期刊
NATURE CHEMICAL BIOLOGY
卷 14, 期 1, 页码 50-+出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/NCHEMBIO.2521
关键词
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资金
- NIH [5R01GM081051-09]
- Harry and Dianna Hind Professorship in Pharmaceutical Sciences
- Helen Hay Whitney Foundation [F-1112]
- Burroughs Wellcome Fund [1017065]
- NATIONAL CANCER INSTITUTE [R01CA191018] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM081051] Funding Source: NIH RePORTER
Enzyme-catalyzed peptide ligation is a powerful tool for site-specific protein bioconjugation, but stringent enzyme-substrate specificity limits its utility. We developed an approach for comprehensively characterizing peptide ligase specificity for N termini using proteome-derived peptide libraries. We used this strategy to characterize the ligation efficiency for >25,000 enzyme-substrate pairs in the context of the engineered peptide ligase subtiligase and identified a family of 72 mutant subtiligases with activity toward N-terminal sequences that were previously recalcitrant to modification. We applied these mutants individually for site-specific bioconjugation of purified proteins, including antibodies, and in algorithmically selected combinations for sequencing of the cellular N terminome with reduced sequence bias. We also developed a web application to enable algorithmic selection of the most efficient subtiligase variant(s) for bioconjugation to user-defined sequences. Our methods provide a new toolbox of enzymes for site-specific protein modification and a general approach for rapidly defining and engineering peptide ligase specificity.
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