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

C.L.PADILLA FRANZOTTI; M.F. RAYA TONETTI; L. ARCE; RODRIGUEZ VAQUERO; M.G.VIZOSO PINTO

Ginebra

Congreso; European Congress on Biotechnology 2018; 2018

European Federation of Biotechnology

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 fortwo decades to design mucosal vaccines consisting of LysM-taggedrecombinant proteins anchored to bacterium like particles (BLP) ascarriers. Surprisingly, less attention has been paid to apply the LysMmotif to protein purification of recombinant proteins. Thus, ourgoal was to determine if the LysM motif is suitable for recombinantprotein purification.We obtained the BLPs by treating overnight cultures of lactobacilli with acid and heat to get rid of other cell wall componentsthat 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 transmissionelectron microscopy. We constructed a fusion protein consistingof the yellow fluorescent protein Venus fused to a module containing five LysM motifs derived from a Lactobacillus sp. strain. Therecombinant protein was expressed in E. coli Rossetta using standard procedures, and the supernatant containing the fusion proteinwas incubated with BLPs for binding. We evaluated the effectiveness of binding by fluorescent microscopy and SDS-PAGE. Afterbinding, the complex was washed several times, and the elutionof the protein was tested by changing pH, ionic strength and buffercomposition. As a conclusion, we demonstrate that the LysM motifcan be used as novel tag to purify recombinant proteins by affinityusing an economical matrix, obtaining similar yields to the NiNTAsystem for protein purification.