Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Summary

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

Abstract

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 µg/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per µg of protein produced or $0.019 per µL of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Introduction

Cell-free Protein Synthesis (CFPS) has emerged as a technology that has unlocked a number of new opportunities for protein production, functional genomics, metabolic engineering, and more within the last 50 years^1,2. Compared to standard in vivo protein expression platforms, CFPS provides three key advantages: 1) the cell-free nature of the platform enables the production of proteins that would be potentially toxic or foreign to the cell^3,4,5,

Protocol

1. Media Preparation and Cell Growth

1. Day 1
   1. Streak E. coli BL21*(DE3) cells from a glycerol stock onto an LB agar plate and incubate for at least 18 h at 37 °C.
   2. Prepare 50 mL of LB media and autoclave the solution on a liquid cycle for 30 min at 121 °C. Store at room temperature.
2. Day 2
   1. Prepare 750 mL of 2x YTP media and 250 mL of 0.4 M D-Glucose solution as described in the supplemental information.
   2. Pour the 2x YTP media into an autoclaved 2.5 L baffled flask and the D-Glucose solution into an autoclaved 500 mL glass bottle. Autoclave b....

Results

We have presented a sonication-based E. coli extract preparation protocol that can be completed over a four-day span, with Figure 1 demonstrating the procedural breakdown over each day. There is malleability to the steps that can be completed in each day with various pausing points, but we have found this workflow to be the most effective to execute. Additionally, both the cell pellets (step 1.3.18) and fully prepared extract (step 2.10) are stable a.......

Discussion

Cell-free protein synthesis has emerged as a powerful and enabling technology for a variety of applications ranging from biomanufacturing to rapid prototyping of biochemical systems. The breadth of applications is supported by the capacity to monitor, manipulate, and augment cellular machinery in real-time. In spite of the expanding impact of this platform technology, broad adaptation has remained slow due to technical nuances in the implementation of the methods. Through this effort, we aim to provide simplicity and cla.......

Materials

Name	Company	Catalog Number	Comments
Luria Broth	ThermoFisher	12795027
Tryptone	Fisher Bioreagents	73049-73-7
Yeast Extract	Fisher Bioreagents	1/2/8013
NaCl	Sigma-Aldrich	S3014-1KG
Potassium Phosphate Dibasic	Sigma-Aldrich	60353-250G
Potassium Phosphate Monobasic	Sigma-Aldrich	P9791-500G
D-Glucose	Sigma-Aldrich	G8270-1KG
KOH	Sigma-Aldrich	P5958-500G
IPTG	Sigma-Aldrich	I6758-1G
Mg(OAc)2	Sigma-Aldrich	M5661-250G
K(OAc)	Sigma-Aldrich	P1190-1KG
Tris(OAc)	Sigma-Aldrich	T6066-500G
DTT	ThermoFisher	15508013
tRNA	Sigma-Aldrich	10109541001
Folinic Acid	Sigma-Aldrich	F7878-100MG
NTPs	ThermoFisher	R0481
Oxalic Acid	Sigma-Aldrich	P0963-100G
NAD	Sigma-Aldrich	N8535-15VL
CoA	Sigma-Aldrich	C3144-25MG
PEP	Sigma-Aldrich	860077-250MG
K(Glu)	Sigma-Aldrich	G1501-500G
NH4(Glu)	MP Biomedicals	02180595.1
Mg(Glu)2	Sigma-Aldrich	49605-250G
Spermidine	Sigma-Aldrich	S0266-5G
Putrescine	Sigma-Aldrich	D13208-25G
HEPES	ThermoFisher	11344041
Molecular Grade Water	Sigma-Aldrich	7732-18-5
L-Aspartic Acid	Sigma-Aldrich	A7219-100G
L-Valine	Sigma-Aldrich	V0500-25G
L-Tryptophan	Sigma-Aldrich	T0254-25G
L-Phenylalanine	Sigma-Aldrich	P2126-100G
L-Isoleucine	Sigma-Aldrich	I2752-25G
L-Leucine	Sigma-Aldrich	L8000-25G
L-Cysteine	Sigma-Aldrich	C7352-25G
L-Methionine	Sigma-Aldrich	M9625-25G
L-Alanine	Sigma-Aldrich	A7627-100G
L-Arginine	Sigma-Aldrich	A8094-25G
L-Asparagine	Sigma-Aldrich	A0884-25G
Glycine	Sigma-Aldrich	G7126-100G
L-Glutamine	Sigma-Aldrich	G3126-250G
L-Histadine	Sigma-Aldrich	H8000-25G
L-Lysine	Sigma-Aldrich	L5501-25G
L-Proline	Sigma-Aldrich	P0380-100G
L-Serine	Sigma-Aldrich	S4500-100G
L-Threonine	Sigma-Aldrich	T8625-25G
L-Tyrosine	Sigma-Aldrich	T3754-100G
Fisherbrand Premium Microcentrifuge Tubes: 2.0 mL	Fisher Scientific	05-408-138
Fisherbrand Premium Microcentrifuge Tubes: 1.5 mL	Fisher Scientific	05-408-129
Fisherbrand Premium Microcentrifuge Tubes: 0.6 mL	Fisher Scientific	05-408-120
PureLink HiPure Plasmid Prep Kit	ThermoFisher	K210007
Ultrasonic Processor	QSonica	Q125-230V/50Hz	3.175 mm diameter probe
Avanti J-E Centrifuge	Beckman Coulter	369001
JLA-8.1000 Rotor	Beckman Coulter	366754
1L Centrifuge Tube	Beckman Coulter	A99028
Tunair 2.5L Baffeled Shake Flask	Sigma-Aldrich	Z710822
Microfuge 20	Beckman Coulter	B30134
New Brunswick Innova 42/42R Incubator	Eppendorf	M1335-0000
Cytation 5	BioTek
Strep-Tactin XT Starter Kit	IBA	2-4998-000
pJL1-sfGFP	Addgene	69496
BL21(DE3)	New England BioLabs