Product Class: Kit

NEBExpress® Cell-free E. coli Protein Synthesis System


Catalog #E5360

Product Introduction

The NEBExpress® Cell-free E. coli Protein Synthesis System is an extract-based transcription/translation system derived from E. coli cells that have been engineered for high in vitro synthesis performance. It is designed to synthesize proteins ranging in size from 17-230 kDa in just 2-4 hours under the control of T7 RNA Polymerase.

  • Synthesize high yields of protein (typically 0.5 mg/ml) 
  • Protein can be synthesized and visualized in approximately 2 - 4 hours
  • Synthesize various target proteins ranging from 17 to 230 kDa
  • Templates can be plasmid DNA, linear DNA or mRNA.
  • Minimal protease activity ensures stability of desired target protein
  • RNase contamination can be inhibited by the supplied RNase inhibitor, eliminating clean-up steps
  • Protein can be isolated with affinity purification or subject to direct functional analysis
  • Reactions can be directly loaded onto an SDS-PAGE gel without the need for acetone or TCA precipitation
  • Flexible reaction conditions achieve maximum yield; protein synthesis can be sustained for 10 hours at 37°C or up to 24 hours at lower temperatures
  • Reactions can be miniaturized or scaled up
  • Compatible with the PURExpress Disulfide Bond Enhancer (NEB #E6820) and NEBExpress GamS Nuclease Inhibitor (NEB #P0774)

Product Information

Description

The NEBExpress® Cell-free E. coli Protein Synthesis System is a coupled transcription/translation system designed to synthesize proteins encoded by a DNA template under the control of a T7 RNA Polymerase promoter. The system offers high expression levels, the ability to produce high molecular weight proteins, scalability, and is cost-effective for high throughput expression applications.  The speed and robustness of the system facilitates protein synthesis in applications such as protein engineering, mutagenesis studies and enzyme screening. In addition, it can be used to generate proteins for biophysical and structure-function analyses.

The NEBExpress® Cell-free E. coli Protein Synthesis System contains all the components required for protein synthesis, except for the target template DNA. It is a combination of a highly active cell extract from a genetically engineered strain, a reaction buffer, and an optimized T7 RNA Polymerase, which together yield robust expression of a wide variety of protein targets ranging from 17 to 230 KDa.

Protein synthesis is achieved with a short incubation and synthesized protein is compatible with downstream purification or analysis by SDS-PAGE, Western Blot or direct functional assay. The novel formulation of this system allows samples to be loaded directly onto SDS-PAGE, without the need for acetone or TCA precipitation.  Additionally, synthesized protein can be isolated from the reaction mixture using affinity purification techniques, such as immobilized metal affinity chromatography (IMAC), for further structural and/or functional characterization.

Figure 1: Protein synthesis using the NEBExpress®Cell-free E. coli Protein Synthesis System

 
50 µl reactions containing 250 ng template DNA were incubated at 37°C for 3 hours. 2 µl of each reaction were analyzed by SDS-PAGE using a 10–20% Tris-glycine gel. The red dot indicates the protein of interest. M = Unstained Protein Standard, Broad range (NEB #P7717), “Neg” = negative control, no DNA.


This product is related to the following categories:
NEBExpress® Cell-free E. coli Protein Synthesis System,
Cell-Free Protein Expression Products,
Protein Expression Products,
This product can be used in the following applications:
NEBExpress® Cell-free E. coli Protein Synthesis System,
Cell-Free Protein Expression,
High-throughput cloning and automation solutions,
Protein Expression

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E5360S     -80    
      NEBExpress™ S30 Synthesis Extract P0864SVIAL -80 1 x 0.125 ml Not Applicable
      Protein Synthesis Buffer (2X) B0864SVIAL -80 1 x 0.25 ml 2 X
      RNase Inhibitor, Murine M1018AVIAL -20 1 x 0.015 ml 40,000 units/ml
      T7 RNA Polymerase M1019AVIAL -20 1 x 0.015 ml Not Applicable
      NEBExpress® Control DHFR-His Plasmid N3273AVIAL -20 1 x 0.01 ml 125 ng/µl
  • E5360L     -80    
      NEBExpress™ S30 Synthesis Extract P0864SVIAL -80 10 x 0.125 ml Not Applicable
      Protein Synthesis Buffer (2X) B0864SVIAL -80 10 x 0.25 ml 2 X
      RNase Inhibitor, Murine M1018AAVIAL -20 1 x 0.15 ml 40,000 units/ml
      T7 RNA Polymerase M1019AAVIAL -20 1 x 0.15 ml Not Applicable
      NEBExpress® Control DHFR-His Plasmid N3273AVIAL -20 1 x 0.01 ml 125 ng/µl

Properties & Usage

Features

  • Quickly generate analytical amounts of protein for further characterization.  
  • Synthesizes various target proteins ranging in size from 17 to 230 kDa
  • Templates can be either plasmid DNA, linear DNA, or mRNA.
  • Minimal protease activity ensures stability of desired target protein
  • Plasmid template does not require additional clean up steps, RNase contamination can be inhibited by the supplied RNase inhibitor 
  • Protein can be isolated with affinity purification or subject to direct functional analysis
  • Reactions can be directly loaded onto an SDS-PAGE gel without the need for acetone or TCA precipitation
  • Flexible reaction conditions to achieve maximum yield: protein synthesis can be sustained for 10 hours at 37°C or up to 24 hours at lower temperatures without supplementation
  • Synthesis reactions can be easily miniaturized or scaled up to yield milligram quantities of protein
  • Kit contains an optimized T7 RNA Polymerase for maximum performance

Application Features

  • High-throughput screening and liquid handling
  • Mutation studies: effect of point mutations, deletions and insertions; rapidly identify active domains and functional residues
  • Epitope mapping and protein folding
  • Expression of toxic proteins

Product Notes

  1. A detailed product manual, with protocols can be found here.
  2. The typical incubation temperature is 37°C. Lowering the incubation temperature can help the folding of some target proteins. The reaction can be incubated at temperatures from 16°C to 42°C and protein synthesis will continue steadily for 10 hours at 37°C or up to 24 hours at lower temperatures, depending on DNA input and aeration.
  3. Plasmid DNA prepared from some commercial kits may contain trace amounts of RNase A. The RNase A contamination can be inhibited by the supplied RNase Inhibitor, and therefore no additional clean-up steps are necessary.
  4. In vitro protein synthesis reactions produced by the NEBExpress Cell-Free E. coli Protein Synthesis System can be directly loaded onto an SDS-PAGE gel without the need for acetone or TCA precipitation.
  5. The system is compatible with linear DNA. To achieve expression levels comparable to a plasmid template, include NEBExpress GamS Nuclease Inhibitor (NEB #P0774S).
  6. The protein synthesis reaction can be scaled up; all reaction components, including the DNA template, must be scaled proportionally. The system has been tested with reaction volumes as small as 1 µl and as large as 2 mL. Protein synthesis yields are comparable within this range. For small volumes, such as 1 µl, an acoustic droplet ejection liquid handler (or similar) should be used. For large volumes use a vessel with adequate headspace to allow for aeration and incubate with agitation.

Protocols, Manuals & Usage

Protocols

  1. Quick Start Protocol for NEBExpress® Cell-free E. coli Protein Synthesis System
  2. NEBExpress Cell-free E.coli Protein Synthesis SDS-PAGE Protocol
  3. Typical Protocol for NEBExpress® GamS Nuclease Inhibitor when used with the NEBExpress® Cell-free E. coli Protein Synthesis System (NEB #E5360)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Application Notes

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. Based on my application, should I use the NEBExpress® Cell-free E. coli Protein Synthesis System (NEB #E5360) or the PURExpress® In Vitro Protein Synthesis Kit (NEB #E6800)? 
  2. What is the difference between NEBExpress® Cell-free E. coli Protein Synthesis System and the PURExpress In Vitro Protein Synthesis Kit?
  3. Are the yields with NEBExpress® Cell-free E. coli Protein Synthesis System comparable with the PURExpress In Vitro Protein Synthesis Kit?
  4. Can I express large proteins with NEBExpress Cell-free E. coli Protein Synthesis System?
  5. Can I use the NEBExpress Cell-free E. coli Protein Synthesis System to incorporate unnatural amino acids or radiolabeled amino acids?
  6. Can the reaction be scaled to smaller or larger volumes?
  7. What methods can be used to purify the synthesized protein?
  8. Can I co-express different protein targets from different plasmids? 
  9. Do I need to purify my plasmid or linear DNA template prior to synthesis?
  10. Are genes under bacterial promoter control expressed with the NEBExpress® Cell-free E. coli Protein Synthesis System?
  11. What systems does NEB offer for protein expression and purification?
  12. Will the target protein remain stable during the synthesis incubation at 37°C?
  13. Following the NEBExpress cell-free protein synthesis reaction, how should I analyze the protein? Is it possible to use native SDS-PAGE or other functional assays to determine the activity of the expressed protein?
  14. Can I add an RNA polymerase (RNAP) other than T7 RNAP to my NEBExpress Cell-free E. coli Protein Synthesis reactions?
  15. Can I use a different concentration or formulation of T7 RNA Polymerase in my NEBExpress Cell-free E. coli Protein Synthesis reactions?
  16. What size is the NEBExpress® Control DHFR-His Plasmid?
  17. The protocol recommends shaking the reactions vigorously. Is this necessary?
  18. What size is the NEBExpress® Control DHFR-His Plasmid?
  19. The protocol recommends shaking the reactions vigorously. Is this required?

Troubleshooting

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Licenses

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc. For more information about commercial rights, please email us atgbd@neb.com While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
 

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