Efficient Multiple Domain Ligation for Proteins Using Asparaginyl Endopeptidase by Selection of Appropriate Ligation Sites Based on Steric Hindrance.

Autor: Okuda A; Institute for Integrated Radiation and Nuclear Science, Kyoto University, Kumatori, Sennan-gun, Osaka 590-0494, Japan., Shimizu M; Institute for Integrated Radiation and Nuclear Science, Kyoto University, Kumatori, Sennan-gun, Osaka 590-0494, Japan., Inoue R; Institute for Integrated Radiation and Nuclear Science, Kyoto University, Kumatori, Sennan-gun, Osaka 590-0494, Japan., Urade R; Institute for Integrated Radiation and Nuclear Science, Kyoto University, Kumatori, Sennan-gun, Osaka 590-0494, Japan., Sugiyama M; Institute for Integrated Radiation and Nuclear Science, Kyoto University, Kumatori, Sennan-gun, Osaka 590-0494, Japan.
Jazyk: angličtina
Zdroj: Angewandte Chemie (International ed. in English) [Angew Chem Int Ed Engl] 2023 Jan 02; Vol. 62 (1), pp. e202214412. Date of Electronic Publication: 2022 Dec 01.
DOI: 10.1002/anie.202214412
Abstrakt: Three domain fragments of a multi-domain protein, ER-60, were ligated in two short linker regions using asparaginyl endopeptidase not involving denaturation. To identify appropriate ligation sites, by selecting several potential ligation sites with fewer mutations around two short linker regions, their ligation efficiencies and the functions of the ligated ER-60s were examined experimentally. To evaluate the dependence of ligation efficiencies on the ligation sites computationally, steric hinderances around the sites for the ligation were calculated through molecular dynamics simulations. Utilizing the steric hindrance, a site-dependent ligation potential index was introduced as reproducing the experimental ligation efficiency. Referring to this index, the reconstruction of ER-60 was succeeded by the ligation of the three domains for the first time. In addition, the new ligation potential index well-worked for application to other domain ligations. Therefore, the index may serve as a more time-effective tool for multi-site ligations.
(© 2022 Wiley-VCH GmbH.)
Databáze: MEDLINE
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