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. 2018 Jul 12;9:2686. doi: 10.1038/s41467-018-05110-x

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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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Fig. 2 — Extract from glyco-optimized chassis strain supports CFGpS. a (left) Western blot analysis of scFv13-R4^DQNAT produced by crude CLM24 extract supplemented with purified CjPglB and organic solvent-extracted (solv-ext) CjLLOs, and primed with plasmid pJL1-scFv13-R4^DQNAT. (right) Western blot analysis of in vitro glycosylation reaction using purified scFv13-R4^DQNAT acceptor protein that was incubated with purified CjPglB and organic solvent-extracted (solv-ext) CjLLOs. Control reactions (lane 1 in each panel) were performed by omitting purified CjPglB. b (left) Western blot analysis of scFv13-R4^DQNAT produced by crude CLM24 extract selectively enriched with CjPglB from heterologous overexpression from pSF-CjPglB. (right) Western blot analysis of scFv13-R4^DQNAT produced by crude CLM24 extract selectively enriched with CjLLOs from heterologous overexpression from pMW07-pglΔB. Reactions were primed with plasmid pJL1-scFv13-R4^DQNAT and supplemented with purified CjPglB and organic solvent-extracted (solv-ext) CjLLOs as indicated. Control reactions (lane 1 in each panel) were performed by omitting solv-ext CjLLOs in (left) or purified CjPglB (right) in (b). Blots were probed with anti-hexa-histidine antibody (anti-His) to detect the acceptor protein or hR6 serum (anti-glycan) to detect the N-glycan. Arrows denote aglycosylated (g0) and singly glycosylated (g1) forms of scFv13-R4^DQNAT. Molecular weight (MW) markers are indicated at left. Results are representative of at least three biological replicates