Product Class: Kit

PURExpress® Disulfide Bond Enhancer

Product Introduction

The PURExpress® Disulfide Bond Enhancer is a proprietary blend of proteins and buffer components formulated to correctly fold target proteins with multiple disulfide bonds.

  • Promotes a proper disulfide bond pattern by assisting with the oxidation of cysteine thiols and correcting mis-oxidized substrates
  • Increase yields of soluble and active proteins relying on correct folding via disulfide bonding
  • Compatible for both PURExpress and other in vitro protein synthesis systems
  • Convenient to use by adding to an in vitro protein synthesis reaction at the time of set up
Bioz Badge Exists : True
Catalog # Size Concentration
E6820S 50 reactions

Product Information

Description

The PURExpress® Disulfide Bond Enhancer (PDBE) is a proprietary blend of proteins and buffer components designed to correctly fold target proteins with multiple disulfide bonds produced in PURExpress reactions. Added at the beginning of a reaction, the components promote a proper disulfide bond pattern by assisting with the oxidation of cysteine thiols and correcting mis-oxidized substrates. These enhancements can increase the yield of soluble and functionally active protein.

Figure 1: PURExpress Disulfide Bond Enhancer (PDBE) promotes proper folding of active vtPA

Line graph and gel depicting effect of PURExpress Disulfide Bond Enhancer (PDBE) on vtPA folding 
PURExpress Disulfide Bond Enhancer (PDBE) promotes proper folding of active vtPA, a truncated version of tissue plasminogen activator with 9 disulfide bonds, 8 of which are non-consecutive.

(A.) Reactions were set-up according to PURExpress specifications with the vtPA template DNA. After a two-hour incubation at 37°C, 5 µl of each reaction was used in an activity assay and cleavage of the chromogenic substrate was monitored for one hour. (B.) 2.5 µl of each reaction was resolved by SDS-PAGE and the gel stained with Coomassie Blue. The vtPA target protein is marked by a red arrow.

The addition of PDBE generates active protein. The difference in observed activity is due to disulfide bond folding, as measured by functional activity of the target, and is not due to differences in the amount of protein produced by PURExpress.


Figure 2: PURExpress Disulfide Bond Enhancer optimization
Line graphs depicting PURExpress Disulfide Bond Enhancer (PDBE) with optimization various supplier kit 
PURExpress Disulfide Bond Enhancer is optimized for use in PURExpress but also shows utility in S30 lysate-based IVTT kits from other suppliers. Reactions were set-up according to manufacturer's specifications with equivalent amounts of template DNA encoding a truncated version of tissue plasminogen activator (9 disulfide bonds, 8 non-consecutive). After a two-hour incubation, 5 µl of each reaction was used in an activity assay and cleavage of the chromogenic substrate was monitored for one hour.


Figure 3: PURExpress Disulfide Bond Enhancer promotes proper folding of active Gaussia Luciferase (GLuc)

Bar graph depicting PURExpress® Disulfide Bond Enhancer effect on folding of Gaussia Luciferase (GLuc) 

PURExpress Disulfide Bond Enhancer promotes proper folding of active Gaussia Luciferase (GLuc) which contains 5 probable disulfide bonds. Reactions were set-up according to PURExpress specifications with equivalent amounts of template DNA encoding GLuc. After a two-hour incubation at 37°C, triplicate aliquots (2.5 µl) of each reaction were incubated with 50 µl of the GLuc substrate solution and the relative luminescence recorded after a 4 second counting window.
This product is related to the following categories:
NEBExpress® Cell-free E. coli Protein Synthesis System,
PURExpress® In Vitro Protein Synthesis,
Cell-Free Protein Expression,
Protein Expression,
This product can be used in the following applications:
NEBExpress® Cell-free E. coli Protein Synthesis System,
PURExpress,
Cell-Free Protein Expression,
Expression of Difficult Proteins,
Disulfide-bonded Protein Expression,
Protein Expression

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • NEB # Stored at (°C)
    E6820S -20
    Component Name Component # Stored at (°C) Amount Concentration
    PURExpress Disulfide Bond Enhancer 1 P6821AVIAL -20 1 x 0.05 ml 25 X
    Enhancer 2 B6822AVIAL -20 1 x 0.05 ml 25 X

Properties & Usage

Product Notes

  1. PURExpress Disulfide Bond Enhancer has been optimized for use with NEB PURExpress Systems (NEB #E6800, E3313). However, it is compatible with bacterial S30 lysate-based IVTT systems from other suppliers (Fig. 2). As the formulation of other systems varies, all guidelines, protocols and FAQs related to PDBE are for use with the PURExpress System.

References

  1. Harada R, Furumoto S, Yoshikawa T, Ishikawa Y, Shibuya K, Okamura N, Ishiwata K, Iwata R, Yanai K (2016). Synthesis and Characterization of 18F-Interleukin-8 Using a Cell-Free Translation System and 4-18F-Fluoro-L-Proline.. Journal of Nuclear Medicine. 57 (4), 634-639. PubMedID: 26742712
  2. Horiya S, Bailey JK, Krauss IJ (2017). Directed Evolution of Gycopeptides Using mRNA Display.. Methods in Enzymology . PubMedID: 28935113
  3. Huang A, P.Q. Nguyen, J.C. Stark, M.K. Takahashi, N. Donghia, T. Ferrante, A.J. Dy, K.J. Hsu, R.S. Dubner, K. Pardee, M.C. Jewett and J.J. Collins (2018). Biobits™ Explorer: A modular synthetic biology education kit. Science Advances. 4 (8),
  4. Mankowska, S.A., P. Gatti-Lafranconi, M. Chodorge, S. Sridharan, R.R. Minter and F. Hollfelder (2016). A Shorter Route to Antibody Binders via Quantitative in vitro Bead-Display Screening and Consensus Analysis. Scientific Reports. 6,
  5. Pardee K, Slomovic S, Nguyen PQ, Lee JW, Donghia N, Burrill D, Ferrante T, McSorley FR, Furuta Y, Vernet A, Lewandowski M, Boddy CN, Joshi NS, Collins JJ (2016). Portable, On-Demand Biomolecular Manufacturing. Cell. 248-259 e212. PubMedID: 27662092
  6. Price A.K., MacConnell A.B., Paegel B.M. (2014). Microfluidic bead suspension hopper. Analytical Chemistry. 86 (10), 5039-5044. PubMedID: 24761972
  7. Price A.K., MacConnell A.B., Paege B.M. (2016). hnuSABR: Photochemical Dose-Response Bead Screening in Droplets.. Analytical Chemistry. 88 (5), 2904-2911.
  8. Ren G, Ke N, Berkmen M (2016). Use of the SHuffle Strains in Production of Proteins. Current Protocols in Protein Science. 85, PubMedID: 27479507
  9. Tran D.T., Cavett V.J., Dang V.Q., Torres H.L., Paegel B.M. (2016). Evolution of a mass spectrometry-grade protease with PTM-directed specificity. Proceedings of the National Acadmey of Sciences of the United States of America. 113 (51), 14686-14691. PubMedID: 27940920

FAQs & Troubleshooting

FAQs

  1. Is the PURExpress® In Vitro Protein Synthesis Kit capable of dealing with disulfide bonds? If not, can you recommend something else to use post synthesis?

Tech Tips

Titration of solutions 1 and 2 for optimal protein folding may be required. Start with 1 ul of each.
Can be used with S30 lysates as well as PURExpress.

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]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

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.

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