Send Orders of Reprints at bspsaif@emirates.net.ae Protein & Peptide Letters, 2013, 20, 133-139 133 Cloning and Purification of IpaC Antigen from Shigella flexneri: Proposal of a New Methodology Cristiane Mobilon 1 , Marcelo Augusto Szymanski de Toledo 2 , Fernanda Laroza Paganelli 3 , Clelton Aparecido dos Santos 2 , Fernanda de Pace 4 , Jacqueline Boldrin de Paiva 1 , Eliana Guedes Stehling 5 , Gerson Nakazato 6 , Aline Gambaro Balieiro 7 , Flávia Pereira da Silva Airoldi 7 , Francisco de Assis Machado Reis 7 , Wanderley Dias da Silveira 1, * 1 Department of Genetics, Evolution and Bioagents, State University of Campinas, Campinas, SP, Brazil; 2 Center of Mo- lecular Biology and Genetics Engineering, State University of Campinas, Campinas, SP, Brazil; 3 Department of Medi- cal Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands; 4 Department of Internal Medicine, State University of Campinas, Campinas, SP, Brazil; 5 Department of Toxicological and Bromatologic Clinical Analyses, University of São Paulo, Ribeirão Preto, SP, Brazil; 6 Department of Microbiology, Londrina State University, Londrina, PR, Brazil; 7 Department of Organic Chemistry, Institute of Chemistry, State University of Campinas, Campinas, SP, Brazil Abstract: Shigella flexneri is a Gram-negative bacillus that is responsible for a severe form of dysentery called Shigello- sis, which mainly affects children and the elderly in both underdeveloped and developed countries. Pathogenic S. flexneri strains possess a large virulence plasmid that codes for effector proteins that are required for the entry and spread of the bacteria into colonocytes. Among these proteins is the translocator IpaC, which plays an important role in the invasion process; IpaC is implicated in pore formation in the host cell membrane and induces cytoskeletal rearrangements in macrophages and epithelial cells, thereby promoting bacterial entry. The ability of IpaC to insert into the plasma mem- brane is due to a large nonpolar region of the protein structure. This characteristic also renders difficulties in recovery and purification when the protein is expressed in E. coli. Several works have considered different methodologies for the im- proved production and purification of IpaC. Herein, we propose an alternative method that is based on changes in the in- duction temperature and extraction buffer to facilitate the accumulation of high yields of soluble proteins for their further processing and ultimate use in biotechnological approaches. Keywords: Shigella flexneri, IpaC production, purification, shigellosis, vaccines. INTRODUCTION Shigella flexneri is a Gram-negative bacillus that is re- sponsible for a severe form of dysentery called shigellosis, which mainly affects children and the elderly in both under- developed and developed countries. The infection process of S. flexneri involves the bacterial invasion of the colonic epi- thelium after oral contamination [1], the intracellular move- ment and multiplication of the pathogen [2, 3] and the infec- tion of neighboring epithelial cells [2, 4, 5]. Pathogenic S. flexneri strains possess a large virulence plasmid that codes for effector proteins that are required for the entry and spread of the bacteria into colonocytes. Within this large plasmid is a 31-kb region which encodes structural components of the Mxi-Spa type III secretion system (TTSS) needed for its assembly and function in addition to the mem- brane-related translocators IpaB, IpaC and IpaD, the effector proteins IcsB, IpgB1, IpgD and IpaA, the chaperones IpgA, IpgC, IpgE and Spa15, and the transcription activators VirB and MxiE [6]. The membrane-related translocator IpaC is *Address correspondence to this author at the Institute of Biology, PC6109, Department of Genetics, Evolution and Bioagents, State University of Campinas, São Paulo, Brazil; Tel: 55(19)35216268; Fax: 55(19)35216185; E-mail: wds@unicamp.br considered to be essential to the invasion process of Shigella and is the first protein that is recognized by the host immune system [7-12]. Together with IpaB, the IpaC antigen is im- plicated in pore formation in the host cell membrane to per- mit the passage of other Shigella proteins that are essential to the invasion process [13, 14]. IpaC also induces cytoskeletal rearrangements in macrophages and epithelial cells that pro- mote bacterial entry [9, 12, 15, 16]. Two predicted trans- membrane domains that are located within a large nonpolar region give IpaC the ability to facilitate its insertion into the plasma membrane but lead to difficulties in its purification following heterologous expression [15, 17]. When IpaC and IpaB are in the Shigella cytoplasm, they are maintained in a soluble state due to their association with IpgC, which is the IpaB/IpaC-specific chaperone [18]. Several works have considered different methodologies for the better production and purification of IpaC, including the addition of denaturing reagents to buffers, changes in temperature and shaker rotation during induction or the co- expression of IpaC chaperone to promote solubility [7, 17, 19]. In this work, ipaC was cloned and expressed in E. coli with the pET system to produce a high yield of soluble IpaC 1875-5305/13 $58.00+.00 © 2013 Bentham Science Publishers