Co-expression of an Elastin-Like Protein as a strategy to improve accumulation of a full antibody in N. benthamiana leaf tissue

ACEVEDO, GONZALO; PETRUCCELLI, SILVANA

Verona

Congreso; Plant Based Vaccines, Antibodies and Biologics PBVA 2013; 2013

Universidad de Verona

n the recent years, Elastin-Like Proteins (ELP) have been studied as fusion partners for recombinant proteins, in order to allow an easy and effective way to recover them from diverse organisms. When ELP fusion constructs are transiently expressed in Nicotiana benthamiana leaf tissue, aggregation occurs within the endoplasmic reticulum with the formation of structures known as protein bodies (PB). In this work we hypothesize that the formation of PB could modify cell folding capacity and consequently have an effect on the accumulation of other proteins in the secretory pathway. To test this hypothesis a hydrophobic ELP [(VPGXG)36 , where X is V or P] with a low phase transition temperature, consequently insoluble under normal plant growing conditions, was constructed. The ELP construct was co-expressed with a fluorescent ER marker protein (GFP or RFP), to analyze the effect on accumulation (in trans effect). Using a confocal microscope, formation of highly mobile punctuate fluorescent structures: the PB, were observed 3-6 days after agroinfiltration. Co-infiltrations performed with sialyl-transferase reporter construct, showed that the PB structures do not co-localize with the Golgi apparatus. When, ER-RFP is used as reporter an ?overflow? of RFP into central vacuole is observed, that is not detected with ER-GFP. Immunoblot assays revealed high amount of GFP on leaves co-infiltrated with ELP as compared with the one obtained in control leaves without ELP. Immunoblot of microsomal fractions performed with his-tag specific antibody demonstrate that ELP is inside the membranous system of the plant cell. To examine the effect of ELP on synthesis of other proteins we assess the consequences of PB formation on the accumulation of the full length recombinant antibody (14D9) in N. benthamiana leaves. Preliminary results indicate that the ELP enhances the accumulation of the light (kappa) and heavy (gamma) chains of this antibody, as shown by confocal microscopy performed on leaf tissue co-expressing their respective fusions to fluorescent reporters. In conclusion, this work shows the expression of ELP construct in leaves besides inducing PB formation causes increased yields of ER-GFP, ER-RFP and a full length monoclonal antibody not fused to this polymer fusion tag.