Figure 1.
The scheme of rationale and the construction of a library of vectors: (A) the physiological role and cleavage site of Kex2; (B) the construction of pPICZαA-CDS libraries with all 20 natural amino acids at Kex2 P1’ site and the pPICZαA-S/G-Venus-Kex2 with additional Kex2 copy.
Figure 2.
Fluorescence/luminescence measurements of Venus/luciferase libraries with variable P1’ site.
Relative fluorescence/luminescence units (RFU/RLU) were normalized to the OD600 of the corresponding cultures and then analyzed and compared with the measurements of recombinant yeast strains with the same range of zeocin resistance. pPICZαA: empty vector control; single letters: the single-letter codes of the corresponding P1’ residues. (A) Venus library with zeocin resistance ranging 500-1,000 µg/ml and (B) 200-500 µg/ml; (C) luciferase library with zeocin resistance ranging 500-1,000 µg/ml and (D) 200-500 µg/ml. Each value representing the average of 5-6 individual colonies for each vector with the same resistance range for zeocin was presented as mean±SEM.
Figure 3.
Luciferase protein determination with western blotting analysis of luciferase library.
(A) A typical western result of TCA precipitated yeast culture supernatants of luciferase library with variable P1’ site, all tested recombinant yeast strains were randomly picked with zeocin resistance ranging 500-1,000 µg/ml, 5 µg of supernatant proteins were loaded for the SDS-PAGE and (B) the western gray-scale intensity of luciferase library. pPICZαA: empty vector control; single letters: the single-letter codes of the corresponding P1’ residues.
Figure 4.
Scale-up expression, purification and mass spectrometric analysis of SCF (about 20 KDa).
(A) Time course for the induction of SCF in 12% SDS-PAGE of TCA precipitated yeast culture supernatant, stained with Coomassie R-250; (B) the corresponding time course showing western blotting result; (C) Ni2+-HisTrapTM elution profile of recombinant SCF, the bound protein was eluted with a gradient of 20-500 mM imidazole; and (D) Coomassie-stained 12% SDS-PAGE of collected fractions 1-9, 4.8 mg purified SCF in 6 ml was obtained from 400 ml yeast culture supernatant; (E) MALDI TOF/TOFTM MS analysis of the purified recombinant SCF.
Figure 5.
Integration of additional Kex2 copies and the resulting influence on Venus fluorescence measurements: (A) western blotting (protein loads: 30 μg/well) showing the expression of integrated additional Kex2 copies (with flag tag), P values determined by t-tests of and S, G (Zeo 200-500 µg/ml) are *: 0.0022 and **0.0015 respectively; and (B) comparisons of Venus fluorescence measurements (normalized to the OD600 of the corresponding cultivation) between recombinant strains with zeocin resistance ranging from 200-500 µg/ml.
Each value representing the average of 5-6 individual colonies for each vector with the same resistance range for zeocin was presented as mean±SEM.