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Abstract
BackgroundExpression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed.ResultsA novel redox buffer consisting of MESNA and diMESNA showed a refolding efficiency comparable to that of GSH/GSSG and prevented loss of the protein's thioester functionality. Moreover, introduction of the MESNA/diMESNA redox couple in the cleavage buffer allowed simultaneous on-column refolding of Ribonuclease A and intein-mediated cleavage to yield Ribonuclease A with a C-terminal MESNA-thioester. The C-terminal thioester was shown to be active in native chemical ligation.ConclusionAn efficient method was developed for the production of disulfide bond containing proteins with C-terminal thioesters. Introduction of a MESNA/diMESNA redox couple resulted in simultaneous on-column refolding, purification and thioester generation of the model protein Ribonuclease A.
Highlights
Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester
Using Ribonuclease A as model protein we show that inclusion of diMESNA during intein-mediated cleavage results in simultaneous on column refolding and thioester formation, providing a novel method for the production of correctly folded, disulfide-bridge containing proteins with a C-terminal thioester suitable for native chemical ligation
To investigate the stability of protein thioesters in a glutathione redox buffer, green fluorescent protein (GFP) with C-terminal mercaptoethane sulphonic acid (MESNA) thioester was used as a test protein and incubated for 10 hours in glutathione refolding buffer
Summary
Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Native chemical ligation (NCL) is a chemoselective reaction of a C-terminal thioester with an N-terminal cysteine to yield a native peptide bond under aqueous conditions This method was originally developed by Dawson et al in 1994 [1] to allow the chemical synthesis of proteins from smaller peptide fragments obtained by solid phase peptide synthesis [1,2,3,4].
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