One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester.
BACKGROUND
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. 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.


RESULTS
A 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.


CONCLUSIONS
An 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.
Bastings
Maartje M C
MM
Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, PO Box 513, 5600 MB, Eindhoven, the Netherlands. m.m.c.bastings@tue.nl
van Baal
Ingrid
I
Meijer
E W
EW
Merkx
Maarten
M
D015080:Mesna / Q000031:analogs & derivatives / Q000737:chemistry* / Q000302:isolation & purification; D010084:Oxidation-Reduction; D020011:Protective Agents / Q000737:chemistry / Q000302:isolation & purification; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012259:Ribonuclease, Pancreatic / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D020011:Protective Agents; D011994:Recombinant Proteins; C027032:2,2'-dithiodiethanesulfonic acid; D012259:Ribonuclease, Pancreatic; D015080:Mesna