Purification of Myristoylated and Nonmyristoylated Neuronal Calcium Sensor-1 Using Single-Step Hydrophobic Interaction Chromatography Jon R. Fisher,* Yogendra Sharma,† ,1 Sheila Iuliano,* Robert A. Piccioti,* Dimitri Krylov,‡ James Hurley,‡ John Roder,§ and Andreas Jeromin§ *Tosohaas, 156 Keystone, Montgomeryville, Pennsylvania 18936; †Centre for Cellular and Molecular Biology, Hyderadad, India; ‡HHMI, University of Washington, Seattle, Washington 98195; and §Mt. Sinai Hospital, SLRI, Toronto, Ontario M5G 1X5, Canada Received April 3, 2000 Neuronal calcium sensors (NCSs) belong to a family of Ca 2 -binding proteins, which serve important functions in neurotransmission, and are highly conserved from yeast to humans. Overexpression of the neuronal cal- cium sensor-1, called frequenin in the fruit fly and in frog, increases the release of neurotransmitters. Study- ing the functional role of frequenin in mammals and understanding its structural dynamics is critically de- pendent on the availability of active purified protein. Neuronal calcium sensors like other members of the family share common structural features: they contain four EF-hands as potential binding sites for Ca 2 and an N-terminal consensus sequence for myristoylation. Pre- viously, recoverin, distantly related to NCSs, has been expressed and purified from Escherichia coli, involving a combination of different chromatographic steps. NCS-1 has earlier been purified adopting a two-step pro- cedure used for recoverin purification. We have overex- pressed NCS-1 from rat in its myristoylated and nonmy- ristoylated form in E. coli and purified it from crude lysates using a single-step hydrophobic interaction chromatography. The purified protein was identified by Western blotting and mass spectrometry and assayed for its ability to bind Ca 2 using a Ca 2 shift assay, terbium fluorescence, and Stains-all binding. The present proto- col provides a rapid, more efficient and simplified, single-step method for purifying NCS-1 for structural and functional studies. This method can also be applied to purify related proteins of the superfamily. © 2000 Academic Press Neuronal calcium sensor-1 (NCS-1) 2 belongs to a growing family of Ca 2+ -binding proteins, which share characteristic structural features like the EF-hands or helix–loop– helix motifs (commonly found in potential Ca 2+ -binding sites of proteins) and a N-terminal my- ristoylation motif. Each of the EF-hands consists of a loop of 12 amino acids flanked by two -helices. This structural principle was first identified in the crystal structure of the carp parvalbumin and is designated EF-hand after the E- and F-helices of parvalbumin (1). All members of this family contain an N-terminal my- ristoylation motif and in the case of the distantly re- lated members recoverin (2), neurocalcin (3), and hip- pocalcin (4) only the myristoylated and not the nonmyristoylated form shows Ca 2+ -dependent binding to membranes. From its biochemical analysis and NMR structure, recoverin has been described as a cal- cium–myristoyl switch (5,6). Ca 2+ binding to two of the four EF-hand-like domains of recoverin leads to the exposure of an N-terminal myristoyl group, allowing membrane attachment. This movement of the myris- toyl group also exposes a hydrophobic pocket that may interact with target proteins (5). Overexpression of NCS-1 in Drosophila (7) and in Xenopus (8) has been shown to increase synaptic effi- cacy. (We refer to members of the family as neuronal calcium sensors (NCSs). NCS-1 has been termed fre- quenin in Drosophila and Xenopus because of its phys- iological properties.) Overexpression of NCS-1 in the neuronal cell line PC12 led to an enhancement of se- 1 To whom correspondence should be addressed at Centre for Cel- lular and Molecular Biology, Uppal Road, Hyderabad-500 007, India. Fax: +91-40-717-1195. E-mail: yogendra@ccmb.ap.nic.in. 2 Abbreviations used: NCS-1, neuronal calcium sensor; Stains-all, 1-ethyl-2-[3-(1-ethylnaphthol[1,2-d]thiazolin-2-ylidene)-2-methyl- propenyl]naphthol[1,2-d]thiazolium bromide; CD, circular dichro- ism; HIC, hydrophobic interaction chromatography; DTT, dithiothre- itol. Protein Expression and Purification 20, 66 –72 (2000) doi:10.1006/prep.2000.1298, available online at http://www.idealibrary.com on 66 1046-5928/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.