Screening for functional IRESes using α-complementation system of β-galactosidase in Pichia pastoris

Figures

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  Fig. 1. Background activity of LacZ gene in P. pastoris. a Schematic diagram of expression vectors with or without AOX1 promoter. b Analysis of enzymatic activity of β-galactosidase on BMMY plates containing X-Gal. The vector pPICZA-LacZ that the transcription of LacZ gene is driven by AOX1 promoter was used as positive control, and empty vector pPICZA was used as negative control. c Quantitative analysis of enzymatic activity of β-galactosidase

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  Fig. 2. Development of the α-complementation of β-galactosidase in P. pastoris. a Schematic diagram of vectors containing α-peptides and ω-peptides with different lengths. b Detection of β-galactosidase activity on plates containing X-gal. Empty vector pAO815 and pPICZA as negative controls; full-length LacZ gene (LacZ WT) as a positive control. c The relative activities of β-galactosidase in the α-complementation systems with different lengths of α-peptide and ω-peptide. Normalization was performed with full-length β-galactosidase (LacZ WT) activity at 100%

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  Fig. 3. Screening for functional IRESes in P. pastoris. a Schematic diagram of the plasmid vectors. b Screening on the X-gal plate for functional IRESes in P. pastoris. Three different colonies were shown for each IRES. c Quantitative detection of β-galactosidase activity for seven functional IRESes in P. pastoris. The strains transformed with empty vector were used as negative control (NC)

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  Fig. 4. Exclusion of read-through of bicistronic mRNA. a Schematic diagram of proteins produced by non-read-through and read-through of bicistronic mRNA. b The fusion protein produced by read-through of bicistronic mRNA was excluded by western blot

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  Fig. 5. Detection of cryptic splicing in bicistronic mRNA by RT-PCR. a Schematic diagram of cryptic splicing and primer design. b Detection of RT-PCR products on agarose gel. NC, PCR negative control reaction without template. ∓, reactions without or with reverse transcriptase, respectively

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  Fig. 6. Detection of cryptic promoter using promoterless vector. a Schematic diagram of producing LacZ (1–92) transcript with 5′ cap by a cryptic promoter harbor in IRES sequence. b Schematic diagram of promoterless vector removed the AOX1 promoter. c Detection of expression of the second reporter LacZ (1–92) with or without AOX1 promoter