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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.
Reagents Supplied:
PURExpress Disulfide Bond Enhancer 1
PURExpress Disulfide Bond Enhancer 2
Reaction Conditions:
Add 1 µl of each component to a PURExpress reaction (as part of the user-added 7.5 µl volume for templates and additives) and incubate reaction at 37°C for at least 2 hours.
Figure 1: 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 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) 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.
Source:
Each of the recombinant proteins present in the PURExpress Disulfide Bond Enhancer has been expressed in E. coli.
Applications:- Formation of properly folded proteins containing multiple disulfide bonds when produced by PURExpress.
Reaction & Storage Conditions
Incubate at 37°C.
Storage Conditions:
50 mM HEPES
100 mM KCl
1 mM EDTA
10% Glycerol
Storage Temperature:
-20°C
Notes
Usage notes:- 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.
Protocols
Protocols for PURExpress® Disulfide Bond Enhancer
Companion Products
PURExpress® Δ Ribosome Kit
PURExpress® In Vitro Protein Synthesis Kit
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