Expression and purification of recombinant annexin V for the detection of membrane alterations on apoptotic cells Gabriela Brumatti 1 , Clare Sheridan 1 , Seamus J. Martin * Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland Accepted 10 November 2007 Abstract Apoptosis is a mode of cell death that is accompanied by specific alterations to the plasma membrane that promote the recognition and engulfment of these cells by phagocytes. Although several such membrane alterations have been defined, redistribution of phospha- tidylserine from the inner to the outer plasma membrane leaflet has become one of the most widely used markers for apoptotic cells in mammals. This is largely due to the availability of a sensitive and specific probe for this event in the form of the phosphatidylserine-bind- ing protein, annexin V. Here, we describe methods for the expression and purification of recombinant polyhistidine-tagged annexin V from Escherichia coli. Recombinant annexin V is highly soluble and is thus readily expressed and purified to high yields; typically in the region of 4 lg of protein per ml of bacterial culture. We also describe methods for conjugation of this protein to the FITC fluoro- phore and for its use for the detection of apoptotic cells by flow cytometry or fluorescence microscopy. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Annexin V; Apoptosis; Bacterial expression; Phosphatidylserine; Plasma membrane changes 1. Introduction Annexin V belongs to the family of phospholipid-bind- ing annexins that share similarity in structure and function [1]. There are 12 known vertebrate members of the annexin family and all are characterised by an ability to bind phos- pholipids in a reversible and Ca 2+ -dependent manner. Annexins have been implicated in several physiological processes, such as Ca 2+ regulated exocytosis, endocytosis, mitogenic signal transduction, differentiation and coagula- tion, which exploits their shared ability to interact with membrane phospholipids [1,2]. Annexin V (also known as annexin A5) binds most effi- ciently to the negatively charged phospholipid, phosphati- dylserine (PS), with modest interaction also detected with phosphatidlycholine (PC) or sphingomyelin. Phosphatidyl- serine resides constitutively in the inner plasma membrane leaflet but is rapidly externalized on the outer leaflet of the plasma membrane in response to increases in intracellular Ca 2+ , cell injury or apoptosis-inducing agents [3–5]. Annexin V has also been shown to play an important role in blood coagulation and efficient recognition and removal of apoptotic cells [6–9]. As an anti-coagulant factor, annexin V prevents the interaction of exposed phosphatid- lyserine on platelets with coagulation factors such as pro- thrombin. Annexin V binds as a triad to the negatively charged PS moiety and inhibits the interaction with other proteins. Disruption of annexin V binding to PS results in an increase in PS exposure and deregulation of blood coagulation as in the condition anti-phospholipid syn- drome [10]. PS externalization is a relatively early event in apoptosis and occurs before plasma membrane integrity is compro- mised [11]. PS serves as a trigger for the recognition of apoptotic cells by macrophages and liposomes containing this phospholipid inhibit the engulfment of apoptotic cells by phagocytes [9]. Thus, PS externalization during apopto- sis promotes the clearance of apoptotic cells, thereby 1046-2023/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2007.11.010 * Corresponding author. E-mail address: martinsj@tcd.ie (S.J. Martin). 1 These authors share first authorship. www.elsevier.com/locate/ymeth Available online at www.sciencedirect.com Methods 44 (2008) 235–240