Affinity purification of recombinant proteins using a LysM domain and bacterium like particles

Authors
Padilla Franzotti, Carla Luciana; Raya Tonetti, María Fernanda; Arce, Lorena Paola; Rodriguez Vaquero, Maria Jose; Vizoso Pinto, María Guadalupe
Publication Year
2018
Language
English
Format
article
Status
Published version
Description
The lysin motif (LysM) is a ubiquitous motif across kingdoms, which in bacteria allows cell wall degrading enzymes to bind noncovalently to peptidoglycan. This property has been exploited for two decades to design mucosal vaccines consisting of LysM-tagged recombinant proteins anchored to bacterium like particles (BLP) as carriers. Surprisingly, less attention has been paid to apply the LysM motif to protein purification of recombinant proteins. Thus, our goal was to determine if the LysM motif is suitable for recombinant protein purification.We obtained the BLPs by treating overnight cultures of lactobacilli with acid and heat to get rid of other cell wall components that may interfere with binding. To select the best binding matrix, we generated BLPs from 3 different Lactobacillus species: L. rhamnosus, L. fermentum, and L. vaginalis and checked them by transmission electron microscopy. We constructed a fusion protein consisting of the yellow fluorescent protein Venus fused to a module containing five LysM motifs derived from a Lactobacillus sp. strain. The recombinant protein was expressed in E. coli Rossetta using standard procedures, and the supernatant containing the fusion protein was incubated with BLPs for binding. We evaluated the effectiveness of binding by fluorescent microscopy and SDS-PAGE. After binding, the complex was washed several times, and the elution of the protein was tested by changing pH, ionic strength and buffer composition. As a conclusion, we demonstrate that the LysM motif can be used as novel tag to purify recombinant proteins by affinity using an economical matrix, obtaining similar yields to the NiNTA system for protein purification.
Fil: Padilla Franzotti, Carla Luciana. Universidad Nacional de Tucumán. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Fil: Raya Tonetti, María Fernanda. Universidad Nacional de Tucumán. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Fil: Arce, Lorena Paola. Universidad Nacional de Tucumán. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Fil: Rodriguez Vaquero, Maria Jose. Universidad Nacional de Tucuman. Facultad de Bioquímica, Química y Farmacia. Instituto de Microbiología "luis Verna". Cátedra de Microbiología Gral.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán; Argentina
Fil: Vizoso Pinto, María Guadalupe. Universidad Nacional de Tucumán. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Subject
recombinant proteins
Lactobacillus species
Biología Celular, Microbiología
Ciencias Biológicas
CIENCIAS NATURALES Y EXACTAS
Access level
Restricted access
License
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repository
CONICET Digital (CONICET)
Institution
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identifier
oai:ri.conicet.gov.ar:11336/81334