Isolation, folding and structural investigations of the amino acid transporter OEP16

Da Qun Ni, James Zook, Douglas A. Klewer, Ronald A. Nieman, J. Soll, Petra Fromme

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Membrane proteins compose more than 30% of all proteins in the living cell. However, many membrane proteins have low abundance in the cell and cannot be isolated from natural sources in concentrations suitable for structure analysis. The overexpression, reconstitution, and stabilization of membrane proteins are complex and remain a formidable challenge in membrane protein characterization. Here we describe a novel, in vitro folding procedure for a cation-selective channel protein, the outer envelope membrane protein 16 (OEP16) of pea chloroplast, overexpressed in Escherichia coli in the form of inclusion bodies. The protein is purified and then folded with detergent on a Ni-NTA affinity column. Final concentrations of reconstituted OEP16 of up to 24 mg/ml have been achieved, which provides samples that are sufficient for structural studies by NMR and crystallography. Reconstitution of OEP16 in detergent micelles was monitored by circular dichroism, fluorescence, and NMR spectroscopy. Tryptophan fluorescence spectra of heterologous expressed OEP16 in micelles are similar to spectra of functionally active OEP16 in liposomes, which indicates folding of the membrane protein in detergent micelles. CD spectroscopy studies demonstrate a folded protein consisting primarily of α-helices. 15N-HSQC NMR spectra also provide evidence for a folded protein. We present here a convenient, effective and quantitative method to screen large numbers of conditions for optimal protein stability by using microdialysis chambers in combination with fluorescence spectroscopy. Recent collection of multidimensional NMR data at 500, 600 and 800 MHz demonstrated that the protein is suitable for structure determination by NMR and stable for weeks during data collection.

Original languageEnglish
Pages (from-to)157-168
Number of pages12
JournalProtein Expression and Purification
Volume80
Issue number2
DOIs
Publication statusPublished - Dec 2011

Keywords

  • Chloroplast outer envelope
  • Membrane protein
  • Protein expression and purification
  • Protein folding
  • Protein solution NMR

ASJC Scopus subject areas

  • Biotechnology

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