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

Bastings MM, van Baal I, Meijer EW, Merkx M - BMC Biotechnol. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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

ABSTRACT

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.

Conclusion: 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.

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On-column refolding and thioester generation. Schematic representation of the simultaneous on-column refolding and thioester generation method. Following capture on a chitin resin incubation of RNase A – intein – CBD with MESNA and diMESNA results in simultaneous refolding of the RNase A and cleavage of the RNase A from the intein domain, generating a C-terminal MESNA thioester. The order of refolding and thioester generation is arbitrary.
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Figure 4: On-column refolding and thioester generation. Schematic representation of the simultaneous on-column refolding and thioester generation method. Following capture on a chitin resin incubation of RNase A – intein – CBD with MESNA and diMESNA results in simultaneous refolding of the RNase A and cleavage of the RNase A from the intein domain, generating a C-terminal MESNA thioester. The order of refolding and thioester generation is arbitrary.

Mentions: We next tested whether protein refolding and MESNA-induced cleavage of immobilized RNase A-intein-CBD could be performed simultaneously (Figure 4). Raines and coworkers previously reported the expression of an RNase A-intein-CBD fusion protein in E. coli [18]. Because of the reducing environment of the E. coli cytoplasm, the RNase A domain was not correctly folded and inactive, but the fusion to the intein-CBD prevented its aggregation. The soluble fraction obtained after cell lysis that contained the RNase A-intein-CBD fusion protein was loaded onto a chitin column and washed to remove contaminating E. coli proteins. While keeping the ratio between MESNA and diMESNA constant at 3:1 (the ratio found in the cell's periplasm), three different concentrations of MESNA/diMESNA were tested, 3/1 mM, 30/10 mM and 75/25 mM. The highest RNase A activity was obtained with 30 mM MESNA and 10 mM diMESNA, which probably represents a compromise between thiol-induced cleavage, which is optimal at high concentrations, and the efficiency of refolding, which is optimal at low mM concentrations. As expected, incubation with only reduced MESNA (75 mM) yielded no active RNase A (Figure 5).


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

Bastings MM, van Baal I, Meijer EW, Merkx M - BMC Biotechnol. (2008)

On-column refolding and thioester generation. Schematic representation of the simultaneous on-column refolding and thioester generation method. Following capture on a chitin resin incubation of RNase A – intein – CBD with MESNA and diMESNA results in simultaneous refolding of the RNase A and cleavage of the RNase A from the intein domain, generating a C-terminal MESNA thioester. The order of refolding and thioester generation is arbitrary.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570673&req=5

Figure 4: On-column refolding and thioester generation. Schematic representation of the simultaneous on-column refolding and thioester generation method. Following capture on a chitin resin incubation of RNase A – intein – CBD with MESNA and diMESNA results in simultaneous refolding of the RNase A and cleavage of the RNase A from the intein domain, generating a C-terminal MESNA thioester. The order of refolding and thioester generation is arbitrary.
Mentions: We next tested whether protein refolding and MESNA-induced cleavage of immobilized RNase A-intein-CBD could be performed simultaneously (Figure 4). Raines and coworkers previously reported the expression of an RNase A-intein-CBD fusion protein in E. coli [18]. Because of the reducing environment of the E. coli cytoplasm, the RNase A domain was not correctly folded and inactive, but the fusion to the intein-CBD prevented its aggregation. The soluble fraction obtained after cell lysis that contained the RNase A-intein-CBD fusion protein was loaded onto a chitin column and washed to remove contaminating E. coli proteins. While keeping the ratio between MESNA and diMESNA constant at 3:1 (the ratio found in the cell's periplasm), three different concentrations of MESNA/diMESNA were tested, 3/1 mM, 30/10 mM and 75/25 mM. The highest RNase A activity was obtained with 30 mM MESNA and 10 mM diMESNA, which probably represents a compromise between thiol-induced cleavage, which is optimal at high concentrations, and the efficiency of refolding, which is optimal at low mM concentrations. As expected, incubation with only reduced MESNA (75 mM) yielded no active RNase A (Figure 5).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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

ABSTRACT

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.

Conclusion: 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.

Show MeSH