Purification of Human and Mammalian Membrane Proteins Expressed in Xenopus laevis Frog Oocytes for Structural Studies

Methods Mol Biol. 2016;1432:223-42. doi: 10.1007/978-1-4939-3637-3_14.

Abstract

This protocol describes the isolation of recombinant human and mammalian membrane proteins expressed in Xenopus laevis frog oocytes for structural studies. The cDNA-derived cRNA of the desired genes is injected into several hundreds of oocytes, which are incubated for several days to allow protein expression. Recombinant proteins are then purified via affinity chromatography. The novelty of this method comes from the design of a plasmid that produces multi-tagged proteins and, most importantly, the development of a protocol for efficiently discarding lipids, phospholipids, and lipoproteins from the oocyte egg yolk, which represent the major contaminants in protein purifications. Thus, the high protein purity and good yield obtained from this method allows protein structure determination by transmission electron microscopy of single detergent-solubilized protein particles and of 2D crystals of membrane protein embedded in lipid bilayers. Additionally, a radiotracer assay for functional analysis of the expressed target proteins in oocytes is described. Overall, this method is a valuable option for structural studies of mammalian and particularly human proteins, for which other expression systems often fail.

Keywords: 2D crystallization; Human; Mammalian; Membrane protein; Protein structure; Single particle analysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromatography, Affinity
  • Cloning, Molecular
  • DNA, Complementary / genetics
  • Female
  • Genetic Vectors / administration & dosage
  • Humans
  • Mammals / genetics
  • Mammals / metabolism*
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Oocytes / metabolism
  • Plasmids / genetics
  • Protein Conformation
  • RNA, Complementary / genetics
  • Recombinant Proteins / metabolism
  • Xenopus laevis / genetics*
  • Xenopus laevis / metabolism

Substances

  • DNA, Complementary
  • Membrane Proteins
  • RNA, Complementary
  • Recombinant Proteins