A high-throughput assay of membrane protein stability.
The preparation of purified, detergent-solubilized membrane proteins in a monodisperse and stable form is usually a prerequisite for investigation not only of their function but also for structural studies by X-ray crystallography and other approaches. Typically, it is necessary to explore a wide range of conditions, including detergent type, buffer pH, and the presence of additives such as glycerol, in order to identify those optimal for stability. Given the difficulty of expressing and purifying membrane proteins in large amounts, such explorations must ideally be performed on as small a scale as practicable. To achieve this objective in the UK Membrane Protein Structure Initiative, we have developed a rapid, economical, light-scattering assay of membrane protein aggregation that allows the testing of 48 buffer conditions in parallel on 6 protein targets, requiring less than 2 mg protein for each target. Testing of the assay on a number of unrelated membrane transporters has shown that it is of generic applicability. Proteins of sufficient purity for this plate-based assay are first rapidly prepared using simple affinity purification procedures performed in batch mode. Samples are then transferred by microdialysis into each of the conditions to be tested. Finally, attenuance at 340 nm is monitored in a 384-well plate using a plate reader. Optimal conditions for protein stability identified in the assay can then be exploited for the tailored purification of individual targets in as stable a form as possible.
Postis
Vincent L G
VL
Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
Deacon
Sarah E
SE
Roach
Peter C J
PC
Wright
Gareth S A
GS
Xia
Xiaobing
X
Ingram
Jean C
JC
Hadden
Jonathan M
JM
Henderson
Peter J F
PJ
Phillips
Simon E V
SE
McPherson
Michael J
MJ
Baldwin
Stephen A
SA
D002021:Buffers; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003460:Crystallization; D003470:Culture Media; D003902:Detergents; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification; D005990:Glycerol; D006863:Hydrogen-Ion Concentration; D008027:Light; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000302:isolation & purification; D017551:Microdialysis; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D012542:Scattering, Radiation
D002021:Buffers; D003470:Culture Media; D003902:Detergents; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D005990:Glycerol