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. 2016 Jan 12;6:18379. doi: 10.1038/srep18379

Figure 3. Inhibition of flagellin proteolysis by domain stabilization.

Figure 3

(a) CFPS total and full-length yields and (b) SDS-PAGE autoradiogram analysis of flagellin produced by 6 h reactions using three different E. coli extracts (KC6 extract, BL21 extract, and BL21 extract with overexpressed GroEL/S). (c) Positions of newly introduced disulfide bonds (SS1 though SS6) at or near the D0 domain. (d) Non-reducing SDS-PAGE analysis of flagellin after purification and oxidation by 20 mM diamide for 3 hours. The flagellin mutants were synthesized using CFPS with BL21 extract containing overexpressed GroEL/S. (e) Stability analysis (by SDS PAGE) of flagellin mutants SS1 through SS6) produced by CFPS using the KC6 extract. The purified and oxidized flagellin mutants were incubated in a CFPS mixture at 30 °C for 6 h and analyzed by autoradiography at 0, 3, and 6 h. (f) Bioactivity of flagellin mutants. Five measurements were taken on different days; error bars indicate the standard error of the mean (SEM).