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. Author manuscript; available in PMC: 2018 May 1.
Published in final edited form as: Biotechnol J. 2017 Apr 20;12(5):10.1002/biot.201600696. doi: 10.1002/biot.201600696

Figure 3.

Figure 3

Evaluation of bioconjugation enzyme activity on yeast using pCL vectors. (A, B) Schematic of protein expression and bioconjugation reactions on the yeast surface for pCL vectors co-expressing sortase A (SrtA) 7M, and its peptide substrate, LPETGG. (C) Detection of SrtA bioconjugation activity on yeast using pCL-Srt-LS (long linker) and pCL-Srt-SS (short linker) under various conditions. Samples were stained with avidin-PE to detect Azp conjugation followed by biotin Click chemistry, and also stained with AlexaFluor 488-conjugated antibodies to measure yeast expression levels. Histograms of PE signal (enzyme activity) as a percentage of the maximum cell number shows readily apparent sortase-active populations in each sample. (D) Detection of SrtA bioconjugation activity on yeast by using pCL-Srt-cGFP-LS (yEGFP plus long linker) with and without biotin-DBCO. Co-expression of yEGFP and SrtA eliminates the need for antibody-staining of epitope tags to quantify yeast surface expression levels.