Phospholipid Analysis and Fractional Reconstitution of the Ice Nucleation Protein Activity Purified from Escherichia coli Overexpressing the inaZ Gene of Pseudomonas syringae M. A. Palaiomylitou,* A. Kalimanis,² A. I. Koukkou,² C. Drainas,² E. Anastassopoulos,‡ N. J. Panopoulos,‡ L. V. Ekateriniadou,* and D. A. Kyriakidis* *Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece; ² Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece; and ‡Biology Department, University of Crete, Heraklion and Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Crete, Greece Ice nucleation protein was partially purified from the membrane fraction of E. coli carrying inaZ from Pseudomonas syringae. The ice nucleation protein was totally localized in the bacterial envelope and was extracted by either salt (0.25 M NH 4 Cl) or the nonionic detergent Tween 20. The extracted protein was partially purified by sequential passage through DEAE-52 cellulose and Sephacryl-S400 columns. The activity of the purified protein was lost after treatment with phospholipase C, and its activity was subsequently restored by addition of the naturally occurring lipid phosphatidylethanolamine. These results suggest that ice nucleation proteins have a requirement for lipids that reconstitute a physiological hydrophobic environment similar to the one existing in vivo, to attain and maintain a structure that enables ice catalysis. © 1998 Academic Press Key Words: ice nucleation protein; inaZ gene; E. coli; reconstitution. During the past 15 years work conducted by S. E. Lindow, G. Warren, P. Wolber, and their colleagues (13, 18, 24, 25, 26) has addressed many of the features of bacterial ice nucleation proteins. Ice nucleation proteins, products of single genes isolated and characterized at the nucleotide level from various gram-negative plant pathogenic or epiphytic bacteria of the genera Pseudomonas, Erwinina, and Xan- thomonas, have been identified as outer mem- brane proteins (14). Their main function is to orient molecules of water at subzero tempera- tures into an array that can create an ice embryo (18, 24). The ice nucleation protein has been purified from Escherichia coli cells overexpressing the ice nucleation gene (inaZ) cloned from Pseudo- monas syringae and was identified as a 150- to 180-kDa protein (4, 14, 26). 3D structural anal- ysis of this protein is hampered by the lack of crystals. However, 3D structural models have been proposed based on the predicted amino acid sequence (9, 24). The phospholipid re- quirement for ice nucleation activity has been investigated to some extent in ice nuclei prep- arations recovered from bacterial membranes (8). In these studies it was shown that phos- phatidylethanolamine and phosphatidylinositol restored the ice nucleation activity of membrane preparations that was lost upon delipidation (8). Phospholipid analysis and reconstitution exper- iments using purified ice nucleation protein have not been previously reported, perhaps be- cause expression of ice nucleation genes in E. coli from strong promoters led to the accumu- lation of ice nucleation protein in inclusion bod- ies or because purified protein had lost its ice nucleation activity (25, 26). However, the many applications of ice nucleation protein in the artificial snow and frozen food industries (13) would benefit from further research in order to elucidate the mode of interaction with phospho- lipids that may result in efficient reconstitution of warm temperature cell-free ice nuclei for these applications. In the present report we describe the purifi- cation of active ice nucleation protein from membranes of recombinant E. coli cells over- Received January 14, 1998; accepted May 19, 1998. CRYOBIOLOGY 37, 67–76 (1998) ARTICLE NO. CY982102 67 0011-2240/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.