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. 2019 Oct 18;9:15007. doi: 10.1038/s41598-019-50941-3

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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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Design and purification of the 14-3-3-pB6 chimera. (A) Schematics showing the primary structure of both individual proteins and the chimera. The N-terminal hexahistidine-tag cleavable by 3C protease is shown. (B) Expression (Exp.) and chromatographic (IMAC1, IMAC2, SEC) purification of the unphosphorylated chimera as analyzed by SDS-PAGE. Qualitatively similar results were obtained after co-expression with PKA, but with a lower yield (not shown). Non-induced (−I), induced (+I), soluble (S), flowthrough (F), wash (W), eluted (E) fractions, and fractions collected during SEC (SEC peak) are indicated below the gel. Protein markers (m) with the corresponding masses (in kDa) are also indicated. Position of the chimera band is shown by an arrow to the left. Note the shift after the 3C proteolysis (3C: “−”, “+”). (C) Kinetics of in vitro phosphorylation of the purified chimera by PKA analyzed by native-PAGE. Note the downward shift corresponding to phosphate group incorporation.