The fusion to a transmembrane domain as strategy to sort a monoclonal antibody to vacuoles

SCABONE, CAMILA MARIA; PETRUCCELLI, SILVANA

La Habana

Congreso; Biotechnology Havana? 2011; 2011

CIGB

Low protein yield is a significant problem for the commercial exploitation of plant expression systems. Increasing the stability of the recombinant protein by sorting to different compartments might be a strategy to improve the efficacy of plant platforms. Plant cells can storage different metabolic products in certain compartments such as plant storage vacuoles. The aim of this work was to develop a new strategy to target recombinant proteins to the vacuolar storage system in transgenic plants by direct fusion of the interest protein to the transmembrana (TM) and cytosolic tail (CT) domains of AtRMR1 (Arabidopsis Receptor-like Membrane Ring-H2). We propose that this fusion could be an efficient way to sort protein of interest to vacuoles, avoiding the requirement of interaction between the cargo protein and the receptor, which is limited by the affinity of the receptor for its target and it is susceptible of saturation. A secreted version of RFP (secRFP) and a fusion of it to the TM and CT domains of AtRMR1 (RFPTMCT) were produced and studied both in transient and stable expression assays. The fusion of TMCT to secRFP is sufficient to sort RFP to central vacuoles in leaves and roots and to PSV in cotyledons of mature embryos. The full-length RFP-TMCT was found in the vacuolar lumen and not associated with the vacuolar membrane suggesting its internalization; supported by the fact that RFP-TMCT in transit through the secretory pathway was found associated with microsomes membranes. In order to test the TMCT fusion strategy, this sorting signal was fused to the monoclonal antibody (mAb) 2G3 that recognized the autoantigen of celiac disease the human-tissue transglutaminasa (htTG). The TMCT signal was introduced at the C-terminal ends of the LC and HC. In addition, a vacuolar sorting signal (VSS) of a cargo protein was also tested. The obtained fusions were cloned into the pGWB2 binary vector, introduced into Agrobacterium tumefaciens GV3101 and temporal expressions in N. benthamiana leaves were carried out. Different combinations of theses constructs and also secretory controls were analyzed 3-4 days after agroinfiltration. Assemble and functionality of 2G3-RFPTMCT was confirmed by enzyme-linked immunosorbent assay using recombinant htTG and localization was confirmed by confocal scanning microscopy. Based on the results presented here, we propose that the TMCT domain could be used to sort protein of interest to vacuoles.