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. 2017 Sep 20;7:11991. doi: 10.1038/s41598-017-11819-4

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© The Author(s) 2017

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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Analysis of fermentation supernatants during the three-phase fermentation process for the production of VAMAX1. (a) Proteins in 15 µL supernatant were separated by LDS-PAGE and stained with SimplyBlue SafeStain. Numbered arrows indicate two impurities that were more abundant in the supernatant when the three-phase strategy was used instead of the two-phase strategy. (b) The proteins separated above were further analyzed by immunoblotting using the reduction-sensitive mAb 4G2. Times above the lanes indicate the elapsed hours of each process phase. M: protein marker. EoB: end of batch-phase. GlyFeed: glycerol fed-batch phase. Numbered arrows: samples analyzed by mass spectrometry. V1: target protein VAMAX1.