Journal Article

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

2023

Overview

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 o ptimized h uman potassium channels, oh ERG and oh Slick (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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Analysis / Animals / Biology and Life Sciences / Brewer's yeast / Cells / Channel gating / Codon / Control / Crystallography / E coli / Electrophysiology / Enzymes / Escherichia coli / Ethylenediaminetetraacetic acid / Expression vectors / Gametocytes / Glycerol / Humans / Identification and classification / Ion channels / Mammalian cells / Mammals / Membrane proteins / Membrane Proteins - metabolism / Membranes / Methods / Neomycin / Oocytes / Oocytes - metabolism / Organisms / Plasmids / Potassium / Potassium channels / Proteins / Research and Analysis Methods / RNA polymerase / Saccharomyces cerevisiae / Scientific equipment and supplies industry / Structure / Tetracycline / Tetracyclines / X-ray crystallography / Xenopus / Xenopus laevis / Yeast / Yeasts