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. 2019 Jan 22;9:310. doi: 10.1038/s41598-018-36559-x

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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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Recombinant proteins used in this study and GFP 1–10 pre-maturation scheme. (a) Schematic representation of the domains present in the proteins used in this study: (α) GFP 1–10 (β) His₆-Z_GFP 11, and (γ) EPO_GFP 11. Both His₆-Z_GFP 11 and EPO_GFP 11 have a short GS-linkers before GFP 11. (b) Illustration of the GFP 1–10 pre-maturation process on a solid support containing His₆-Z_GFP 11. (1) The 11th sheet of GFP fused to the Z domain derived from protein A (His₆-Z_GFP 11) is covalently coupled to Sepharose beads. The remaining part of GFP (GFP 1–10) is supplemented to the beads, (2) whereupon the two subunits self-associate and thereby complete the GFP structure. This initiates the relatively slow rearrangement of covalent bonds, which is necessary for the chromophore present in GFP 1–10 to mature. (3) Eventually, GFP 1–10 bound to the beads via GFP 11 will start to fluoresce as their chromophores have been maturated. Unbound GFP 1–10 and other impurities are washed away (4) before the bound GFP 1–10^mat is released from GFP 11 by low pH and the eluate is neutralized. (5) The released GFP 1–10^mat now comprises a maturated chromophore and is therefore prone to start fluorescing more rapidly when re-encountering GFP 11.