pXOOY: A dual-function vector for expression of membrane proteins in Saccharomyces cerevisiae and Xenopus laevis oocytes
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pXOOY : A dual-function vector for expression of membrane proteins in Saccharomyces cerevisiae and Xenopus laevis oocytes. / Vold, Victoria Amstrup; Glanville, Sebastian; Klaerke, Dan Arne; Pedersen, Per Amstrup.
In: PLoS ONE, Vol. 18, No. 2, e0281868, 2023.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - pXOOY
T2 - A dual-function vector for expression of membrane proteins in Saccharomyces cerevisiae and Xenopus laevis oocytes
AU - Vold, Victoria Amstrup
AU - Glanville, Sebastian
AU - Klaerke, Dan Arne
AU - Pedersen, Per Amstrup
N1 - Publisher Copyright: © 2023 Vold et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2023
Y1 - 2023
N2 - 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.
AB - 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.
U2 - 10.1371/journal.pone.0281868
DO - 10.1371/journal.pone.0281868
M3 - Journal article
C2 - 36809531
AN - SCOPUS:85148679283
VL - 18
JO - PLoS ONE
JF - PLoS ONE
SN - 1932-6203
IS - 2
M1 - e0281868
ER -
ID: 338983635