A simple strategy towards membrane protein purification and crystallization.
A simple and cost-efficient detergent screening strategy has been developed, by which a number of detergents were screened for their efficiency to extract and purify the recombinant ammonium/ammonia channel, AmtB, from Escherichia coli, hence selecting the most efficient detergents prior to large-scale protein production and crystallization. The method requires 1 ml cell culture and is a combination of immobilized metal ion affinity chromatography and filtration steps in 96-well plates. Large-scale protein purification and subsequent crystallization screening resulted in AmtB crystals diffracting to low resolution with three detergents. This strategy allows exclusion of detergents with the lowest probability in yielding protein crystals and selecting those with higher probability, hence, reducing the number of detergents to be screened prior to large-scale membrane protein purification and perhaps also crystallization.
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Damian
D
Niegowski
Marie
M
Hedrén
Pär
P
Nordlund
Said
S
Eshaghi
D027682:Cation Transport Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods; D003902:Detergents / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
C111096:AmtB protein, E coli; D027682:Cation Transport Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins