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. 2018 Mar 15;8:4612. doi: 10.1038/s41598-018-22860-2

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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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Fusion of the M domain increases the expression levels of recombinant protein. (a) Schematic representation of the constructs. All target sequences were expressed under the control of the cauliflower mosaic virus (CaMV) 35 S promoter and heat shock protein (HSP) terminator. ER-targeted proteins contain the leader sequence of BiP. Recombinant proteins targeted to the chloroplast or mitochondria contain the transit peptide of Cab or the presequence of the F1-ATPase gamma subunit, respectively. Enk, enterokinase. (b) Western blot analysis of various recombinant proteins. Protein extracts from protoplasts transformed with the indicated constructs were analyzed by western blotting using anti-HA antibody. Actin (detected using anti-actin antibody) was used as a loading control. EeLepf and EeLepfM are ER-targeted proteins, CeLepf and CeLepfM are chloroplast-targeted proteins, and FeLepf and FeLepfM are mitochondria-targeted proteins. (c) Quantification of protein levels. The intensity of protein bands from Fig. 2(b) was quantified using the software provided with the LAS4000 image analyzer; values relative to that of ER-targeted EeLepf are shown. Error bars, standard deviation (n = 3). Means with different letters indicate significant difference (Tukey’s test P < 0.05). (d) Schematic representation of unglycosylated mutant protein M1234. Four Asn residues were substituted with Gln residues as indicated. (e) Analysis of N-glycosylation. Protoplasts transformed with the indicated constructs were incubated for 24 h with or without tunicamycin (10 μg/mL). Protein extracts from transformed protoplasts were analyzed by western blotting using anti-HA antibody. (f) Quantification of protein levels. To quantify protein levels, the signal intensity of each band in Fig. 2(e) was measured using the software provided with the LAS4000 system; values relative to that of tunicamycin-untreated EeLepf (EeLepf -) are shown. Three independent experiments were performed. Error bar, standard deviation (n = 3). Means with different letters indicate significant difference (Tukey’s test P < 0.05).