Codon usage bias regulates gene expression and protein conformation in yeast expression system P. pastoris

Figures

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  Fig. 1. The codon bias profile in P. pastoris genome. a Correlation between ω and tAI values in P. pastoris and S. cerevisiae. b Rare codon frequency at the 5′ end of all protein coding genes in P. pastoris (continuous line) and S. cerevisiae (dashed line). c Rare codon frequency at the 5′ end of genes coding for secreted proteins (dashed line) and all protein coding genes (continuous line) in P. pastoris

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  Fig. 2. Synonymous codon replacement does not affect the secretion ability of signal peptides. CAI, extracellular & intracellular specific activity of amylase reporter, as well as secretion ability of SP9 (a) and SP13 (b) when codons were de-optimized or optimized. c CAI plots of codon de-optimized and optimized α-mating factor sequences. d Top, western blow showing protein levels of extracellular & intracellular protein levels of amylase reporter in codon de-optimized and optimized strains; middle and bottom, densitometric quantification of western blot results. 6 × His tag antibody and β-Actin antibody were used as primary antibodies to detect AmyA and β-actin (loading control), respectively. The level of aFori-AmyA was set as 1. For enzyme activity and western blot quantification, error bars were calculated as standard deviation of three independent experiments. *, p < 0.05

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  Fig. 3. Codon usage bias are negatively correlated with protein structural disorder in a significant number of P. pastoris genes. a The distribution of CAI and disorder score correlation of all protein coding genes in P. pastoris. b The distribution of CAI and disorder score correlation of protein coding genes longer than 600aa in P. pastoris. c, d, CAI score plot of PAS_chr2-2_0376, PAS_chr2-2_0432 and their codon optimized sequences, respectively. e Disorder score plot of PAS_chr2-2_0376 and PAS_chr2-2_0432

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  Fig. 4. The expression profiles of two gene candidates after codon optimization. a, b Top, western blot showing protein levels of PAS_chr2-2_0376 (a), PAS_chr2-2_0432 (b) before and after codon optimization; bottom, densitometric quantification of western blot results. 6 × His tag antibody and β-Actin antibody were used as primary antibodies to detect target proteins and β-actin (loading control), respectively. c qPCR showing RNA levels of PAS_chr2-2_0376 before and after codon optimization. d, e, qPCR showing RNA levels of PAS_chr2-2_0432 before and after codon optimization with 5′ primer sets (d) and 3′ primer sets (e). Error bars were calculated as standard deviation of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001

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  Fig. 5. Codon optimization altered protein stability upon trypsin digestion and freeze–thaw cycles. a, b Top, western blot showing 0376 (a) and 0432 (b) protein sensitivity towards trypsin digestion before and after codon optimization; bottom, densitometric analyses of the western blot results. The protein levels at time point 0 were set as 1. c Top, western blot slowing 0376 protein degradation after freeze and thaw cycles before and after codon optimization; bottom, densitometric analyses of the western blot results. The protein levels at freeze–thaw cycle 0 were set as 1. 6 × His tag antibody was used as primary antibody to detect target protein. Error bars were calculated as standard deviation of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001

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  Fig. 6. Circular Dichroism (CD) spectra of 0376ori and 0376opt proteins under different temperatures. a, b Protein purification of 0376ori (a) and 0376opt (b) by affinity chromatography. In a: lane 1, flow through; lane 2, wash with 10 mM imidazole; lane 3, wash with 20 mM imidazole; lane 4, marker; lane 5, elution with 150 mM imidazole. In b: lane 1, flow through; lane 2, wash with 10 mM imidazole; lane 3, wash with 20 mM imidazole; lane 4, elution with 150 mM imidazole; lane 5, marker. Unlabeled lanes are irrelevant to this experiment. c, d CD spectra of purified 0376ori (c) and 0376opt (d) proteins under 25 °C and 95 °C. The difference in between is shadowed by grey. e Temperature mediated molecular ellipticity (208 nm) decay of 0376ori and 0376opt. Error bars were calculated as standard deviation of three independent experiments

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  Fig. 7. Circular Dichroism (CD) spectra of 0376ori (a) and 0376opt (b) proteins with urea addition. Urea concentrations used in each curve are indicated at the bottom