Internal ribosome entry site mediates protein synthesis ... - Springer Link

Biotechnol Lett (2012) 34:957–964 DOI 10.1007/s10529-012-0862-2

ORIGINAL RESEARCH PAPER

Internal ribosome entry site mediates protein synthesis in yeast Pichia pastoris Shuli Liang • Ying Lin • Cheng Li • Yanrui Ye

Received: 8 January 2012 / Accepted: 18 January 2012 / Published online: 28 January 2012 Ó Springer Science+Business Media B.V. 2012

Abstract The imitation of translation, as mediated by internal ribosome entry sites, has not yet been reported in Pichia pastoris. An IRES element from Saccharomyces cerevisiae was demonstrated to direct the translation of a dicistronic mRNA in P. pastoris. The 50 -untranslated region of GPR1 mRNA, termed GPR, was cloned into a dual reporter construct containing an upstream Rhizomucor miehei lipase (RML) and a downstream b-galactosidase gene (lacZ) from Escherichia coli BL21. After being transformed into P. pastoris, the RML gene and lacZ were simultaneously expressed. The possibility of DNA rearrangement, spurious splicing, or cryptic promoter in the GPR sequence were eliminated, indicating that expression of a second ORF was IRES-dependent. These findings strongly suggested that the IRES-

Electronic supplementary material The online version of this article (doi:10.1007/s10529-012-0862-2) contains supplementary material, which is available to authorized users. S. Liang Y. Lin C. Li Y. Ye (&) School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, Guangdong, China e-mail: [email protected] S. Liang e-mail: [email protected] Y. Lin e-mail: [email protected] C. Li e-mail: [email protected]

dependent translation initiation mechanism is conserved in P. pastoris and provides a useful means to express multiple genes simultaneously. Keywords Internal ribosome entry sites Pichia pastoris Translation initiation mechanism Co-expression

Introduction Most eukaryotic mRNAs are translated in a capdependent manner. Under conditions when capdependent translation initiation is compromised, an alternative initiation pathway, internal ribosome entry site (IRES), is used for protein synthesis. This translation initiation mechanism is independent of the cap-structure and allows the 40S ribosome to be directly recruited to the vicinity of the initiation codon (Komar and Hatzoglou 2011). IRES elements were first described in polio virus and encephalomyocarditis virus (EMCV) mRNAs (Jang et al. 1988; Pelletier and Sonenberg 1988). Subsequently, a number of mammalian mRNAs were found to contain IRES elements (Van Eden et al. 2004). The EMCV IRES functionally mediates translation of the second ORF of a bicistronic construct in stable transgenic plants (Urwin et al. 2000). In Saccharomyces cerevisiae, the IRES element of crucifer-infecting tobamovirus (crTMV) (Dorokhov et al. 2002), IRESs from URE2 (Komar et al. 2003)

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and seven yeast genes required for invasive growth (Gilbert et al. 2007), and an artificial IRES (SIRES) (Paz et al. 1999) are active. The methylotrophic yeast Pichia pastoris is widely used for high-level production of recombinant industrial and biopharmaceutical proteins. However, neither an IRES-dependent translation initiation mechanism nor endogenous IRES elements have been reported in P. pastoris. This might be because of a deficiency in UTR information for P. pastoris genes. Co-expression of two or more genes, which is achieved using functional IRES element in mammalian cells (Wan and Flavell 2005), is an important and useful method for improving recombinant protein production, monitoring the expression of recombinant proteins, and metabolic engineering in P. pastoris. Consequently, surveying the IRES-dependent translation initiation mechanism in P. pastoris is necessary for further development of the P. pastoris expression platform. In this study, the IRES activity of GPR sequence from S. cerevisiae was investigated using bicitronic assays containing an upstream Rhizomucor miehei lipase (RML) cistron and a downstream LacZ ORF in P. pastoris. The results showed that IRES-mediated translation initiation was functional in P. pastoris.

E. coli BL21 genomic DNA with primers L1/L2 into the KpnI-NotI sites of plasmid pGAPZA (Invitrogen). The GPR fragment was amplified from S. cerevisiae BY4743 genomic DNA with primers G1/G2 and digested with PstI/HindIII. The lacZ gene was amplified from plasmid pLacZ using primers L3/L2 and digested with HindIII/Not I. These two fragments were ligated into PstI/NotI sites in plasmid pRml, generating the plasmid pRml-GPR-LacZ. To simplify detection of RML expression, a sequence encoding FlagTag (DDDDK-Tag) was placed in front of the RML gene ORF. To ensure translational termination of RML and to prevent reinitiation of lacZ from leaky ribosome scanning, three additional stop codons (TAATGAT AA) were added immediately downstream of RML. The reverse complementary sequence of GPR was obtained using primers G3/G4 and used to replace the GPR sequence in plasmid pRml-GPR-LacZ, for the negative control pRml-Null-LacZ. For pGAP-LacZHIS, the HIS4 sequence was obtained from plasmid pPIC9K and introduced into the BamHI site of the pLacZ plasmid. To generate the promoterless vector pGPR-LacZ-HIS, the GAP promoter was replaced in the BglII/BstBI site by a GPR segment obtained with primers G5/G6. All plasmids were transformed into E. coli TOP10F0 chemically competent cells and confirmed by DNA sequencing.

Materials and methods

Yeast transformation

Strains, media and growth conditions

Plasmids were linearized with BlnI (Takara, Japan), which cuts in the GAP promoter, or BspEI, which cuts in the HIS4 sequence, and transformed into P. pastoris X33 competent cells by the lithium chloride transformation method. The transformed cells were selected on YPDSZ agar plates.

Escherichia coli TOP10F0 cells were cultivated in low-salt LB medium. Bacterial plasmid selection and maintenance was with 25 mg zeocin/l. P. pastoris strain X33 (Invitrogen, USA) was used as host and cultivated in YPD medium. Transformants of P. pastoris were selected on YPDSZ agar plates. Strains used in present study are shown in Supplementary Table 1. Construction of vectors The RML gene was amplified from plasmid pPICR (Han et al. 2009) with primers R1/R2 (Supplementary Table 1) and cloned into the EcoRI-KpnI sites in plasmid pGAPZaA (Invitrogen) to create plasmid pRml. The plasmid pLacZ was constructed by inserting the b-galactosidase gene (lacZ) amplified from

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Detection of RML and b-galactosidase on plates To detect the expression of RML, positive clones were spotted onto MDT plates: 5 g (NH4)2SO4/l, 3 g yeast extract/l, 1 g polyvinyl alcohol (PVA)/l, 20 g agar/l, 5 ml tributyrin/l (Acros Organics, USA) and 100 ml 1 M phosphate buffer, pH 6.0/l. Clones were simultaneously spotted on MDX plates: 5 g (NH4)2SO4/l, 3 g yeast extract/l, 20 g agar/l, 4 ml X-gal/l (Takara, Japan, 20 mg/m buffered by dimethyl formamide) and 100 ml 1 M phosphate buffer, pH 6.0/l, to determine b-galactosidase activity.


SDS-PAGE and western blot Recombinant P. pastoris strains were cultured in YPD medium for 72 h. Intracellular proteins were extracted by the freeze–thaw cycles method in the Yeast Protocols Handbook (Clontech, USA). Supernatant and intracellular proteins were separated by SDSPAGE and analyzed by western blot. Briefly, 20 ll protein samples from each strain were resolved by 10% SDS-PAGE gel and transferred onto a nitrocellulose membrane (Bio-Rad, USA) in electroblotting buffer (25 mM Tris/HCl, 190 mM glycine, and 20% methanol) at 100 V for 3 h. The membrane was immersed in blocking buffer (3% bovine serum albumin fraction V, 20 mM Tris/HCl, 0.9% NaCl, and 0.1% Tween 20, pH 7.4) at 4°C overnight, followed by incubation with anti-DDDDK-Tag or anti-b-galactosidase antibody for 2 h. Both antibodies (MBL, Japan) were used at 1:1,000. After washing three times, membranes were probed with horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (Sigma, 1:5,000) at room temperature for 1 h and visualized using Super ECL Plus Detection Reagent (Applygen Technologies, China). Extraction of genomic DNA and PCR identification Genomic DNA was extracted from P. pastoris using the Yeast DNAiso-Kit (Takara, Japan) according to the manufacturer’s manual. Subsequently, PCR identifications were performed with the primer sets R1?R2, R1?G2 and R1?L2. Total RNA isolation and RT-PCR Total RNA was isolated from P. pastoris strains using the hot acidic phenol method. Cells were collected by centrifugation at 12,0009g for 5 min and directly suspended in lysis solution [400 ll AE (50 mM sodium acetate, 10 mM EDTA, pH 5.2), 40 ll 10% (v/v) SDS, 300 ll water-saturated phenol and 300 ll chloroform], vortexed for 15 min at 65°C and held for 5 min on ice. The mixture was centrifuged at 12,0009g at room temperature for 7 min, the aqueous phase was transferred to a new tube and precipitated with 1 ml ethanol at -20°C for 1 h and centrifuged for 10 min at 12,0009g at 4°C. The pellet was washed with 1 ml 75% (v/v) ethanol, centrifuged for 5 min at

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12,0009g at 4°C and resuspended in 50 ll RNase-free water. RNA integrity was monitored by gel electrophoresis under denaturing conditions and quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, USA). Genomic DNA was removed and RT-PCR assay was carried out using the PrimeScript RT reagent Kit With gDNA Eraser (Perfect Real Time) (Takara, Japan) according to the manufacturer’s protocol. Using the primer sets R1?R2 and L1?L2, the transcription of the RML and lacZ genes were detected with cDNA as template.

Results GPR mediates cap-independent translation initiation To assess whether GPR mediates IRES-dependent translation initiation in P. pastoris, a dicistronic vector pRml-GPR-LacZ was constructed (Fig. 1a). In this context, translation of the upstream cistron relies on cap-dependent scanning, whereas the downstream lacZ reporter can be translated only if the IRES in GPR functions efficiently in P. pastoris. Plasmid pRml-GPR-LacZ and the corresponding control plasmids (Supplementary Table 1; Fig. 1a) were transformed into P. pastoris X33. The host strain X33, and recombinant P. pastoris strains with the above integrated reporter vectors were cultivated on MDT hydrolysis plates and MDX chromogenic plates. To test whether the chimeric constructs had undergone DNA rearrangements during transformation, genomic DNA from pRml-GPR-LacZ strain was used as the template for PCR identifications. When amplified by three pairs of primer sets (R1?R2, R1?G2, R1?L2), three corresponding bands were observed (Fig. 1b) and compared to the PCR products amplified from plasmid pRml-GPR-LacZ. These data clearly demonstrated the all three regions of the binary reporter constructs were present on contiguous genomic DNA fragments. The expression of RML was monitored by hydrolysis of tributyrin on the MDT plate. As shown in Fig. 1d, both the pRml-Null-LacZ strain and pRmlGPR-LacZ strain produced active RML, similar to the positive control pRml strain. These results revealed that the expression of the first cistron was not affected

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Biotechnol Lett (2012) 34:957–964 1 GPR1 IRES mediates LacZ protein synthesis in a bicistronic expression cassette. a Schematic representation of vectors constructed for investigating IRES activity of GPR (50 UTR of GPR1). Bicistronic vectors contain an upstream Rhizomucor miehei lipase (RML) gene and a downstream Escherichia coli BL21 b-galactosidase gene (lacZ). Null represents the reserve complementary sequence of GPR and arrows represent primers used to construct vectors or PCR identification. b Identification of constructs in genomic DNA from pRml-GPR-LacZ strain. c Transcription analysis of the RML and lacZ genes. d Detection of RML by MDT plate and western blot with anti-DDDDK-Tag antibody. e Detection of bgalactosidase by MDX plate and western blot with anti-bgalactosidase antibody

b Fig.

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secreted RML was detectable in the supernatant of strains transformed with pRml-Null-LacZ, pRmlGPR-LacZ or pRml, as analyzed by western blot using anti-DDDDK-Tag antibody (Fig. 1d). Strains X33 and pLacZ secreted no RML protein. Intracellular proteins were also extracted and detected by anti-bgalactosidase antibody. b-galactosidase was efficiently synthesized in the pRml-GPR-LacZ strain, comparable to expression from a monocistronic transcript pLacZ. No b-galactosidase was produced in the pRml-Null-LacZ strain or the X33 and pRml strains (Fig. 1e). Transcription from the RML and lacZ genes was examined in all strains. We obtained cDNA from total RNA and RT-PCR was performed using primer sets R1?R2 or L1?L2. Figure 1c shows that the pRml-Null-LacZ strain and the pRml-GPR-LacZ strain yielded transcripts of both the RML and lacZ genes, and sizes were consistent with transcripts from pRml and pLacZ. These data suggested that GPR provided efficient translation of the downstream cistron. b-Galactosidase expression in pRml-GPR-LacZ strain is not due to spurious splicing

Fig. 2 Test of spurious splicing events. PCR identifications were performed with genomic DNA or cDNA from the pRmlGPR-LacZ strain as template. The position of primer P1 is the end of the RML gene. P2 is located in the beginning of lacZ

by the transcription of the second ORF. The translation of the second cistron was detected as expression of the lacZ gene, a classical histochemical reporter gene encoding b-galactosidase. b-Galactosidase activity was detected in the pRml-GPR-LacZ strain and the corresponding control pLacZ strain (Fig. 1e). However, no b-galactosidase activity was detectable from pRml-Null-LacZ, indicating a requirement for the GPR in the translation of the downstream ORF. This observation was further studied by western blot analysis and RT-PCR. After 72 h in YPD,

Previous studies reported that the putative eIF4G, NF-kappa B repressing factor NRF, RNA-binding motif protein 3, and X-linked inhibitor of apoptosis protein IRESs (Holcik et al. 2005; Baranick et al. 2008; Saffran and Smiley 2009) are spliced and generate monocistronic mRNA in a classical dicistronic reporter system, resulting in the expression of the second cistron. If a receptor site is located in GPR, a splicing event will remove the additional three stop codons and create an ORF encoding a hybrid protein with a part of b-galactosidase fused to RML. The integrity of bicistronic mRNAs from pRmlGPR-LacZ was analyzed by RT-PCR with primer sets P1?P2. Primer P1 binds the end of the RML gene, while P2 binds the beginning of lacZ. With this pair of primers, a spliced mRNA generates a small PCR product or possibly no PCR fragment. However, we observed only one PCR product (Fig. 2), the size of which was similar to the corresponding band when genomic DNA was used as template. This strongly suggested that the integrity of pRml-GPR-LacZ transcript was preserved in vivo, ruling out the possibility of an RNA splicing event.

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GPR contains no cryptic promoter Identification of IRES in eukaryotes is challenged by cryptic promoter activity in the DNA sequence corresponding to the 50 -UTR in the commonly used dicistronic test method (Kozak 2005). Some candidate IRESs are eliminated because the sequence harbors a transcriptional promoter, such as in the 50 -UTR of the hepatitis C virus (Dumas et al. 2003), S. cerevisiae TIF4631 (Verge et al. 2004), and the mice serine/ threonine kinase pim-1 (Wang et al. 2005). In these cases, the transcription and translation of the downstream ORF result from the cryptic promoter and not IRES activity. We tested whether the GPR1 50 -UTR has an IRES element or a cryptic promoter using the promoterless monocistronic vector pGPR-LacZ-HIS (Fig. 3a). To detect lacZ expression, positive clones were cultivated on MDX plates containing X-gal as a color-developing substrate. Reporter LacZ gene expression is in Fig. 3b, showing that lacZ was efficiently expressed in the pGAP-LacZ-HIS strain, making the clones blue. However, X33 and pGPR-LacZ-HIS clones remained white, indicating that no endogenous proteins processed X-gal and that the pGPR-LacZ-HIS strain did not drive lacZ expression. These results were consistent with western blot analysis using anti-galactosidase antibody (Fig. 3c). After overnight in YPD, intracellular proteins were analyzed by western blot. As shown in Fig. 3c, b-galactosidase was detected in the pGAP-LacZ-HIS strain and no LacZp was detectable in X33 and pGPR-LacZ-HIS strains. These data suggested that GPR did not contain a cryptic promoter Fig. 3 GPR has no cryptic promoter activity. a Construction of a promoterless vector and corresponding control vector. The insertion direction of HIS4 was confirmed by PCR. bgalactosidase expression was detected by MDX plates (b) and western blot using anti-b-galactosidase antibody (c)

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that directed aberrant transcription and subsequent cap-dependent translation. These combined data demonstrated that insertion of the GPR upstream of the b-galactosidase coding sequence resulted in translation of the second ORF. The expression of RML resulted from a cap-dependent ribosome scanning mechanism while the production of b-galactosidase reflected the IRES activity of GPR. Detection of b-galactosidase in the pRml-GPR-LacZ strain but not in the pRml-Null-LacZ strain suggested that the translation of b-galactosidase was mediated by sequences within GPR. Since the RML ORF was terminated by additional stop codons and the possibilities of a cryptic promoter and RNA splicing event were ruled out, the translation of b-galactosidase in the pRml-GPR-LacZ strains was dependent on the IRES mechanism. Collectively, these data showed that an IRES-dependent translation initiation mechanism existed in P. pastoris.

Discussion IRES elements are demonstrated in mRNAs from viruses, and mammalian, vertebrate and yeast cells. Detailed data are at the IRES website. However, an IRES-dependent translation initiation mechanism has not previously been demonstrated in P. pastoris. In S. cerevisiae, which shares many morphological and physiological similarities with P. pastoris, 50 -UTRs of GPR1 and other genes required for invasive growth mediate cap-independent translation (Gilbert et al. 2007). We used the IRES of GPR1 from S. cerevisiae


to investigate IRES-dependent translation initiation mechanisms in P. pastoris. GPR mediated the translation of a second ORF in recombinant P. pastoris transformed with a bicistronic vector pRmlGPR-LacZ. These findings strongly suggested that an IRES-dependent translation initiation mechanism is conserved in P. pastoris. The conservation of an IRES-dependent translation initiation mechanism in P. pastoris suggests IRES elements in the 50 -UTR of P. pastoris genes and corresponding IRES-transacting factors in IRES-mediated translation. The identification of endogenous IRES elements will be a major field in the study of P. pastoris IRES-dependent translation initiation mechanism. In mammalian systems, IRES elements have been successfully introduced into vectors used for coexpression of two or more recombinant proteins (Mountford and Smith 1995). This application of IRES is also feasible in P. pastoris with our discovery of an IRES-dependent translation initiation mechanism. Methods used for co-expression in P. pastoris including using two individual plasmids (Gasser et al. 2006; Gasser et al. 2007; Stadlmayr et al. 2010), introducing multiple expression cassettes into a single plasmid (Bhataya et al. 2009), and constructing fusion proteins (Hu et al. 2011; Broger et al. 2011). These approaches usually require double transformation and selection, relatively large plasmids or treatment with Kex2 protease to produce target proteins. These drawbacks can be circumvented with the use of an IRES-mediated dicistronic vector, indicating that the GPR IRES element could be a useful method for co-expression in an efficient P. pastoris expression system. Acknowledgment This work was supported by National Natural Science Foundation of China (no. 20976062).

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