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. Author manuscript; available in PMC: 2016 Jul 13.
Published in final edited form as: Angew Chem Int Ed Engl. 2015 Jun 10;54(29):8390–8394. doi: 10.1002/anie.201501186

Figure 2.

Figure 2

In-cell expression of MCoTI-I based cyclotides in S. cerevisiae cells using Npu DnaE intein-mediated PTS. A. SDS-PAGE analysis of the recombinant expression of cyclotide precursors 1a using a high-copy µ episomal plasmid under the control of the GAL1 (inducible, left) or GPD (constitutive, right) promoters (M stands for protein markers, TP for total cell lysate, P and S for insoluble and soluble cell lysate fractions, and B for fraction captured by Ni-NTA-agarose beads). B. Analytical HPLC-MS/MS traces of the soluble cell extract (left) or trypsin pull-down (right) fraction from S. cerevisiae cells expressing precursor 1a under the control of a GAL1 inducible promoter. The peak marked with an asterisk corresponds to folded cyclotide MCoTI-I. MS/MS analysis was performed using the ion (m/z = 871.6) for both Q1 and Q3 (I and cps stand for intensity and ion counts per second, respectively).