Yeast signal peptide screen YSST was used to verify the secretion function of signal peptide. The yeast strain selected in the experiment is YTK12, which lacks the sucrose invertase gene and cannot grow on a culture medium with sucrose as the sole carbon source. The vector plasmid selected was pSUC2 containing tryptophan synthesis gene and a sucrose invertase gene (SUC2) lacking signal peptide and starting codon ATG. When a functional signal peptide is connected to the sucrose invertase gene of pSUC2, the secretion function of sucrose invertase can be restored, so that YTK12 strain can grow on the defective medium with raffinose as the carbon source. In addition, TTC color reaction is also commonly used to verify the results.
1. The signal peptide region of the candidate secretory protein was predicted by SignalP, and primers were designed to construct the pSUC2 vector. The vector resistance was ampicillin resistance.
2. The YTK12 strain was cultured on YPDA culture, and the constructed corresponding vector was introduced into YTK12 and coated on CMD-W (achromatic amino acid) medium plate for cultivation.
3. Select positive clones and culture them on YPRAA medium with raffinose as the carbon source. If the transferred yeast cells can grow on YPRAA medium, then it proves that signal peptide has secretory function; If it cannot grow, it indicates that signal peptide has no secretion function.
4. Cultivate the positive transformation strains in the above steps with CMD-W culture medium, collect cell suspension and centrifuge to remove the supernatant. Resuspension the precipitate with resuspension and incubate at 37 ℃ for 10 minutes. After centrifugation, the supernatant was added to TTC in a test tube, and the untransformed YTK12 yeast was used as a control. The color changes were observed and recorded by photography. If the color turns red, it indicates that signal peptide has secretory function.
Verification of signal peptide secretion activity by yeast signal peptide tracking system
（Xu et al., Nat. Commun., 2019）
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