International Journal of Biological Macromolecules 39 (2006) 83–87 A simple strategy towards membrane protein purification and crystallization  Damian Niegowski a,b , Marie Hedr´ en a , P¨ ar Nordlund a , Said Eshaghi a,∗ a Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden b Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden Received 11 November 2005; received in revised form 13 December 2005; accepted 9 February 2006 Available online 23 February 2006 Abstract 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. © 2006 Elsevier B.V. All rights reserved. Keywords: Membrane proteins; Detergent screen; High-throughput 1. Introduction The recombinant expression and subsequent purification of integral membrane proteins are considered major challenges, and together with the crystallization step, the major hurdles towards routine structure determination of membrane proteins. Consequently, the number of membrane proteins with known structure has remained negligible as compared to those of solu- ble proteins [1,2]. This in turn causes serious lack of information in the field of drug discovery, since membrane proteins already cover more than 50% of the current drug targets [3,4]. There- fore, it is essential to improve the success rate in membrane proteins structural determination, by dealing with experimental difficulties in the production of these proteins. Experimental procedures for handling and isolating integral membrane proteins are generally more challenging than their soluble counterparts, since the former requires purification in detergent. General experiences from workers in the field with  This paper was presented at the “Challenging Proteins Workshop” in Paris, October 17–18, 2005. ∗ Corresponding author. Tel.: +46 8 524 86863; fax: +46 8 524 868650. E-mail address: said.eshaghi@ki.se (S. Eshaghi). the problematic experimental behaviour of integral membrane proteins have lead to the expectation that these proteins are dra- matically harder to produce than soluble proteins. One of the reasons may be the usage of the wrong detergent during extrac- tion and purification. There are dozens of different detergents that are commonly used, dozens more that are less character- ized but still probably useful, and many novel detergents under development. It has also been reported that some compartments of the cell membrane show resistance towards certain detergents [5]. Moreover, mixtures of detergents are sometimes used dur- ing purification and crystallization [6,7]. Altogether, the size of the detergent parameter space becomes very large. There- fore, it is crucial to choose the right detergent for an efficient extraction and purification of the membrane protein of interest. Recently, we reported an efficient strategy, by which we could screen the expression of a number of membrane proteins in a high-throughput manner [8]. Here, we report the application of this strategy to design downstream protocols for large-scale pro- duction of the recombinantly expressed ammonium transporter AmtB from Escherichia coli, by which 26 detergents, 4 types of chromatography columns and various buffer conditions have been screened using a 96-well plate format. The method is very cost efficient and may easily be applied to other membrane pro- 0141-8130/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2006.02.011