Functionality of two vacuolar sequences in different tissues of Nicotiana tabacum and their use in the production of immunoglobulins in plants

LAREU, FABRICIO; PETRUCCELLI, SILVANA

La habana

Congreso; Biotechnology Havana’ 2011; 2011

CIGB

The increase in the global demand for recombinant proteins for a variety of industrial, diagnostic and therapeutic applications is encouraging the improvement of the large scale production platforms. One important limitation of plant expression systems is the low protein yield. A general strategy to augment protein accumulation is to retain it inside the endoplasmic reticulum, but this approach is not adequate for protein that requires N-glycosylation. Since secreted proteins are frequently produced with low yields, we focus on study vacuolar sorting signals as strategy to increase protein stability. The aim of this work was first to test the hypothesis that heterologous proteins can be stored in vacuoles without affecting plant survival. The second aim was to evaluate the stability and proper folding of the vacuolar sorted recombinant proteins in different tissues. Finally, the strategy was tested using full length immunoglobulin. To reach the first goal, constructs consisting in green fluorescent protein- β-glucuronidase (GFP-GUS) reporter fused to two vacuolar sorting signals of the 11S amaranth globulin: the C-terminal KISIA pentapeptide (GG-AmhCt) and the GNIFRGF internal sequence (GG-Amh-H1N) were introduced into Nicotiana tabacum.  GUS activity in leaves, roots and seeds of the GG-AmhCt and GG-AmhH1N transgenic tobacco plants, was analyzed with X-Gluc histochemical. A typical 35S expression pattern was obtained in the different tissues analyzed confirming the proper folding and stability of the enzyme. Localization of the GFP reporter was studied by confocal laser scanning microscopy. In epidermis of GG-Amh-H1N leaves, GFP was found in lytic and central vacuoles, as confirmed when co expression with mCherry-ﻻ-TIP marker was analyzed.  GG-Amh-H1N transgenic tobacco root tip cells showed GFP in central vacuoles that are stained with neutral red. GG-Amh-Ct plants showed green fluorescent in storage vacuoles both in mesophyll of leaves and meristem root tips. In seeds both signals sorted GFP-GUS to PSV (protein storage vacuole). Finally, Ct-Amh was fused the 14D9 gamma sequence (ﻻ) to obtain ﻻ-Ct-Amh and ﻻ-RFP-Ct-Amh. These constructs were agroinfiltrated in leaves of 14D9 kappa transgenic tobacco plants and analyzed by ELISA, Western Blot and confocal scanning microscopy 72hs after infiltration. A full length functional 14D9 mAb was detected confirming the value of this strategy.