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. Author manuscript; available in PMC: 2021 Jul 1.
Published in final edited form as: Metab Eng. 2020 Mar 26;60:37–44. doi: 10.1016/j.ymben.2020.03.009

Fig. 2. In vitro biosynthesis of valinomycin using CFPS system.

Fig. 2.

(A) SDS-PAGE (7.5% gel) analysis of Vlm1 and Vlm2 expressed individually or together. T, total protein. S, soluble protein. M, protein molecular weight marker. NC, negative control without plasmid in the reaction. (B) SDS-PAGE (4–12% gel) analysis of coexpressed proteins. 1, coexpression of Vlm1, Vlm2, and Sfp. 2, coexpression of Vlm1, Vlm2, Sfp, and TEII. (C) LC-MS analysis of valinomycin produced by the CFPS reaction. Insert, MS spectrum of valinomycin peak. EIC, extracted ion chromatogram. a, negative control without plasmid in the reaction. b, valinomycin standard. c, CFPS reaction sample. (D) Valinomycin yields from different CFPS reaction conditions. 1, valinomyicn synthesis by coexpression of Vlm1, Vlm2, and Sfp. Valinomyicn synthesis by coexpression of Vlm1, Vlm2, Sfp, and TEII for a total time of (2) 20 h reaction and (3) 24 h reaction, respectively. Values show means with error bars representing standard deviations (S.D.) of at least three independent experiments.