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. 2018 Apr 20;8:6333. doi: 10.1038/s41598-018-24568-9

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© The Author(s) 2018

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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Characterization of single and “two-warhead” evasins. (A) Arrangement of the 2-warhead expression constructs P1243:G4S:P1156 and P1156:G4S:P1243 (not to scale). P1243 was engineered in-frame with a GGGGS (G4S) flexible linker to P1156. The construct was tagged at the C-terminus with a StrepII:8xHis purification tag (S2:HIS). (B) Colloidal Coomassie stained SDS-polyacrylamide gels showing size exclusion column fractions of P1243:G4S:P1156 (left) and P1156:G4S:P1243 (right) obtained following nickel affinity chromatography. Molecular weight ladder (kDa) is indicated in lane 1. The gel images were cropped for clarity and the uncropped images are provided in Fig.S5. (C) Binding affinities (K_d, M) and target residence times (RT, minutes) of P1243, P1243:G4S:P1156 and P1156:G4S:P1243 to human CC-chemokines using biolayer interferometry. High affinity binding is indicated as shades of green, medium affinity as yellow, and low affinity as shades of orange. Chemokines are arranged by sequence-similarity based phylogeny. A dash (-) indicates that binding was either not detected (CCL2) or that K_d could not be determined. Affinity and target residence time in each case was calculated on data obtained at five or more chemokine concentrations.