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. 2019 Dec 20;9:19558. doi: 10.1038/s41598-019-55923-z

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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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The gluzincin core of MecR1 is membrane-embedded. (a) The modeled gluzincin domain is shown, with hydrophobic residues displayed in light grey, polar residues in green and the putative metal ligands (H204, H208, E245 and D249) in red. (b) MecR1^GLZ.E205A (19.8 kDa) was expressed in E. coli BL21 Star (DE3). IPTG-induced cells (WC, whole cell extract) were sonicated and centrifuged to separate the pellet (P; inclusion bodies and large fragments of membrane) from the fraction containing soluble proteins and membrane vesicles. The latter was subjected to ultracentrifugation to separate soluble proteins (S) from membrane proteins. Lanes labelled KCl, Na₂CO₃ and Triton correspond to fractions of proteins solubilized from the membrane preparation with 1 M KCl, 0.1 M Na₂CO₃ and 0.155% Triton X-100, respectively. Lane Pu is the pellet remaining after ultracentrifugation of the membranes treated with Triton X-100. (c) Detergent screening to evaluate conditions to solubilize MecR1^GLZ.E205A from membranes. MecR1^GLZ was found in the membrane protein fraction (M). The fractions corresponding to solubilized proteins are shown. Both the soluble and insoluble fractions are shown in Fig. S8. MecR1^GLZ.E205A could only be solubilized from membranes with the detergents C7Bz0, ASB-14, Brij10, Nonidet and LDAO, being the membrane solubilizing zwitterionic detergent ASB-14 (amidosulfobetaine-14) the one that allowed the highest level of protein recovery. In (b,c), MecR1^GLZ.E205A was detected by Western blot using a His-tag specific HRP-conjugated antibody. Numbers on the right indicate the positions of migration of the molecular mass markers (kDa).