A protocol for high throughput methods for the expression and purification of inner membrane proteins.
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.
McLuskey
Karen
K
Department of Chemistry, Faculty of Biological Life Sciences, University of Glasgow, Glasgow, Scotland, UK.
Gabrielsen
Mads
M
Kroner
Frank
F
Black
Isobel
I
Cogdell
Richard J
RJ
Isaacs
Neil W
NW
D018929:Cell Culture Techniques; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003902:Detergents; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
D003902:Detergents; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006639:Histidine