期刊
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
卷 53, 期 24, 页码 6101-6104出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.201402519
关键词
enzymes; peptides; protein engineering; protein modifications; protein stability
资金
- BBSRC
- Sekisui Diagnostics
- Clarendon Fund
- New College Oxford
- Worcester College Oxford
- Oxford University Department of Biochemistry
- Biotechnology and Biological Sciences Research Council [1243565] Funding Source: researchfish
SpyTag is a peptide that spontaneously forms an amide bond with its protein partner SpyCatcher. SpyTag was fused at the N terminus of beta-lactamase and SpyCatcher at the C terminus so that the partners could react to lock together the termini of the enzyme. The wild-type enzyme aggregates above 37 degrees C, with irreversible loss of activity. Cyclized beta-lactamase was soluble even after heating at 100 degrees C; after cooling, the catalytic activity was restored. SpyTag/SpyCatcher cyclization led to a much larger increase in stability than that achieved through point mutation or alternative approaches to cyclization. Cyclized dihydrofolate reductase was similarly resilient. Analyzing unfolding through calorimetry indicated that cyclization did not increase the unfolding temperature but rather facilitated refolding after thermal stress. SpyTag/SpyCatcher sandwiching represents a simple and efficient route to enzyme cyclization, with potential to greatly enhance the robustness of biocatalysts.
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