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. 2019 Jul 25;9:10820. doi: 10.1038/s41598-019-47273-7

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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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An intrinsically disordered protein with AEP recognition residues can be backbone cyclised. (a) Protein sequence of the MSP2_AEP produced recombinantly. The N-terminal recognition motif (NTRM; ML) and C-terminal recognition motif (CTRM; GLPSLAA) are shown in bold. The NTRM was altered to ML to accommodate the initiating methionine. ▾Denotes the target AEP processing site which is followed by the CTRM and a C-terminal 6xHis tag (shown in italics). ∇Denotes potential off-target AEP processing sites, as determined by similarity to the native minimal CTRM, N/GL. (b) SDS PAGE analysis of MSP2 (3 µg) incubated with and without recombinant OaAEP1_b. The assays were conducted in activity buffer without reducing agent (50 mM sodium acetate buffer, pH 5.0, 50 mM NaCl, 1 mM EDTA). The dominant processing product indicated by ◂ was purified by size exclusion chromatography and confirmed to be the cyclic product by ESI-MS. MSP2 only and enzyme only controls are also shown. A single representative experiment of two technical replicates is shown. The gel has been cropped and an uncropped image is shown in Supplementary Fig. S8. (c) SDS PAGE analysis of MSP2 (3 µg) incubated with recombinant OaAEP1_b, OaAEP3, OaAEP4 and OaAEP5 at 0.132 µM for 30 min. Enzyme only controls are also shown. ◂Denotes the product assigned as backbone cyclised MSP2. The grouping of different parts of the same gel is indicated by the white space. A single representative experiment of two technical replicates is shown. The gel has been cropped and an uncropped image is shown in Supplementary Fig. S8. (d) An anti-MSP2 monoclonal antibody 6D8 was allowed to interact with immobilised MSP2 in the presence or absence of soluble MSP2 (linear or cyclic). Both cyclic and linear soluble MSP2 proteins inhibited the interaction of MAb 6D8 with immobilised MSP2. A control with no soluble MSP2 added was set as 100% 6D8 binding, and the impact of the addition of linear or cyclic MSP2 is reported relative to this. The average of two technical replicates is shown and error bars report the range.