Vacuolar sorting as strategy to increase recombinant protein accumulation levels in plants

PETRUCCELLI, SILVANA

La Habana

Congreso; Biotechnology Havana? 2011; 2011

CIGB

Currently high-capacity production systems are required to fulfil the needs of numerous  biopharmaceutical proteins, among them antibodies. Of the large-scale production systems, plants offer several advantages, such as the low cost of materials, ease of scale-up and availability of natural protein storage organs. In addition, plants are the safest biological system therefore many research groups are developing systems based on different plant species and tissues for production. Seeds are convenient tissues to express recombinant proteins because they accumulate proteins in relatively large amounts in a stable environment for long periods of time. In dicotyledonous seeds, proteins accumulate in a special compartment, which has not equivalent in yeast and mammalians, named Protein Storage Vacuoles (PSV). The transport pathway to PSV has been studied only in few plant species and the sorting machinery involved in the traffic and the transport route are scarcely known. To explore the feasibility accumulated proteins in PSV, the monoclonal antibody 14D9 (mAb14D9) fused to Ct-KDEL sequence was expressed in tobacco seeds. The KDEL sequence follows the rules for CtVSS (vacuolar sorting signals). In seeds, mAb-KDEL was both partially sorted to PSV and also secreted. In contrast, mAb-KDEL in leaves was efficiently retained in the endoplasmic reticulum. Glycosylation pattern of mAb-KDEL purified from leaves was consistent with ER retention/retrieve from the cis Golgi, while seed purified mAb-KDEL has trans-Golgi N-glycan modifications. Accumulation level of mAb-KDEL in seeds was higher than that of the secreted mAb. In order to increase the efficiency of vacuolar sorting, VSS derived from cargo vacuolar proteins and also vacuolar sorting receptors were identified and their ability to sort reporter proteins (FPs) to vacuoles in different plant organs was evaluated using both transient and stable expression assays. FPs fused to different VSS were sort to central vacuoles in leaves and roots and to PSV in cotyledons of mature embryos. The identified VSSs were fused to full length immunoglobulins and expression of mAbs-VSS was studied using transient expression assays. Vacuolar localization of the mAb-VSS was confirmed by confocal scanning microscopy. In addition, assembly, functionality and stability were studied by enzyme-linked immunosorbent assay and Western blot. The obtained results established the feasibility of vacuolar sorting as alternative strategy to accumulate proteins in plants.