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. 2020 Jul 21;10:12117. doi: 10.1038/s41598-020-69079-8

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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SDS-PAGE and Superdex 75 profiles. After refolding and concentration by ultrafiltration, µPlg wild-type and mutants were purified on a Superdex 75 column as described⁴⁷. (A) An example of the wild-type and 5 alanine mutants. In the SEC graph, the first peak is the unfolded aggregates, and the second peak (red arrows) is the refolded peak. A non-reduced SDS-PAGE of the purified proteins is shown in the insert. (b-1) SDS-PAGE of 26 samples of the purified alanine mutants along with wild-type human and mouse samples. M is a molecular weight marker. 1. wild type; 2. W761A; 3. G762A; 4. R719A; 5. G695A; 6. G739A; 7. G739A; 8. G739A; 9. G764A; 10. T688A; 11. T688A; 12. L626A; 13. G693A; 14. K645A; 15. V624A; 16. H621A; 17. T691A; 18. V720A; 19. R610A; 20. Mouse-wild; 21. G690A; 22. L696A; 23. D740A; 24. F692A; 25. S760A; 26. L763A; 27. R582A; 28. P642A. (b-2) SDS-PAGE of purified F587 mutants. 1. F587H; 2. F587I; 3. F587K; 4. F587L; 5. F587M; 6. F587N; 7. F587P; 8. F587Q; 9. F587R; 10. F587S; 11. F587T; 12. F587Y; 13. F587W; 14. F587V; 15. Wild-type (F); 16. F587C; 17. F587D; 18. F587E; 19. F587G. (c) (c-1–c-4) are Superdex 75 purification profile and SDS-PAGE of F587 saturation mutagenesis proteins. (c-1) 1. Wild-type; 2. F587A; 3. F587C; 4. F587D; 5. F587E; 6. F587G. (c-2) 7. F587H; 8. F587I; 9. F587K; 10. F587L; 11. F587M; 12.F587N. (c-3) 13. F587P; 14. F587Q; 15. F587R; 16. F587S; 17. F587T; 18. F587V. (c-4) 19. F587W; 20. F587Y. Full-length gels are presented in Supplementary Fig. 4.