Towards higher-throughput membrane protein production for structural genomics initiatives.
Integral membrane proteins present unparalleled challenges for structural genomics programs. Samples from this class of proteins are not only difficult to produce in quantities sufficient for analysis by X-ray diffraction or NMR, but their hydrophobic properties add extra dimension to their purification and subsequent crystallization. New systems that seek to tackle the production problems are in development. In our laboratory, one such strategy exploits the unique physiology of the Rhodobacter species of photosynthetic bacteria where we have designed an overexpression system that coordinates the heterologous production of targeted hydrophobic proteins with nascent, unfilled membranes that can be used to harbor them. In this study, we describe the means by which purification of recombinant membrane proteins produced in such a fashion can be purified efficiently from Rhodobacter membranes using relatively higher-throughput, semi-automated methods. These protocols utilize a state-of-the-art FPLC system for affinity chromatography, followed by gel filtration or ion exchange chromatography to enhance purity for crystallization attempts. The Rhodobacter expression system coupled with the semi-automation of purification steps represents an advance towards the development of a strategy for obtaining structures for membrane proteins at a more rapid pace.
Laible
Philip D
PD
Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439, USA. laible@anl.gov
Scott
Heather N
HN
Henry
Lynda
L
Hanson
Deborah K
DK
D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D015394:Molecular Structure; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012242:Rhodobacter sphaeroides / Q000235:genetics / Q000378:metabolism
D008565:Membrane Proteins; D011994:Recombinant Proteins