Engineering Intracellular Protein Sensors in Mammalian Cells

Summary

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.

Abstract

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.

Introduction

The study and modulations of cellular responses via controllable engineered gene circuits are major goals in synthetic biology^1,2,3 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....

Protocol

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 tested^28,29,30<.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
AlexaFluor 647 mouse anti-human HLA-A, B, C antibody clone W6/32	Biolegend	311414	Antibodies
Annexin V	LifeTechnologies	A35122	Apoptosis marker
Attractene	Qiagen	301005	Transfection reagent
BD Falcon Round-Bottom Tubes	BD Biosciences	352053	FACS tubes
Doxycycline	Clonetech	-	Cell Culture: Drugs
Dulbecco's modified Eagle medium	Cellgro	10-013-CM	Cell Culture: Medium
Evos Cell Imaging System	Life Technology	EVOS M5000	Imaging systems; Infectious molecular clones
FACSDiva8 software	BD Biosciences	659523	FACS software
Fast SYBR Green Master Mix	ThermoFisher Scientific	4385612	qPCR reaction
FBS (Fetal Bovin Serum)	Atlanta BIO	S11050	Cell Culture: Medium
Gateway System	Life Technologies	-	Plasmid Construction
Golden Gate System	in-house	-	Plasmid Construction
HEK 293FT	Invitrogen	R70007	Cell Culture: Cells
Infusion Cloning System	Clonetech	638920	Plasmid Construction
JetPRIME reagent	Polyplus transfection	114-15	Transcfection reagent
Jurkat Cells	ATCC	TIB-152	Cell Culture: Cells
L-Glutamine	Sigma-Aldrich	G7513-100ML	Cell Culture: Medium
Lipofectamine LTX with Plus Reagent	Thermo Fisher Scientific	15338030	Transfection reagent
LSR Fortessa flow cytometer (405, 488, and 561 nm lasers)	BD Biosciences	649225	Flow cytometer
MicroAmp Fast Optical 96-Well Reaction Plate (0.1 mL)	ThermoFisher Scientific	4346907	qPCR reaction
Neon Transfection System	Life Technologies	MPK10025	Transfection reagent
Non-essential amino acids	HyClone	SH3023801	Cell Culture: Medium
Opti-MEM I reduced serum medium	Life Technologies	31985070	Transfection medium
Penicillin/Streptomycin	Sigma-Aldrich	P4458-100ML	Cell Culture: Medium
QuantiTect Reverse Transcription Kit	Qiagen	205313	Rev Transcriptase kit
RNeasy Mini Kit	Qiagen	74106	RNA extraction kit
RPMI-1640	ATCC	ATCC 30­2001	Cell Culture: Medium
Shield	Clonetech	632189	Cell Culture: Drugs
SpheroTech RCP-30-5-A beads	Spherotech	RCP-30- 5A-2	Compensation set up
StepOnePlus 7500 Fast machine	Applied Biosystems	4351106	qPCR reaction