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. 2019 Mar 4;9:3331. doi: 10.1038/s41598-019-39219-w

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© The Author(s) 2019

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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rHRNR_2576–2707 is highly sensitive towards proteolysis. 10 µg rHRNR_2576–2707 (for AA-sequence see Supplementary Table 2) each was incubated for 18 h at 37 °C with the respective protease in appropriate digestion buffers. Thereafter samples were subjected to a SDS-PAGE analysis and rHRNR_2576–2707 and its fragments were detected by a HRNR-Western-Blot analysis using an in-house affinity-purified polyclonal α-HRNR_2591–2662-antibody. We used (a) 0,1 U to 0,0001 U enterokinase EKMax, (b) 1 ng to 1 pg α-chymotrypsin, (c) 100 pg to 0,1 pg trypsin (not modified), (d) 10 ng to 10 pg thermolysin and (e) 10 ng to 10 pg Lys-C. Note the extraordinary sensitivity of µg-amounts rHRNR_2576–2707 towards non-modified trypsin (c). All data shown are representatives (n = 3).