Cloning and purification of IpaC antigen from Shigella flexneri: proposal of a new methodology.
Shigella flexneri is a Gram-negative bacillus that is responsible for a severe form of dysentery called Shigellosis, 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 onto the plasma membrane 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 improved production and purification of IpaC. Herein, we propose an alternative method that is based on changes in the induction temperature and extraction buffer to facilitate the accumulation of high yields of soluble proteins for their further processing and ultimate use in biotechnological approaches.
Department of Genetics, Evolution and Bioagents, State University of Campinas, Campinas, SP, Brazil.
Cristiane
C
Mobilon
Marcelo Augusto Szymanski
MA
de Toledo
Fernanda Laroza
FL
Paganelli
Clelton Aparecido
CA
dos Santos
Fernanda
F
de Pace
Jacqueline Boldrin
JB
de Paiva
Eliana Guedes
EG
Stehling
Gerson
G
Nakazato
Aline Gambaro
AG
Balieiro
Flavia Pereira da Silva
FP
Airoldi
Francisco de Assis Machado
Fde A
Reis
Wanderley Dias
WD
da Silveira
D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004405:Dysentery, Bacillary / Q000276:immunology / Q000382:microbiology; D004797:Enzyme-Linked Immunosorbent Assay; D012763:Shigella flexneri / Q000276:immunology / Q000378:metabolism*
D000942:Antigens, Bacterial; C092342:IpaC protein, Shigella