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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Genetics

Engineering Intracellular Protein Sensors in Mammalian Cells

Published: April 28th, 2020

DOI:

10.3791/60878

1Istituto Italiano di Tecnologia, 2Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology

Here, we present a protocol for engineering genetically-encoded intracellular protein sensor-actuator(s). The device specifically detects target proteins through intracellular antibodies (intrabodies) and responds by switching on gene transcriptional output. A general framework is built to rapidly replace intrabodies, enabling rapid detection of any desired protein, without altering the general architecture.

Proteins can function as biomarkers of pathological conditions, such as neurodegenerative diseases, infections or metabolic syndromes. Engineering cells to sense and respond to these biomarkers may help the understanding of molecular mechanisms underlying pathologies, as well as to develop new cell-based therapies. While several systems that detect extracellular proteins have been developed, a modular framework that can be easily re-engineered to sense different intracellular proteins was missing.

Here, we describe a protocol to implement a modular genetic platform that senses intracellular proteins and activates a specific cellular response. The device operates on intracellular antibodies or small peptides to sense with high specificity the protein of interest, triggering the transcriptional activation of output genes, through a TEV protease (TEVp)-based actuation module. TEVp is a viral protease that selectively cleaves short cognate peptides and is widely used in biotechnology and synthetic biology for its high orthogonality to the cleavage site. Specifically, we engineered devices that recognize and respond to protein-biomarkers of viral infections and genetic diseases, including mutated huntingtin, NS3 serine-protease, Tat and Nef proteins to detect Huntington’s disease, hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections, respectively. Importantly, the system can be hand tailored for the desired input-output functional outcome, such as fluorescent readouts for biosensors, stimulation of antigen presentation for immune response, or initiation of apoptosis to eliminate unhealthy cells.

The study and modulations of cellular responses via controllable engineered gene circuits are major goals in synthetic biology1,2,3 for the development of prospective tools with relevant biological or medical applications in cancer4, infections5,  metabolic diseases6, and immunology7.

Reprogramming cell functions in response to specific signals requires the design of smart interfaces that link sensing of extracellular or intracellular dynamic change....

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1. Design principles for construction and test the sensor-actuator device

  1. Select a protein of interest.
    NOTE: We designed a system for proteins located in the cytoplasm or shuttling between the cytoplasm and other compartments.
  2. Select two intrabodies binding different epitopes of the target protein. In our study we selected proteins for which the intrabodies were already developed and tested28,29,30<.......

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An architecture for modular intracellular protein detection
As shown in Figure 1, the device is composed of: 1) intrabody 1 connected to the membrane-tethered fluorescent marker (mKate) and TEVp cleavage site (TCS), followed by a transcription activator GAL4VP16 (TF); 2) intrabody 2 fused to TEV protease (TEVp), free in the cytosol; 3) a synthetic promoter responsive to GAL4VP16, driving the expression of a reporter gene. The modularity is guaranteed by intrabodies tha.......

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Until recently, interrogating cells based on intracellular environment was performed with systems developed de novo for specific targets. The present protocol describes an example of the most recent, cell engineering approach for protein sensing and actuating in one device, that can be rapidly adapted to new desired biomarkers.

This pioneering system sense intracellular proteins and provide a specific output to detect or neutralize the disease. The advantage of this class of genetic circuits i.......

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This work was supported by the Istituto Italiano di Tecnologia.

....

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NameCompanyCatalog NumberComments
AlexaFluor 647 mouse anti-human HLA-A, B, C antibody clone W6/32Biolegend311414Antibodies
Annexin VLifeTechnologiesA35122Apoptosis marker
AttracteneQiagen301005Transfection reagent
BD Falcon Round-Bottom TubesBD Biosciences352053FACS tubes
DoxycyclineClonetech-Cell Culture: Drugs
Dulbecco's modified Eagle mediumCellgro10-013-CMCell Culture: Medium
Evos Cell Imaging SystemLife TechnologyEVOS M5000Imaging systems; Infectious molecular clones
FACSDiva8 softwareBD Biosciences659523FACS software
Fast SYBR Green Master MixThermoFisher Scientific4385612qPCR reaction
FBS (Fetal Bovin Serum)Atlanta BIOS11050Cell Culture: Medium
Gateway SystemLife Technologies-Plasmid Construction
Golden Gate Systemin-house-Plasmid Construction
HEK 293FTInvitrogenR70007Cell Culture: Cells
Infusion Cloning SystemClonetech638920Plasmid Construction
JetPRIME reagentPolyplus transfection114-15Transcfection reagent
Jurkat CellsATCCTIB-152Cell Culture: Cells
L-GlutamineSigma-AldrichG7513-100MLCell Culture: Medium
Lipofectamine LTX with Plus ReagentThermo Fisher Scientific15338030Transfection reagent
LSR Fortessa flow cytometer (405, 488, and 561 nm lasers)BD Biosciences649225Flow cytometer
MicroAmp Fast Optical 96-Well Reaction Plate (0.1 mL)ThermoFisher Scientific4346907qPCR reaction
Neon Transfection SystemLife TechnologiesMPK10025Transfection reagent
Non-essential amino acidsHyCloneSH3023801Cell Culture: Medium
Opti-MEM I reduced serum mediumLife Technologies31985070Transfection medium
Penicillin/StreptomycinSigma-AldrichP4458-100MLCell Culture: Medium
QuantiTect Reverse Transcription KitQiagen205313Rev Transcriptase kit
RNeasy Mini KitQiagen74106RNA extraction kit
RPMI-1640ATCCATCC 30­2001Cell Culture: Medium
ShieldClonetech632189Cell Culture: Drugs
SpheroTech RCP-30-5-A beadsSpherotechRCP-30- 5A-2Compensation set up
StepOnePlus 7500 Fast machineApplied Biosystems4351106qPCR reaction

  1. Bernardo, D., Marucci, L., Menolascina, F., Siciliano, V. Predicting Synthetic Gene Networks. Synthetic Gene Networks: Methods and Protocols. 813, 57-81 (2012).
  2. Krams, R., et al. Mammalian synthetic biology: emerging medical applications. J. R. Soc. Interface. 12, (2015).
  3. MacDonald, J. T., Siciliano, V. Computational Sequence Design with R2oDNA Designer. Methods in molecular biology. 1651, 249-262 (2017).
  4. Zah, E., Lin, M. -. Y., Silva-Benedict, A., Jensen, M. C., Chen, Y. Y. T cells expressing CD19/CD20 bi-specific chimeric antigen receptors prevent antigen escape by malignant B cells. Cancer Immunology Research. 4, 498-508 (2016).
  5. Wu, F., Bethke, J. H., Wang, M., You, L. Quantitative and synthetic biology approaches to combat bacterial pathogens. Current Opinion in Biomedical Engineering. 4, 116-126 (2017).
  6. Ye, H., et al. Pharmaceutically controlled designer circuit for the treatment of the metabolic syndrome. Proceedings of the National Academy of Sciences of the United States of America. 110, 141-146 (2013).
  7. Caliendo, F., Dukhinova, M., Siciliano, V. Engineered Cell-Based Therapeutics: Synthetic Biology Meets Immunology. Frontiers in Bioengineering and Biotechnology. 7, (2019).
  8. Thaker, M. N., Wright, G. D. Opportunities for synthetic biology in antibiotics: Expanding glycopeptide chemical diversity. ACS Synthetic Biology. 4, 195-206 (2015).
  9. Culler, S. J., Hoff, K. G., Smolke, C. D. Reprogramming cellular behavior with RNA controllers responsive to endogenous proteins. Science. 330, 1251-1255 (2010).
  10. Ausländer, S., et al. A general design strategy for protein-responsive riboswitches in mammalian cells. Nature Methods. 11, 1154-1160 (2014).
  11. Cella, F., Siciliano, V. Protein-based parts and devices that respond to intracellular and extracellular signals in mammalian cells. Current Opinion in Chemical Biology. 52, 47-53 (2019).
  12. Miki, K., et al. Efficient Detection and Purification of Cell Populations Using Synthetic MicroRNA Switches. Cell Stem Cell. 16, 699-711 (2015).
  13. Xie, Z., Wroblewska, L., Prochazka, L., Weiss, R., Benenson, Y. Multi-input RNAi-based logic circuit for identification of specific cancer cells. Science. , 1307-1311 (2011).
  14. Sun, J., et al. Engineered proteins with sensing and activating modules for automated reprogramming of cellular functions. Nature Communications. 8, 477 (2017).
  15. Shigeto, H., et al. Insulin sensor cells for the analysis of insulin secretion responses in single living pancreatic β cells. Analyst. 144, 3765-3772 (2019).
  16. Cella, F., Wroblewska, L., Weiss, R., Siciliano, V. Engineering protein-protein devices for multilayered regulation of mRNA translation using orthogonal proteases in mammalian cells. Nature Communications. 9, (2018).
  17. Siciliano, V., et al. MiRNAs confer phenotypic robustness to gene networks by suppressing biological noise. Nature communications. 4, 2364 (2013).
  18. Barnea, G., et al. The genetic design of signaling cascades to record receptor activation. Proceedings of the National Academy of Sciences of the United States of America. 105, 64-69 (2008).
  19. Schwarz, K. A., Daringer, N. M., Dolberg, T. B., Leonard, J. N. Rewiring human cellular input–output using modular extracellular sensors. Nature Chemical Biology. 13, 202-209 (2016).
  20. Wieland, M., Fussenegger, M. Engineering molecular circuits using synthetic biology in mammalian cells. Annual review of chemical and biomolecular engineering. 3, 209-234 (2012).
  21. Siciliano, V., et al. Engineering modular intracellular protein sensor-actuator devices. Nature Communications. 9, 1881 (2018).
  22. Lin, C. . HCV NS3-4A Serine Protease. , (2006).
  23. Romani, B., Engelbrecht, S., Glashoff, R. H. Functions of Tat: the versatile protein of human immunodeficiency virus type 1. Journal of General Virology. 91, 1-12 (2010).
  24. Basmaciogullari, S., Pizzato, M. The activity of Nef on HIV-1 infectivity. Frontiers in microbiology. 5, 232 (2014).
  25. Ross, C. A., Tabrizi, S. J. Huntington's disease: from molecular pathogenesis to clinical treatment. Lancet neurology. 10, 83-98 (2011).
  26. Pfeffer, C. M., Singh, A. T. K. Apoptosis: A Target for Anticancer Therapy. International journal of molecular sciences. 19, 448 (2018).
  27. Guzzo, C., et al. The CD8-derived chemokine XCL1/lymphotactin is a conformation-dependent, broad-spectrum inhibitor of HIV-1. PLoS pathogens. 9, 1003852 (2013).
  28. Marasco, W. A., LaVecchio, J., Winkler, A. Human anti-HIV-1 tat sFv intrabodies for gene therapy of advanced HIV-1-infection and AIDS. Journal of Immunological Methods. 231, 223-238 (1999).
  29. Gal-Tanamy, M., et al. HCV NS3 serine protease-neutralizing single-chain antibodies isolated by a novel genetic screen. Journal of molecular biology. 347, 991-1003 (2005).
  30. Southwell, A. L., et al. Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity. The Journal of neuroscience the official journal of the Society for Neuroscience. 28, 9013-9020 (2008).
  31. Bouchet, J., et al. Inhibition of the Nef regulatory protein of HIV-1 by a single-domain antibody. Blood. 117, 3559-3568 (2011).
  32. Arold, S., et al. The crystal structure of HIV-1 Nef protein bound to the Fyn kinase SH3 domain suggests a role for this complex in altered T cell receptor signaling. Structure. 5, 1361-1372 (1997).
  33. Beal, J. Signal-to-Noise Ratio Measures Efficacy of Biological Computing Devices and Circuits. Frontiers in Bioengineering and Biotechnology. 3, 93 (2015).
  34. Arold, S., et al. The crystal structure of HIV-1 Nef protein bound to the Fyn kinase SH3 domain suggests a role for this complex in altered T cell receptor signaling. Structure. 5, 1361-1372 (1997).
  35. Tyshchuk, O., et al. Detection of a phosphorylated glycine-serine linker in an IgG-based fusion protein. mAbs. 9, 94-103 (2017).

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