A Step-by-Step Guide for the Production of Recombinant Fluorescent TAT-HA-Tagged Proteins and their Transduction into Mammalian Cells.
DNA molecular assembly
Ni‐NTA‐based protein purification
fluorescent protein
fusion protein
protein transduction
recombinant protein production
tagged protein
Journal
Current protocols
ISSN: 2691-1299
Titre abrégé: Curr Protoc
Pays: United States
ID NLM: 101773894
Informations de publication
Date de publication:
Mar 2024
Mar 2024
Historique:
medline:
21
3
2024
pubmed:
21
3
2024
entrez:
21
3
2024
Statut:
ppublish
Résumé
Investigating the function of target proteins for functional prospection or therapeutic applications typically requires the production and purification of recombinant proteins. The fusion of these proteins with tag peptides and fluorescently derived proteins allows the monitoring of candidate proteins using SDS-PAGE coupled with western blotting and fluorescent microscopy, respectively. However, protein engineering poses a significant challenge for many researchers. In this protocol, we describe step-by-step the engineering of a recombinant protein with various tags: TAT-HA (trans-activator of transduction-hemagglutinin), 6×His and EGFP (enhanced green fluorescent protein) or mCherry. Fusion proteins are produced in E. coli BL21(DE3) cells and purified by immobilized metal affinity chromatography (IMAC) using a Ni-nitrilotriacetic acid (NTA) column. Then, tagged recombinant proteins are introduced into cultured animal cells by using the penetrating peptide TAT-HA. Here, we present a thorough protocol providing a detailed guide encompassing every critical step from plasmid DNA molecular assembly to protein expression and subsequent purification and outlines the conditions necessary for protein transduction technology into animal cells in a comprehensive manner. We believe that this protocol will be a valuable resource for researchers seeking an exhaustive, step-by-step guide for the successful production and purification of recombinant proteins and their entry by transduction within living cells. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA cloning, molecular assembly strategies, and protein production Basic Protocol 2: Protein purification Basic Protocol 3: Protein transduction in mammalian cells.
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e1016Informations de copyright
© 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.
Références
Ai, H., Baird, M. A., Shen, Y., Davidson, M. W., & Campbell, R. E. (2014). Engineering and characterizing monomeric fluorescent proteins for live‐cell imaging applications. Nature Protocols, 9(4), 910–928. https://doi.org/10.1038/nprot.2014.054
Andersen, D. C., & Krummen, L. (2002). Recombinant protein expression for therapeutic applications. Current Opinion in Biotechnology, 13(2), 117–123. https://doi.org/10.1016/S0958‐1669(02)00300‐2
MacKay, A. J., & Szoka, F. C. Jr. (2003). HIV TAT protein transduction domain mediated cell binding and intracellular delivery of nanoparticles. Journal of Dispersion Science and Technology, 24(3–4), 465–473. https://doi.org/10.1081/DIS‐120021802
Anné, J., Maldonado, B., Van Impe, J., Van Mellaert, L., & Bernaerts, K. (2012). Recombinant protein production and streptomycetes. Journal of Biotechnology, 158(4), 159–167. https://doi.org/10.1016/j.jbiotec.2011.06.028
Assenberg, R., Wan, P. T., Geisse, S., & Mayr, L. M. (2013). Advances in recombinant protein expression for use in pharmaceutical research. Current Opinion in Structural Biology, 23(3), 393–402. https://doi.org/10.1016/j.sbi.2013.03.008
Baneyx, F. (1999). Recombinant protein expression in Escherichia coli. Current Opinion in Biotechnology, 10(5), 411–421. https://doi.org/10.1016/S0958‐1669(99)00003‐8
Baneyx, F., & Mujacic, M. (2004). Recombinant protein folding and misfolding in Escherichia coli. Nature Biotechnology, 22(11), 1399–1408. https://doi.org/10.1038/nbt1029
Barnes, L. M., Bentley, C. M., & Dickson, A. J. (2003). Stability of protein production from recombinant mammalian cells. Biotechnology and Bioengineering, 81(6), 631–639. https://doi.org/10.1002/bit.10517
Becker‐Hapak, M., McAllister, S. S., & Dowdy, S. F. (2001). TAT‐mediated protein transduction into mammalian cells. Methods, 24(3), 247–256. https://doi.org/10.1006/meth.2001.1186
Block, H., Maertens, B., Spriestersbach, A., Brinker, N., Kubicek, J., Fabis, R., Labahn, J., & Schäfer, F. (2009). Immobilized‐metal affinity chromatography (IMAC): A review. Methods in Enzymology, 463, 439–473. https://doi.org/10.1016/S0076‐6879(09)63027‐5
Bordat, A., Houvenaghel, M.‐C., & German‐Retana, S. (2015). Gibson assembly: An easy way to clone potyviral full‐length infectious cDNA clones expressing an ectopic VPg. Virology Journal, 12(1), 89. https://doi.org/10.1186/s12985‐015‐0315‐3
Brondyk, W. H. (2009). Selecting an appropriate method for expressing a recombinant protein. Methods in Enzymology, 463, 131–147. https://doi.org/10.1016/S0076‐6879(09)63011‐1
Burnett, M. J. B., & Burnett, A. C. (2020). Therapeutic recombinant protein production in plants: Challenges and opportunities. Plants, People, Planet, 2(2), 121–132. https://doi.org/10.1002/ppp3.10073
Casini, A., Storch, M., Baldwin, G. S., & Ellis, T. (2015). Bricks and blueprints: Methods and standards for DNA assembly. Nature Reviews Molecular Cell Biology, 16(9), 568–576. https://doi.org/10.1038/nrm4014
Chen, R. (2012). Bacterial expression systems for recombinant protein production: E. coli and beyond. Biotechnology Advances, 30(5), 1102–1107. https://doi.org/10.1016/j.biotechadv.2011.09.013
Cohen, S. N. (2013). DNA cloning: A personal view after 40 years. Proceedings of the National Academy of Sciences, 110(39), 15521–15529. https://doi.org/10.1073/pnas.1313397110
Cregg, J. M., Cereghino, J. L., Shi, J., & Higgins, D. R. (2000). Recombinant protein expression in Pichia pastoris. Molecular Biotechnology, 16(1), 23–52. https://doi.org/10.1385/MB:16:1:23
Current Protocols. (2006). Commonly Used Reagents. Current Protocols in Microbiology, 00, A.2A.1–A.2A.15. https://doi.org/10.1002/9780471729259.mca02as00
Eguchi, A., Akuta, T., Okuyama, H., Senda, T., Yokoi, H., Inokuchi, H., Fujita, S., Hayakawa, T., Takeda, K., Hasegawa, M., & Nakanishi, M. (2001). Protein transduction domain of HIV‐1 Tat protein promotes efficient delivery of DNA into mammalian cells. Journal of Biological Chemistry, 276(28), 26204–26210. https://doi.org/10.1074/jbc.M010625200
Farzaneh, M., Hassani, S.‐N., Mozdziak, P., & Baharvand, H. (2017). Avian embryos and related cell lines: A convenient platform for recombinant proteins and vaccine production. Biotechnology Journal, 12(5), 1600598. https://doi.org/10.1002/biot.201600598
Ferro, M. M. M., Ramos‐Sobrinho, R., Xavier, C. A. D., Zerbini, F. M., Lima, G. S. A., Nagata, T., & Assunção, I. P. (2019). New approach for the construction of infectious clones of a circular DNA plant virus using Gibson assembly. Journal of Virological Methods, 263, 20–23. https://doi.org/10.1016/j.jviromet.2018.10.017
Fittipaldi, A., & Giacca, M. (2005). Transcellular protein transduction using the Tat protein of HIV‐1. Advanced Drug Delivery Reviews, 57(4), 597–608. https://doi.org/10.1016/j.addr.2004.10.011
Ford, K. G., Souberbielle, B. E., Darling, D., & Farzaneh, F. (2001). Protein transduction: An alternative to genetic intervention? Gene Therapy, 8(1), 1–4. https://doi.org/10.1038/sj.gt.3301383
Froger, A., & Hall, J. E. (2007). Transformation of plasmid DNA into E. coli using the heat shock method. Journal of Visualized Experiments, 6, e253. https://doi.org/10.3791/253
Gaberc‐Porekar, V., & Menart, V. (2001). Perspectives of immobilized‐metal affinity chromatography. Journal of Biochemical and Biophysical Methods, 49(1), 335–360. https://doi.org/10.1016/S0165‐022X(01)00207‐X
Gibson, D. G., Young, L., Chuang, R.‐Y., Venter, J. C., Hutchison, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343–345. https://doi.org/10.1038/nmeth.1318
Gopal, G. J., & Kumar, A. (2013). Strategies for the production of recombinant protein in Escherichia coli. The Protein Journal, 32(6), 419–425. https://doi.org/10.1007/s10930‐013‐9502‐5
Greenblatt, J., & Schleif, R. (1971). Arabinose C protein: Regulation of the arabinose operon in vitro. Nature New Biology, 233(40), 166–170. https://doi.org/10.1038/newbio233166a0
Gump, J. M., & Dowdy, S. F. (2007). TAT transduction: The molecular mechanism and therapeutic prospects. Trends in Molecular Medicine, 13(10), 443–448. https://doi.org/10.1016/j.molmed.2007.08.002
Gump, J. M., June, R. K., & Dowdy, S. F. (2010). Revised role of glycosaminoglycans in TAT protein transduction domain‐mediated cellular transduction. Journal of Biological Chemistry, 285(2), 1500–1507. https://doi.org/10.1074/jbc.M109.021964
Guo, Q., Zhao, G., Hao, F., & Guan, Y. (2012). Effects of the TAT peptide orientation and relative location on the protein transduction efficiency. Chemical Biology & Drug Design, 79(5), 683–690. https://doi.org/10.1111/j.1747‐0285.2011.01315.x
Helmuth, J. A., Burckhardt, C. J., Greber, U. F., & Sbalzarini, I. F. (2009). Shape reconstruction of subcellular structures from live cell fluorescence microscopy images. Journal of Structural Biology, 167(1), 1–10. https://doi.org/10.1016/j.jsb.2009.03.017
Hopkins, R. F., Wall, V. E., & Esposito, D. (2012). Optimizing transient recombinant protein expression in mammalian cells. Methods in Molecular Biology, 801, 251–268. https://doi.org/10.1007/978‐1‐61779‐352‐3_16
Jäger, V., Büssow, K., & Schirrmann, T. (2015). Transient recombinant protein expression in mammalian cells. In M. Al‐Rubeai (Ed.), Animal Cell Culture. Cell Engineering (Vol. 9, pp. 27–64). Springer. https://doi.org/10.1007/978‐3‐319‐10320‐4_2
Jakobs, S., Subramaniam, V., Schönle, A., Jovin, T. M., & Hell, S. W. (2000). EGFP and DsRed expressing cultures of Escherichia coli imaged by confocal, two‐photon and fluorescence lifetime microscopy. FEBS Letters, 479(3), 131–135. https://doi.org/10.1016/S0014‐5793(00)01896‐2
Jang, C.‐W., & Magnuson, T. (2013). A novel selection marker for efficient DNA cloning and recombineering in E. coli. PLoS ONE, 8(2), e57075. https://doi.org/10.1371/journal.pone.0057075
Joung, Y. H., Park, S. H., Moon, K.‐B., Jeon, J.‐H., Cho, H.‐S., & Kim, H.‐S. (2016). The last ten years of advancements in plant‐derived recombinant vaccines against Hepatitis B. International Journal of Molecular Sciences, 17(10), 1715. https://doi.org/10.3390/ijms17101715
Kabouridis, P. S. (2003). Biological applications of protein transduction technology. Trends in Biotechnology, 21(11), 498–503. https://doi.org/10.1016/j.tibtech.2003.09.008
Kaplan, I. M., Wadia, J. S., & Dowdy, S. F. (2005). Cationic TAT peptide transduction domain enters cells by macropinocytosis. Journal of Controlled Release, 102(1), 247–253. https://doi.org/10.1016/j.jconrel.2004.10.018
Kirchhausen, T. (2009). Imaging endocytic clathrin structures in living cells. Trends in Cell Biology, 19(11), 596–605. https://doi.org/10.1016/j.tcb.2009.09.002
Kostylev, M., Otwell, A. E., Richardson, R. E., & Suzuki, Y. (2015). Cloning should be simple: Escherichia coli DH5α‐mediated assembly of multiple DNA fragments with short end homologies. PLoS ONE, 10(9), e0137466. https://doi.org/10.1371/journal.pone.0137466
Leifert, J. A., Harkins, S., & Whitton, J. L. (2002). Full‐length proteins attached to the HIV tat protein transduction domain are neither transduced between cells, nor exhibit enhanced immunogenicity. Gene Therapy, 9(21), 1422–1428. https://doi.org/10.1038/sj.gt.3301819
Li, C., Ji, C., Huguet‐Tapia, J. C., White, F. F., Dong, H., & Yang, B. (2019). An efficient method to clone TAL effector genes from Xanthomonas oryzae using Gibson assembly. Molecular Plant Pathology, 20(10), 1453–1462. https://doi.org/10.1111/mpp.12820
Li, L., Jiang, W., & Lu, Y. (2018). A modified Gibson assembly method for cloning large DNA fragments with high GC contents. Methods in Molecular Biology, 1671, 203–209. https://doi.org/10.1007/978‐1‐4939‐7295‐1_13
Li, Z.‐N., Jelkmann, W., Sun, P., & Zhang, L. (2020). Construction of full‐length infectious cDNA clones of apple stem grooving virus using Gibson assembly method. Virus Research, 276, 197790. https://doi.org/10.1016/j.virusres.2019.197790
Mattanovich, D., Branduardi, P., Dato, L., Gasser, B., Sauer, M., & Porro, D. (2012). Recombinant protein production in yeasts. Methods in Molecular Biology, 824, 329–358. https://doi.org/10.1007/978‐1‐61779‐433‐9_17
McDonald, D., Vodicka, M. A., Lucero, G., Svitkina, T. M., Borisy, G. G., Emerman, M., & Hope, T. J. (2002). Visualization of the intracellular behavior of HIV in living cells. Journal of Cell Biology, 159(3), 441–452. https://doi.org/10.1083/jcb.200203150
McRae, S. R., Brown, C. L., & Bushell, G. R. (2005). Rapid purification of EGFP, EYFP, and ECFP with high yield and purity. Protein Expression and Purification, 41(1), 121–127. https://doi.org/10.1016/j.pep.2004.12.030
Newman, R. H., Fosbrink, M. D., & Zhang, J. (2011). Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells. Chemical Reviews, 111(5), 3614–3666. https://doi.org/10.1021/cr100002u
Nienhaus, K., & Nienhaus, G. U. (2014). Fluorescent proteins for live‐cell imaging with super‐resolution. Chemical Society Reviews, 43(4), 1088–1106. https://doi.org/10.1039/C3CS60171D
Noguchi, H., & Matsumoto, S. (2006). Protein transduction technology: A novel therapeutic perspective. Acta Medica Okayama, 60(1), 1–11. http://doi.org/10.18926/AMO/30757
Nolte, C., Matyash, M., Pivneva, T., Schipke, C. G., Ohlemeyer, C., Hanisch, U.‐K., Kirchhoff, F., & Kettenmann, H. (2001). GFAP promoter‐controlled EGFP‐expressing transgenic mice: A tool to visualize astrocytes and astrogliosis in living brain tissue. Glia, 33(1), 72–86.
Norris, A. D., Kim, H.‐M., Colaiácovo, M. P., & Calarco, J. A. (2015). Efficient genome editing in Caenorhabditis elegans with a toolkit of dual‐marker selection cassettes. Genetics, 201(2), 449–458. https://doi.org/10.1534/genetics.115.180679
Overton, T. W. (2014). Recombinant protein production in bacterial hosts. Drug Discovery Today, 19(5), 590–601. https://doi.org/10.1016/j.drudis.2013.11.008
Pawson, T., & Nash, P. (2000). Protein‐protein interactions define specificity in signal transduction. Genes & Development, 14(9), 1027–1047. https://doi.org/10.1101/gad.14.9.1027
Porath, J. (1992). Immobilized metal ion affinity chromatography. Protein Expression and Purification, 3(4), 263–281. https://doi.org/10.1016/1046‐5928(92)90001‐D
Porro, D., Sauer, M., Branduardi, P., & Mattanovich, D. (2005). Recombinant protein production in yeasts. Molecular Biotechnology, 31(3), 245–259. https://doi.org/10.1385/MB:31:3:245
Rizzuto, R., Brini, M., Pizzo, P., Murgia, M., & Pozzan, T. (1995). Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Current Biology, 5(6), 635–642. https://doi.org/10.1016/S0960‐9822(95)00128‐X
Roberts, M. A. J. (2019). Recombinant DNA technology and DNA sequencing. Essays in Biochemistry, 63(4), 457–468. https://doi.org/10.1042/EBC20180039
Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: Advances and challenges. Frontiers in Microbiology, 5, 172. https://www.frontiersin.org/articles/10.3389/fmicb.2014.00172
Rudenko, O., & Barnes, A. C. (2018). Gibson assembly facilitates bacterial allelic exchange mutagenesis. Journal of Microbiological Methods, 144, 157–163. https://doi.org/10.1016/j.mimet.2017.11.023
Salomonnson, E., Mihalko, L. A., Luker, K. E., Verkhusha, V. V., & Luker, G. D. (2012). Cell‐based and in vivo spectral analysis of fluorescent proteins for multiphoton microscopy. Journal of Biomedical Optics, 17(9), 096001. https://doi.org/10.1117/1.JBO.17.9.096001
Sands, B., & Brent, R. (2016). Overview of post Cohen‐Boyer methods for single segment cloning and for multisegment DNA assembly. Current Protocols in Molecular Biology, 113(1), 3.26.1–3.26.20. https://doi.org/10.1002/0471142727.mb0326s113
Schleif, R. (2000). Regulation of the L‐arabinose operon of Escherichia coli. Trends in Genetics, 16(12), 559–565. https://doi.org/10.1016/S0168‐9525(00)02153‐3
Schleif, R., Hess, W., Finkelstein, S., & Ellis, D. (1973). Induction kinetics of the L‐arabinose operon of Escherichia coli. Journal of Bacteriology, 115(1), 9–14. https://doi.org/10.1128/jb.115.1.9‐14.1973
Schwarze, S. R., Hruska, K. A., Dowdy, S. F., Schwarze, S. R., Hruska, K. A., & Dowdy, S. F. (2000). Protein transduction: Unrestricted delivery into all cells? Trends in Cell Biology, 10(7), 290–295. https://doi.org/10.1016/S0962‐8924(00)01771‐2
Shen, Y., Chen, Y., Wu, J., Shaner, N. C., & Campbell, R. E. (2017). Engineering of mCherry variants with long Stokes shift, red‐shifted fluorescence, and low cytotoxicity. PLoS ONE, 12(2), e0171257. https://doi.org/10.1371/journal.pone.0171257
Shokolenko, I. N., Alexeyev, M. F., LeDoux, S. P., & Wilson, G. L. (2005). TAT‐mediated protein transduction and targeted delivery of fusion proteins into mitochondria of breast cancer cells. DNA Repair, 4(4), 511–518. https://doi.org/10.1016/j.dnarep.2004.11.009
Sørensen, H. P., & Mortensen, K. K. (2005). Advanced genetic strategies for recombinant protein expression in Escherichia coli. Journal of Biotechnology, 115(2), 113–128. https://doi.org/10.1016/j.jbiotec.2004.08.004
Subach, F. V., Patterson, G. H., Manley, S., Gillette, J. M., Lippincott‐Schwartz, J., & Verkhusha, V. V. (2009). Photoactivatable mCherry for high‐resolution two‐color fluorescence microscopy. Nature Methods, 6(2), 153–159. https://doi.org/10.1038/nmeth.1298
Sun, X., Chiu, J.‐F., & He, Q.‐Y. (2005). Application of immobilized metal affinity chromatography in proteomics. Expert Review of Proteomics, 2(5), 649–657. https://doi.org/10.1586/14789450.2.5.649
Sunley, K., & Butler, M. (2010). Strategies for the enhancement of recombinant protein production from mammalian cells by growth arrest. Biotechnology Advances, 28(3), 385–394. https://doi.org/10.1016/j.biotechadv.2010.02.003
Thomas, S., Maynard, N. D., & Gill, J. (2015). DNA library construction using Gibson assembly®. Nature Methods, 12(11), i–ii. https://doi.org/10.1038/nmeth.f.384
Wadia, J. S., & Dowdy, S. F. (2002). Protein transduction technology. Current Opinion in Biotechnology, 13(1), 52–56. https://doi.org/10.1016/S0958‐1669(02)00284‐7
Wang, J. W., Wang, A., Li, K., Wang, B., Jin, S., Reiser, M., & Lockey, R. F. (2015). CRISPR/Cas9 nuclease cleavage combined with Gibson assembly for seamless cloning. Biotechniques, 58(4), 161–170. https://doi.org/10.2144/000114261
Wang, Y., Shyy, J. Y. J., & Chien, S. (2008). Fluorescence proteins, live‐cell imaging, and mechanobiology: Seeing is believing. Annual Review of Biomedical Engineering, 10(1), 1–38. https://doi.org/10.1146/annurev.bioeng.010308.161731
Wiedenmann, J., Oswald, F., & Nienhaus, G. U. (2009). Fluorescent proteins for live cell imaging: Opportunities, limitations, and challenges. IUBMB Life, 61(11), 1029–1042. https://doi.org/10.1002/iub.256
Wurm, F. M. (2004). Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnology, 22(11), 1393–1398. https://doi.org/10.1038/nbt1026
Yin, J., Zhu, D., Zhang, Z., Wang, W., Fan, J., Men, D., Deng, J., Wei, H., Zhang, X.‐E., & Cui, Z. (2013). Imaging of mRNA–protein interactions in live cells using novel mCherry trimolecular fluorescence complementation systems. PLoS ONE, 8(11), e80851. https://doi.org/10.1371/journal.pone.0080851
Zhang, H., Ma, Y., Gu, J., Liao, B., Li, J., Wong, J., & Jin, Y. (2012). Reprogramming of somatic cells via TAT‐mediated protein transduction of recombinant factors. Biomaterials, 33(20), 5047–5055. https://doi.org/10.1016/j.biomaterials.2012.03.061