The development, characterization, and demonstration of a novel strategy for purification of recombinant proteins expressed in plants

Abstract

Plants have attracted increasing attention as an expression platform for the production of pharmaceutical proteins due to its unlimited scalability and low cost potential. However, compared to other expression systems, plants accumulate relatively low levels of foreign proteins, thus necessitating the development of efficient systems for purification of foreign proteins from plant tissues. We have developed a novel strategy for purification of recombinant proteins expressed in plants, based on genetic fusion to soybean agglutinin (SBA), a homotetrameric lectin that binds to N-acetyl-D-galactosamine. Previously it was shown that high purity SBA could be recovered from soybean with an efficiency of greater than 90% following one-step purification using N-acetyl-D-galactosamine-agar columns. We constructed an SBA fusion protein containing the reporter green fluorescent protein (GFP) and transiently expressed it in N. benthamiana plants. We achieved over 2.5% of TSP accumulation in leaves of N. benthamiana. Confocal microscopic analysis demonstrated in vivo activity of the fused GFP partner. Importantly, high purity rSBA-GFP was recovered from crude leaf extract with ~90% yield via one-step purification on N-acetyl-D-galactosamine-agar columns, and the purified fusion protein was able to induce the agglutination of rabbit red blood cells. Combined with this, tetrameric assembly of the fusion protein was demonstrated via western blotting. In addition, rSBA-GFP retained its GFP signal on agglutinated red blood cells, demonstrating the feasibility of using rSBA-GFP for discrimination of cells that bear the ligand glycan on their surface. This work validates SBA as an effective affinity tag for simple and rapid purification of genetically fused proteins.

Growing Uses of 2A in Plant Biotechnology

“…Unfortunately, the high cost of enzymes is a major barrier in the biomass-to-fuel industry [128]. Observed results indicate in planta enzyme expression offers a potential method for low cost large-scale enzyme production [129,130]. Current indications are subcellular targeting [131,132] and simultaneous expression of recombinant cellulolytic enzymes [133] are key factors in optimizing their accumulation in transgenic plants.…”

Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming

“…HDEL (His-Asp-Glu-Leu) retention peptide was also shown to be crucial for ER targeting of the IgG-HDEL fusion into transgenic tobacco plants [83]. The histidine tag (his-tag) is widely used for targeting and facilitating the purification of recombinant proteins [80,84]. The ZZ linker from Staphylococcus aureus is also used as an epitope tag in chloroplast expression cassette to facilitate the purification process of synthetic and native IGF-1 [78].…”

Mannose‐specific lectin isolation from Canavalia ensiformis seeds by PHEMA‐based cryogel

“…As lectins have many functions in biological processes, such as host defense mechanisms, inflammation, metastasis, cell differentiation, and development of organisms, they are valuable tools in biochemistry 2, 3. They are used in a variety of biological applications including cell separation, mitogenic stimulation of immune cells, and following changes that occur in cell surface sugars in processes such as development and differentiation 4–6. Lectins are also used for the purification and characterization of glycoconjugates 7–11…”