A protocol for high throughput methods for the expression and purification of inner membrane proteins

• Published in: Molecular membrane biology Vol. 25; no. 8; pp. 599 - 608
• Main Authors: McLuskey, Karen, Gabrielsen, Mads, Kroner, Frank, Black, Isobel, Cogdell, Richard J., Isaacs, Neil W.
• Format: Journal Article
• Language: English
• Published: England Informa UK Ltd 01.01.2008; Taylor & Francis
• Subjects: Cell Culture Techniques; Chromatography, Affinity; Cloning, Molecular; Detergents; Green Fluorescent Proteins - metabolism; high-throughput; Histidine - metabolism; Membrane protein; Membrane Proteins - biosynthesis; Membrane Proteins - chemistry; Membrane Proteins - isolation & purification; protein expression; protein purification; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - isolation & purification; Spectrometry, Fluorescence
• DOI: 10.1080/09687680802510289
• ISSN: 0968-7688

The production of diffraction quality crystals for the structural determination of inner membrane proteins relies on obtaining large amounts of stable protein. Achieving this, by finding the correct parameters to successfully express and purify these proteins is often time-consuming and frustrating. The methods described here examine the most important parameters, in both expression and purification, quickly and simply. They take into account methods previously used in successful structural determinations of inner membrane proteins and collect and analyse data for use in further experiments and to investigate overall trends. These methods make use of histidine-tagged membrane proteins with a green fluorescent protein fusion but could be adapted easily for other proteins.