Enhanced production of recombinant secretory proteins in Pichia pastoris by optimizing Kex2 P1' site

Abstract

Pichiapastoris is one of the most widely used expression systems for the production of recombinant secretory proteins. Its universal application is, however, somewhat hampered by its unpredictable yields for different heterologous proteins, which is now believed to be caused in part by their varied efficiencies to traffic through the host secretion machinery. The yeast endoprotease Kex2 removes the signal peptides from pre-proteins and releases the mature form of secreted proteins, thus, plays a pivotal role in the yeast secretory pathways. In this study, we found that the yields of many recombinant proteins were greatly influenced by Kex2 P1' site residues and the optimized P1's amino acid residue could largely determine the final amount of secretory proteins synthesized and secreted. A further improvement of secretory yield was achieved by genomic integration of additional Kex2 copies, which again highlighted the importance of Kex2 cleavage to the production of recombinant secretory proteins in Pichia yeast.

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⁺-HisTrap^TM 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/TOF^TM 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.