Vold VA et al. (2023), pXOOY: A dual-function vector for expression of...

XB-ART-59600

PLoS One 2023 Jan 01;182:e0281868. doi: 10.1371/journal.pone.0281868.

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pXOOY: A dual-function vector for expression of membrane proteins in Saccharomyces cerevisiae and Xenopus laevis oocytes.

Vold VA , Glanville S , Klaerke DA , Pedersen PA .

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On the quest for solving structures of membrane proteins by X-ray crystallography or cryo-EM, large quantities of ultra-pure protein are a paramount prerequisite. Obtaining enough protein of such high standard is not a trivial task, especially for difficult-to-express membrane proteins. Producing membrane protein for structural studies is often performed in Escherichia coli or Saccharomyces cerevisiae and is frequently complemented with functional studies. Ion channels and electrogenic receptors are traditionally studied in terms of their electrophysiological behavior, which cannot be performed in neither E. coli nor yeast. Therefore, they are frequently characterized in mammalian cells or in Xenopus laevis oocytes. To avoid generating two different plasmids, we here describe the construction of a dual-function plasmid, pXOOY, for membrane protein production in yeast and for electrophysiology in oocytes. pXOOY was constructed such that all elements required for oocyte expression were copied from the dual Xenopus-mammalian vector pXOOM and meticulously introduced into the high-yield yeast expression vector pEMBLyex4. pXOOY is thus designed to preserve the high yield of protein from pEMBLyex4 while simultaneously permitting in vitro transcription for expression in oocytes. We evaluated the performance of pXOOY by comparing expression of two yeast codon optimized human potassium channels, ohERG and ohSlick (Slo2.1) from pXOOY to expression of these channels obtained from the reference vectors pEMBLyex4 and pXOOM. Our proof-of-concept study indicates that accumulation in PAP1500 yeast cells was higher when the channels were expressed from pXOOY, which was verified both qualitatively and quantitatively. Two-electrode voltage clamp measurements in oocytes showed that the pXOOY constructs encoding ohERG and ohSlick gave currents with full preservation of electrophysiological characteristics. Our results show that it is possible to design a dual-function Xenopus-yeast vector without compromising expression in yeast and simultaneously maintaining channel activity in oocytes.

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Species referenced: Xenopus laevis
Genes referenced: cyc1 lgals4.2
GO keywords: membrane [+]

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	Fig 1. Plasmids used for creation of pXOOY. (A) The dual-function pXOOM plasmid created for heterologous protein expression in both mammalian cells and Xenopus laevis oocytes. CMV, cytomegalovirus early enhancer and promoter; T7, T7 RNA polymerase promoter; UTRs, both 5’ and 3’ regions from the Xenopus laevis β-globin gene; polyA consensus, native polyadenylation consensus sequence within the 3’UTR of Xenopus laevis β-globin gene; A34, synthetic polydT tail for in vitro transcription into a polyA tail; SV40, simian virus 40 early enhancer and promoter enabling expression of neo/kana-EGFP fusion gene; EM7, synthetic bacterial promoter directing expression of neo/kana-EGFP fusion gene; neo/kana-EGFP, neomycin resistance-enhanced GFP fusion gene; polyA, simian virus 40 fragment containing a polyadenylation signal. The XbaI, NheI and XhoI restriction sites can be used to linearize the plasmid prior to in vitro transcription. (B) pEMBLyex4 provides the backbone for the new pXOOY vector. URA3, encodes orotidine-5-phosphate decarboxylase; CYC-GAL, a fusion between the CYC1 promoter and the GAL10 upstream activating sequence containing two Gal4 binding sites; MCS, multicloning site; 2μ, yeast two micron origin of replication; leu2-d, poorly expressed allele of the β-isopropylmalate dehydrogenase gene; bla, β-lactamase gene; pMB1, E. coli origin of replication. The restriction sites for BamHI, HindIII and SalI are used to linearize the plasmid at the MCS for insertion of PCR fragments into the vector using homologous recombination. (C) Plasmid map of the new dual-function pXOOY vector for expression of proteins in yeast and in Xenopus laevis oocytes. The expression cassette contains elements from pEMBLyex4 required for protein expression in yeast such as the CYC-GAL promoter and a yeast 5’UTR as well as the selective markers URA3 and leu2-d and the 2μ origin of replication. To enable in vitro transcription, it contains the pXOOM T7 promoter and the 3’UTR from the Xenopus laevis β-globin gene, which encodes a synthetic polyA tail comprised of 34 A-nucleotides. https://doi.org/10.1371/journal.pone.0281868.g001
	Fig 2. Illustration of how pXOOY was constructed by inserting a Xenopus-yeast dual expression cassette into pEMBLyex4. Both forward and reverse primers (pXOOY-fw and pXOOY-rv, respectively) used to amplify the Xenopus-yeast expression cassette are indicated by black arrows. Each primer contained a 5’ extension (Rec and Rec, respectively), which was identical to either side of the MCS in pEMBLyex4. These regions of homology direct correct insertion of the PCR amplified expression cassette. The PCR fragment and pEMBLyex4 linearized at its MCS with BamHI, HindIII and SalI to prevent plasmid recircularization were co-transformed into PAP1500 cells to assemble the final pXOOY plasmid by homologous recombination (shown to the right). https://doi.org/10.1371/journal.pone.0281868.g002
	Fig 3. Detailed nucleotide sequence of the dual Xenopus-yeast expression cassette of pXOOY. Green nucleotides are the CYC-GAL promoter, nucleotides in bold green constitute the two Gal4 binding sites, nucleotides in bold pink are TATA boxes, bold blue nucleotides are the T7 promoter, yellow bold nucleotides are the MCS, nucleotides in red are 3’UTR from Xenopus β-globin, nucleotides written in bold italics in black and red are an XbaI site and an NheI site, respectively. Underlined nucleotides represent the recombination sites used to insert the pXOOY expression cassette into the pEMBLyex4 backbone. The larger letters indicate transcription start sites from the CYC-GAL promoter [29]. https://doi.org/10.1371/journal.pone.0281868.g003
	Fig 4. Illustration of how the ohSlick-TEV-yEGFP-His10 was assembled in pXOOY. The ohSlick cDNA was PCR amplified in two overlapping pieces (a and b) using the indicated primer pairs. A TEV site was introduced into the ohSlick b and yEGFP PCR fragments through 5’ extensions on the indicated PCR primers. A His10 tag was introduced to the yEGFP PCR fragment using a 5’ extension on the reverse PCR primer. The orange Rec and the green Rec are regions identical to either side of the MCS used for homologous recombination into pXOOY. All three PCR fragments were co-transformed into PAP1500 cells together with pXOOY linearized at its MCS with BamHI and HindIII. https://doi.org/10.1371/journal.pone.0281868.g004
	Fig 5. Accumulation of ohERG-TEV-yEGFP-His10 in S. cerevisiae. (A) Fluorescence in 1 OD450 unit as a function of time after induction of ohERG-TEV-yEGFP-His10 production with galactose. The dark red curve and the bright red curve show average fluorescence accumulation in PAP1500 cells expressing ohERG-TEV-yEGFP-His10 from pXOOY or pEMBLyex4, respectively. Averages were calculated from four repetitions of the experiment and error bars indicate ± S.D. The accumulation curves were compared using a two-way ANOVA test where **p = 0.0056. (B) Light microscopy (DIC) and fluorescence microscopy bio-imaging shown pairwise of yeast cells expressing either ohERG-TEV-yEGFP-His10 from pEMBLyex4 (left) or pXOOY (right). (C) In-gel fluorescence from crude yeast membranes isolated from cells harvested at the indicated time points after induction with galactose. The left gel shows 25 μg crude membrane samples from cells expressing ohERG-TEV-yEGFP-His10 from pEMBLyex4 and the right gel shows 25 μg membrane samples from cells expressing the same protein but from pXOOY. The two gels were imaged simultaneously. Uncropped gels can be found in S1, S2 Figs. https://doi.org/10.1371/journal.pone.0281868.g005
	Fig 6. Accumulation of ohSlick-TEV-yEGFP-His10 in S. cerevisiae. (A) Fluorescence accumulation in 1 OD450 unit as a function of time after induction of ohSlick-TEV-yEGFP-His10 production. The bright green curve and the dark green curve show average fluorescence accumulation in PAP1500 cells expressing ohSlick-TEV-yEGFP-His10 from pEMBLyex4 or pXOOY, respectively. Averages were calculated from four repetitions of the experiment and error bars indicate ± S.D. The accumulation curves were compared using a two-way ANOVA test where **p = 0.0012. (B) Light microscopy and fluorescence microscopy bio-imaging of yeast cells expressing either ohSlick-TEV-yEGFP-His10 from pEMBLyex4 (left) or pXOOY (right). (C) In-gel fluorescence of an SDS-PAGE gel of 25 μg yeast membranes purified from yeast cells harvested at the indicated time points after induction with galactose. The left gel shows purified membrane samples from cells expressing ohSlick-TEV-yEGFP-His10 from pEMBLyex4 and the right gel shows membrane samples from cells expressing the same fusion protein but from pXOOY. The two gels were imaged with the same exposure time. Uncropped gels can be found in S3, S4 Figs. https://doi.org/10.1371/journal.pone.0281868.g006
	Fig 7. Expression of ohERG-TEV-yEGFP-His10 in Xenopus laevis oocytes. (A) Representative current traces of ohERG-TEV-yEGFP-His10 measured in Xenopus laevis oocytes as described in the materials and methods section 5 days after injection of 25 ng of polyA RNA in vitro transcribed from pXOOY. The tail currents recorded at -120 mV are shown in S5 Fig. (B) I-V relation of ohERG-TEV-yEGFP-His10 given as the average ± S.E.M (n = 13). (C) Representative confocal images of oocytes incubated with FM 4–64. The upper image shows an uninjected oocyte, and the lower image shows an oocyte expressing ohERG-TEV-yEGFP-His10, which colocalized with FM4-64. https://doi.org/10.1371/journal.pone.0281868.g007
	Fig 8. Expression of ohSlick-TEV-yEGFP-His10 in Xenopus laevis oocytes. (A) Representative traces of ohSlick-TEV-yEGFP-His10 expressed and measured in Xenopus laevis oocytes 5 days after injection of 25 ng of polyA RNA in vitro transcribed from pXOOY. (B) I-V relation of ohSlick-TEV-yEGFP-His10 shown as the average ± S.E.M (n = 11). (C) Representative confocal images of oocytes incubated with FM 4–64. The upper image shows an uninjected control oocyte, and the lower image shows an oocyte expressing ohSlick-TEV-yEGFP-His10, which colocalizes with the FM4-64 dye. https://doi.org/10.1371/journal.pone.0281868.g008

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