PD-10 desalting column GE Healthcare 17085101 Desalting
Q5 Site-Directed Mutagenesis Kit NEB E0554 Introducing the stop codon
BL21 (DE3) cells NEB C2527 Expressing strain
QIAprep Spin Miniprep Kit QIAGEN 27106 Extracting plasmids
Ni-NTA resin QIAGEN 30210 Affinity purification resin
nicotinamide Sigma-Aldrich N3376 Deacetylase inhibitor
β-Mercaptoethanol Sigma-Aldrich M6250 Reducing agent
BugBuster Protein Extraction Reagent Sigma-Aldrich 70584 Breaking cells
Benzonase nuclease Sigma-Aldrich E1014 DNase
ECL Western Blotting Substrate ThermoFisher 32106 Chemiluminescence
Premixed LB Broth VWR 97064 Cell growth medium
Bovine serum albumin VWR 97061-416 western blots blocking
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Riferimenti

  1. Krishna, R. G., Wold, F. Post-translational modification of proteins. Adv Enzymol Relat Areas Mol Biol. 67, 265-298 (1993).
  2. Lothrop, A. P., Torres, M. P., Fuchs, S. M. Deciphering post-translational modification codes. FEBS Lett. 587 (8), 1247-1257 (2013).
  3. Walsh, C. T. . Posttranslational modification of proteins : expanding nature’s inventory. , (2006).
  4. Khoury, G. A., Baliban, R. C., Floudas, C. A. Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database. Sci Rep. 1, (2011).
  5. Grotenbreg, G., Ploegh, H. Chemical biology: dressed-up proteins. Nature. 446 (7139), 993-995 (2007).
  6. Arif, M., Selvi, B. R., Kundu, T. K. Lysine acetylation: the tale of a modification from transcription regulation to metabolism. Chembiochem. 11 (11), 1501-1504 (2010).
  7. Cohen, T., Yao, T. P. AcK-knowledge reversible acetylation. Science’s STKE : signal transduction knowledge environment. (245), pe42 (2004).
  8. Drazic, A., Myklebust, L. M., Ree, R., Arnesen, T. The world of protein acetylation. Biochim Biophys Acta. 1864 (10), 1372-1401 (2016).
  9. Escalante-Semerena, J. C. Nε-acetylation control conserved in all three life domains. Microbe. 5, 340-344 (2010).
  10. Kouzarides, T. Acetylation: a regulatory modification to rival phosphorylation?. The EMBO journal. 19 (6), 1176-1179 (2000).
  11. Soppa, J. Protein acetylation in archaea, bacteria, and eukaryotes. Archaea. , (2010).
  12. Verdin, E., Ott, M. 50 years of protein acetylation: from gene regulation to epigenetics, metabolism and beyond. Nat Rev Mol Cell Biol. 16 (4), 258-264 (2015).
  13. Allfrey, V. G., Faulkner, R., Mirsky, A. E. Acetylation and Methylation of Histones and Their Possible Role in the Regulation of Rna Synthesis. P Natl Acad Sci USA. 51, 786-794 (1964).
  14. Phillips, D. M. The presence of acetyl groups of histones. Biochem j. 87, 258-263 (1963).
  15. Sterner, D. E., Berger, S. L. Acetylation of histones and transcription-related factors. Microbiology and molecular biology reviews: MMBR. 64 (2), 435-459 (2000).
  16. Glozak, M. A., Sengupta, N., Zhang, X., Seto, E. Acetylation and deacetylation of non-histone proteins. Gene. 363, 15-23 (2005).
  17. Close, P., et al. The emerging role of lysine acetylation of non-nuclear proteins. Cell Mol Life Sci. 67 (8), 1255-1264 (2010).
  18. Iyer, A., Fairlie, D. P., Brown, L. Lysine acetylation in obesity, diabetes and metabolic disease. Immunol Cell Biol. 90 (1), 39-46 (2012).
  19. You, L., Nie, J., Sun, W. J., Zheng, Z. Q., Yang, X. J. Lysine acetylation: enzymes, bromodomains and links to different diseases. Essays Biochem. 52, 1-12 (2012).
  20. Bonnaud, E. M., Suberbielle, E., Malnou, C. E. Histone acetylation in neuronal (dys)function. Biomol Concepts. 7 (2), 103-116 (2016).
  21. Fukushima, A., Lopaschuk, G. D. Acetylation control of cardiac fatty acid beta-oxidation and energy metabolism in obesity, diabetes, and heart failure. Biochim Biophys Acta. 1862 (12), 2211-2220 (2016).
  22. Kaypee, S., et al. Aberrant lysine acetylation in tumorigenesis: Implications in the development of therapeutics. Pharmacol Ther. 162, 98-119 (2016).
  23. Tapias, A., Wang, Z. Q. Lysine Acetylation and Deacetylation in Brain Development and Neuropathies. Genomics Proteomics Bioinformatics. 15 (1), 19-36 (2017).
  24. Hu, L. I., Lima, B. P., Wolfe, A. J. Bacterial protein acetylation: the dawning of a new age. Molecular microbiology. 77 (1), 15-21 (2010).
  25. Jones, J. D., O’Connor, C. D. Protein acetylation in prokaryotes. Proteomics. 11 (15), 3012-3022 (2011).
  26. Bernal, V., et al. Regulation of bacterial physiology by lysine acetylation of proteins. New biotechnol. 31 (6), 586-595 (2014).
  27. Hentchel, K. L., Escalante-Semerena, J. C. Acylation of Biomolecules in Prokaryotes: a Widespread Strategy for the Control of Biological Function and Metabolic Stress. Microbiology and molecular biology reviews : MMBR. 79 (3), 321-346 (2015).
  28. Ouidir, T., Kentache, T., Hardouin, J. Protein lysine acetylation in bacteria: Current state of the art. Proteomics. 16 (2), 301-309 (2016).
  29. Wolfe, A. J. Bacterial protein acetylation: new discoveries unanswered questions. Curr Genet. 62 (2), 335-341 (2016).
  30. Albaugh, B. N., Arnold, K. M., Lee, S., Denu, J. M. Autoacetylation of the histone acetyltransferase Rtt109. J Biol Chem. 286 (28), 24694-24701 (2011).
  31. Neumann, H., Peak-Chew, S. Y., Chin, J. W. Genetically encoding Nε-acetyllysine in recombinant proteins. Nat chem biol. 4 (4), 232-234 (2008).
  32. Fan, C., Xiong, H., Reynolds, N. M., Soll, D. Rationally evolving tRNAPyl for efficient incorporation of noncanonical amino acids. Nucleic Acids Res. 43 (22), e156 (2015).
  33. Bryson, D., et al. Continuous directed evolution of aminoacyl-tRNA synthetases to alter amino acid specificity and enhance activity. Nat Chem Biol. , (2017).
  34. Venkat, S., Gregory, C., Sturges, J., Gan, Q., Fan, C. Studying the Lysine Acetylation of Malate Dehydrogenase. J Mol Biol. 429 (9), 1396-1405 (2017).
  35. Venkat, S., Gregory, C., Gan, Q., Fan, C. Biochemical characterization of the lysine acetylation of tyrosyl-tRNA synthetase in Escherichia coli. Chembiochem. 18 (19), 1928-1934 (2017).
  36. Liu, C. C., Schultz, P. G. Adding new chemistries to the genetic code. Annu Rev Biochem. 79, 413-444 (2010).
  37. Mukai, T., Lajoie, M. J., Englert, M., Soll, D. Rewriting the Genetic Code. Annu Rev Microbiol. , (2017).
  38. O’Donoghue, P., Ling, J., Wang, Y. S., Soll, D. Upgrading protein synthesis for synthetic biology. Nat Chem Biol. 9 (10), 594-598 (2013).
  39. Chin, J. W. Expanding and reprogramming the genetic code of cells and animals. Annu Rev Biochem. 83, 379-408 (2014).
  40. O’Donoghue, P., et al. Near-cognate suppression of amber, opal and quadruplet codons competes with aminoacyl-tRNAPyl for genetic code expansion. FEBS Lett. 586 (21), 3931-3937 (2012).
  41. Aerni, H. R., Shifman, M. A., Rogulina, S., O’Donoghue, P., Rinehart, J. Revealing the amino acid composition of proteins within an expanded genetic code. Nucleic Acids Res. 43 (2), e8 (2015).
  42. Huang, Y., et al. A convenient method for genetic incorporation of multiple noncanonical amino acids into one protein in Escherichia coli. Mol Biosyst. 6 (4), 683-686 (2010).
  43. Venkat, S., et al. Genetically encoding thioacetyl-lysine as a nondeacetylatable analog of lysine acetylation in Escherichia coli. FEBS Open. , (2017).
  44. Wan, W., Tharp, J. M., Liu, W. R. Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool. Biochim Biophys Acta. 1844 (6), 1059-1070 (2014).
  45. Gregoretti, I. V., Lee, Y. M., Goodson, H. V. Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis. J Mol Biol. 338 (1), 17-31 (2004).
  46. Weinert, B. T., et al. Acetyl-phosphate is a critical determinant of lysine acetylation in E. coli. Mol cell. 51 (2), 265-272 (2013).

Tags

Site-specific AcetylationProtein AcetylationGenetic Code ExpansionPyrrolysyl-tRNA SynthetaseAmber Stop CodonE. Coli ExpressionSite-directed MutagenesisAcetyllysine Incorporation

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Venkat, S., Gregory, C., Meng, K., Gan, Q., Fan, C. A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli. J. Vis. Exp. (130), e57061, doi:10.3791/57061 (2017).
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