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. 2020 Jan 17;11:336. doi: 10.1038/s41467-019-13654-9

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© The Author(s) 2020

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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Fig. 1 — a Schematic representation of scFv constructs fused with various peptide tags: [1] non-tagged scFv, [2] scFv with N-terminal T7 and EGFP, and C-terminal 6× His (scFv-GFP), [3] scFv with N-terminal s3Flag (s3Flag-scFv), [4] ScFv with N-terminal s3Flag and C-terminal HA (s3Flag-scFv-HA). b Net charge comparison of scFv-A36 and scFv-M4 proteins fused with indicated peptide tags at cytoplasmic pH 6.6 (green), 7.03 (red), and 7.4 (blue), and the isoelectric point (pI) determination (grey circle) using in silico physicochemical analysis. c Intracellular stability comparison of scFv-A36 (left panels) and scFv-M4 (right panels) fused with different peptide tags expressed in HeLa cells using immunocytochemistry; anti-Flag (lower and middle panels) or anti-T7 (upper panels) antibodies were used to detect scFv proteins. Arrows indicate intracellular scFv aggregates. Arrowheads indicate scFvs stably expressed in the cytoplasm. Scale bar, 50 μm. d Percentage of scFv-expressing cells with aggregates in c (upper panel) and total cell and aggregated cell number in 3 independent experiments (bottom panel). Statistical analysis: two-tailed one-way analysis of variance (A36; percentage of aggregation, F (2, 6) = 834.25, P < 0.00001; Tukey’s multiple comparison test, s3Flag-A36-HA vs. s3Flag-A36: P = 0.01614, s3Flag-A36-HA vs. scFv-GFPA36: P < 0.00001, s3Flag-A36 vs. scFv-GFPA36: P < 0.00001; M4; percentage of aggregation, F (2, 6) = 180.65, P < 0.00001; Tukey’s multiple comparison test, s3Flag-M4-HA vs. s3Flag-M4: P = 0.01949, s3Flag-M4-HA vs. scFv-GFPM4: P < 0.00001, s3Flag-M4 vs. scFv-GFPM4: P < 0.00001). Error bars represent standard error of the mean. Source data are provided as a Source Data file.