Purification, characterization, and crystallization of membrane bound Escherichia coli tyrosine kinase Chelsy Chesterman, Zongchao Jia ⇑ Department of Biomedical and Molecular Sciences, Queen’s University, 18 Stuart Street, Kingston, Ontario K7L 3N6, Canada article info Article history: Received 1 July 2015 and in revised form 28 August 2015 Accepted 29 August 2015 Available online 9 September 2015 Keywords: E. coli tyrosine kinase Membrane protein purification Detergent Crystallization abstract Escherichia coli tyrosine kinase (Etk) is a membrane bound kinase in gram-negative bacteria that regulates the export of capsular polysaccharides (CPS). The molecular mechanism behind CPS regulation remains unclear, despite access to a crystal structure of the cytoplasmic kinase domain of Etk. In this study, an efficient protocol to produce full length Etk solubilized in n-dodecyl-b-D-maltoside has been established with high yield. We have determined that detergent solubilized Etk retains kinase activity, but the protein is prone to aggregation, degradation, and has been unsuccessful in protein crystallization trials. In response, we designed and characterized truncations of Etk that do not aggregate and have led to successful crystallization experiments. In this article, we discuss our optimized expression and purifica- tion protocol for Etk, the design of Etk protein truncations, and the behavior of Etk during purification in a range of stabilizing detergents. These efforts have successfully produced protein suitable for crystalliza- tion. Our results will be a useful guide for future structural and functional studies of the bacterial tyrosine kinase family. Ó 2015 Elsevier Inc. All rights reserved. 1. Introduction Although approximately 50% of current drug targets are membrane proteins [1], this class of protein remains severely under-represented in structural databases due to the significant challenges associated with expression and purification. Escherichia coli tyrosine kinase (Etk) is a 726 amino acid (81 kDa) protein imbedded in the inner membrane of E. coli, which contains a large periplasmic domain flanked by two transmembrane helices and a C-terminal cytoplasmic kinase domain [2]. Etk is also a representa- tive member of the bacterial tyrosine kinase family, which is found across gram-negative bacteria. This family of tyrosine kinases in prokaryotes is structurally unique compared to eukaryotic kinases [3]. Therefore, this family is an interesting target for the develop- ment of novel antibiotics. Etk is a member of a protein complex that spans both the inner and outer membranes in E. coli, and is expected to bind the YccZ channel protein in the outer membrane (analogous to the Wzc–Wza complex reported in [4]). This complex is involved in the Wzy-dependent mechanism used for group 4 capsular polysac- charide (CPS) export from the cell [5] and polysaccharides exported by this mechanism play an important role in virulence [6]. Much effort has been exerted to functionally characterize the Wzy-dependent export mechanism, however structural informa- tion about the Wzy complex and its regulation remains limited [5]. Based on studies of the homologous protein Wzc, Etk is expected to have a regulatory role in this complex which involves auto-phosphorylation of a C-terminal tyrosine cluster [3,4,7]. Two crystal structures of the kinase domain [3,8] and a structure for the outer membrane channel protein are available [9], but the trans- mission of regulatory signals from the kinase domain to the chan- nel is unclear. Signal transduction likely involves conformational changes in the kinase domain [3,10] that are transmitted across the inner membrane to Etk’s periplasmic domain. Etk’s periplasmic domain likely interacts directly with the outer membrane channel protein and could be responsible for the opening and closing of this structure [4,9]. The study of signal transduction is hindered by a lack of high-resolution data for Etk’s periplasmic domain. Therefore, it is desirable to study the full-length Etk protein or truncations that contain the N-terminal domain. In order to carry out these experiments, the ability to purify membrane proteins reproducibly in large quantity is critical. Herein we describe two constructs of Etk, the full length pro- tein, and a truncation that contains the periplasmic domain flanked by the two transmembrane helices. We have developed a protocol for the production of large amounts of detergent solubilized mem- brane bound Etk by exploiting different expression temperatures, http://dx.doi.org/10.1016/j.pep.2015.08.029 1046-5928/Ó 2015 Elsevier Inc. All rights reserved. ⇑ Corresponding author. E-mail address: jia@queensu.ca (Z. Jia). Protein Expression and Purification 125 (2016) 34–42 Contents lists available at ScienceDirect Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep