The expression of a xylanase targeted to ER-protein bodies provides a simple strategy to produce active insoluble enzyme polymers in tobacco plants

Background

Xylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera) of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs).

Methodology/Principal Findings

Tobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase.

Conclusion/Significance

In the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low-cost bioreactors for industrial purposes.     

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

The N-terminal proline-rich domain of γ-zein (Zera) plays an important role in protein body (PB) formation not only in the original host (maize seeds) but in a broad spectrum of eukaryotic cells. However, the elements within the Zera sequence that are involved in the biogenesis of PBs have not been clearly identified. Here, we focused on amino acid sequence motifs that could be involved in Zera oligomerization, leading to PB-like structures in Nicotiana benthamiana leaves. By using fusions of Zera with fluorescent proteins, we found that the lack of the repeat region (PPPVHL)₈ of Zera resulted in the secretion of the fusion protein but that this repeat by itself did not form PBs. Although the repeat region containing eight units was the most efficient for Zera self-assembly, shorter repeats of 4–6 units still formed small multimers. Based on site-directed mutagenesis of Zera cysteine residues and analysis of multimer formation, we conclude that the two N-terminal Cys residues of Zera (Cys⁷ and Cys⁹) are critical for oligomerization. Immunoelectron microscopy and confocal studies on PB development over time revealed that early, small, Zera-derived oligomers were sequestered in buds along the rough ER and that the mature size of the PBs could be attained by both cross-linking of preformed multimers and the incorporation of new chains of Zera fusions synthesized by active membrane-bound ribosomes. Based on these results and on the behavior of the Zera structure determined by molecular dynamics simulation studies, we propose a model of Zera-induced PB biogenesis.     

Protein body formation in leaves of Nicotiana benthamiana: a concentration‐dependent mechanism influenced by the presence of fusion tags

Protein bodies (PBs) are endoplasmic reticulum (ER) derived organelles originally found in seeds whose function is to accumulate seed storage proteins. It has been shown that PB formation is not limited to seeds and green fluorescent protein (GFP) fused to either elastin‐like polypeptide (ELP) or hydrophobin (HFBI) fusion tags induce the formation of PBs in leaves of N. benthamiana. In this study, we compared the ELP‐ and HFBI‐induced PBs and showed that ELP‐induced PBs are larger than HFBI‐induced PBs. The size of ELP‐ and HFBI‐induced PBs increased over time along with the accumulation levels of their fused protein. Our results show that PB formation is a concentration‐dependent mechanism in which proteins accumulating at levels higher than 0.2% of total soluble protein are capable of inducing PBs in vivo. Our results show that the presence of fusion tags is not necessary for the formation of PBs, but affects the distribution pattern and size of PBs. This was confirmed by PBs induced by fluorescent proteins as well as fungal xylanases. We noticed that in the process of PB formation, secretory and ER‐resident molecules are passively sequestered into the lumen of PBs. We propose to use this property of PBs as a tool to increase the accumulation levels of erythropoietin and human interleukin‐10 by co‐expression with PB‐inducing proteins.     

Accumulation of rice prolamin–GFP fusion proteins induces ER-derived protein bodies in transgenic rice calli

Key message

We showed that rice prolamin polypeptides formed ER-derived PBs in transgenic rice calli, and that this heterologous transgene expression system is suitable for studying the mechanism of rice PB-I formation.

Abstract

Rice prolamins, alcohol-soluble seed storage proteins, accumulate directly within the rough endoplasmic reticulum (ER) lumen, leading to the formation of ER-derived type I protein bodies (PB-Is) in rice seed. Because rice prolamins do not possess a well-known ER retention signal such as K(H)DEL, or a unique sequence for retention in the ER such as a tandem repeat domain of maize and wheat prolamins, the mechanisms of prolamin accumulation in the ER and PB-I formation are poorly understood. In this study, we examined the formation mechanisms of PBs by expressing four types of rice prolamin species fused to green fluorescent protein (GFP) in transgenic rice calli. Each prolamin–GFP fusion protein was stably accumulated in rice calli and formed ER-derived PBs. In contrast, GFP fused with the signal peptide of prolamin was secreted into the intercellular space in rice calli. In addition, each of the four types of prolamin–GFP fusion proteins was co-localized with the ER chaperone binding protein. These results suggest that the mature polypeptide of prolamin is capable of being retained in the ER and induce the formation of PBs in non-seed tissue, and that the rice callus heterologous transgene expression system is useful for studying the mechanisms of rice PB-I formation.     

Development of patatin knockdown potato tubers using RNA interference (RNAi) technology,for the production of human-therapeutic glycoproteins

Background  

Patatins encoded by a multi-gene family are one of the major storage glycoproteins in potato tubers. Potato tubers have recently emerged as bioreactors for the production of human therapeutic glycoproteins (vaccines). Increasing the yield of recombinant proteins, targeting the produced proteins to specific cellular compartments, and diminishing expensive protein purification steps are important research goals in plant biotechnology. In the present study, potato patatins were eliminated almost completely via RNA interference (RNAi) technology to develop potato tubers as a more efficient protein expression system. The gene silencing effect of patatins in the transgenic potato plants was examined at individual isoform levels.     

Development of the cotyledon cells during olive (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) in vitro seed germination and seedling growth

The structural changes occurred in differentiating olive cotyledon cells into mesophyll cells are described. Using histological and immunocytological methods as well as microscopic observations, we showed that in the cells of mature embryo, large electron-dense proteins bodies (PBs) are surrounded by numerous oil bodies (OBs). After 3 days of in vitro germination, the presence of large PBs originated by fusion of smaller PBs was observed. It was also detected a close spatial proximity between PBs and OBs, likely as a reflection of interconnected metabolic pathways. Between the 3rd and the 12th day of germination, the formation of a large vacuolar compartment takes place accompanied by a decrease in the PBs and OBs number. This was coincident with a progressive decrease in the amount of the 11S-type seed storage proteins (SSPs), showed in situ and after Western blot analysis of crude protein extracts. After 26 days germination, the cellular organization became typical for a leaf mesophyll cell, with well-differentiated chloroplasts surrounding a large central vacuole. Our results suggest that the olive cotyledon storage reserves are mobilized gradually until the seedling becomes autotrophic. Moreover, the specific accumulation of storage proteins in the intravacuolar material suggests that these structures may operate as a shuttle for SSPs and/or products of their degradation into the cytoplasm, where finally they supply amino acids for the differentiating mesophyll cells.     

Quantification of the physiochemical constraints on the export of spider silk proteins by <Emphasis Type="Italic">Salmonella</Emphasis> type III secretion

Background  

The type III secretion system (T3SS) is a molecular machine in gram negative bacteria that exports proteins through both membranes to the extracellular environment. It has been previously demonstrated that the T3SS encoded in Salmonella Pathogenicity Island 1 (SPI-1) can be harnessed to export recombinant proteins. Here, we demonstrate the secretion of a variety of unfolded spider silk proteins and use these data to quantify the constraints of this system with respect to the export of recombinant protein.     

The formation,function and fate of protein storage compartments in seeds

Seed storage proteins (SSPs) have been studied for more than 250 years because of their nutritional value and their impact on the use of grain in food processing. More recently, the use of seeds for the production of recombinant proteins has rekindled interest in the behavior of SSPs and the question how they are able to accumulate as stable storage reserves. Seed cells produce vast amounts of SSPs with different subcellular destinations creating an enormous logistic challenge for the endomembrane system. Seed cells contain several different storage organelles including the complex and dynamic protein storage vacuoles (PSVs) and other protein bodies (PBs) derived from the endoplasmic reticulum (ER). Storage proteins destined for the PSV may pass through or bypass the Golgi, using different vesicles that follow different routes through the cell. In addition, trafficking may depend on the plant species, tissue and developmental stage, showing that the endomembrane system is capable of massive reorganization. Some SSPs contain sorting signals or interact with membranes or with other proteins en route in order to reach their destination. The ability of SSPs to form aggregates is particularly important in the formation or ER-derived PBs, a mechanism that occurs naturally in response to overloading with proteins that cannot be transported and that can be used to induce artificial storage bodies in vegetative tissues. In this review, we summarize recent findings that provide insight into the formation, function, and fate of storage organelles and describe tools that can be used to study them.     

Protein production from recombinant protein bodies

In this study, we describe a process for protein expression and purification from plants and insect cells based on the accumulation of recombinant proteins in protein bodies. This technology is using Zera^®, which sequence has the capacity to trigger in vivo the formation of dense, non-secretory storage protein body-like organelles derived from the endoplasmic reticulum (ER). With this method, recombinant human growth hormone (hGH) was expressed and purified from protein bodies accumulated in plants (Nicotiana benthamiana) and in insect cells (Spodoptera frugiperda). We found that recombinant Zera-hGH are stored in large quantity inside those proteins bodies and can be easily recovered during a one-step process from plant and insect cell biomass. After solubilization of recombinant protein bodies and cleavage of Zera tag from the fusion protein, active hGH was finally purified by a single chromatography step. These results indicate that recombinant proteins derived from Zera-fusion could provide both an efficient protein production system and eased purification downstream process.     

Accumulation and processing of a recombinant protein designed as a cleavable fusion to the endogenous Rubisco LSU protein in Chlamydomonas chloroplast

Background  

Expression of recombinant proteins in green algal chloroplast holds substantial promise as a platform for the production of human therapeutic proteins. A number of proteins have been expressed in the chloroplast of Chlamydomonas reinhardtii, including complex mammalian proteins, but many of these proteins accumulate to significantly lower levels than do endogenous chloroplast proteins. We examined if recombinant protein accumulation could be enhanced by genetically fusing the recombinant reporter protein, luciferase, to the carboxy-terminal end of an abundant endogenous protein, the large subunit of ribulose bisphosphate carboxylase (Rubisco LSU). Additionally, as recombinant proteins fused to endogenous proteins are of little clinical or commercial value, we explored the possibility of engineering our recombinant protein to be cleavable from the endogenous protein in vivo. This strategy would obviate the need for further in vitro processing steps in order to produce the desired recombinant protein. To achieve this, a native protein-processing site from preferredoxin (preFd) was placed between the Rubisco LSU and luciferase coding regions in the fusion protein construct.     

Cell adhesion and signaling on the fibronectin 1st type III repeat; requisite roles for cell surface proteoglycans and integrins

Background  

The first type III repeat of fibronectin is known to be involved in fibronectin matrix assembly, and recombinant proteins from this type III repeat can inhibit cell proliferation, tumor metastasis and angiogenesis. We have analyzed the way rat aortic smooth muscle cells (RASMCs) interact with a recombinant protein encompassing a C-terminal portion of the first type III repeat of fibronectin (protein III1-C).     

An efficient system to generate monoclonal antibodies against membrane-associated proteins by immunisation with antigen-expressing mammalian cells

Background  

The generation of monoclonal antibodies specific for protein antigens usually depends on purified recombinant protein for both immunisation and hybridoma screening. Purification of recombinant protein in sufficient yield and purity is a tedious undertaking and can be demanding especially in the case of membrane proteins. Furthermore, antibodies generated against a purified recombinant protein are frequently incapable of binding to the endogenous protein in its native context.     

Plant-derived protein bodies as delivery vehicles for recombinant proteins into mammalian cells

The encapsulation of biopharmaceuticals into micro- or nanoparticles is a strategy frequently used to prevent degradation or to achieve the slow release of therapeutics and vaccines. Protein bodies (PBs), which occur naturally as storage organelles in seeds, can be used as such carrier vehicles. The fusion of the N-terminal sequence of the maize storage protein, γ-zein, to other proteins is sufficient to induce the formation of PBs, which can be used to bioencapsulate recombinant proteins directly in the plant production host. In addition, the immunostimulatory effects of zein have been reported, which are advantageous for vaccine delivery. However, little is known about the interaction between zein PBs and mammalian cells. To better understand this interaction, fluorescent PBs, resulting from the fusion of the N-terminal portion of zein to a green fluorescent protein, was produced in Nicotiana benthamiana leaves, recovered by a filtration-based downstream procedure, and used to investigate their internalization efficiency into mammalian cells. We show that fluorescent PBs were efficiently internalized into intestinal epithelial cells and antigen-presenting cells (APCs) at a higher rate than polystyrene beads of comparable size. Furthermore, we observed that PBs stimulated cytokine secretion by epithelial cells, a characteristic that may confer vaccine adjuvant activities through the recruitment of APCs. Taken together, these results support the use of zein fusion proteins in developing novel approaches for drug delivery based on controlled protein packaging into plant PBs.     

Fluorescent labeling in semi-solid medium for selection of mammalian cells secreting high-levels of recombinant proteins

Background  

Despite the powerful impact in recent years of gene expression markers like the green fluorescent protein (GFP) to link the expression of recombinant protein for selection of high producers, there is a strong incentive to develop rapid and efficient methods for isolating mammalian cell clones secreting high levels of marker-free recombinant proteins. Recently, a method combining cell colony growth in methylcellulose-based medium with detection by a fluorescently labeled secondary antibody or antigen has shown promise for the selection of Chinese Hamster Ovary (CHO) cell lines secreting recombinant antibodies. Here we report an extension of this method referred to as fluorescent labeling in semi-solid medium (FLSSM) to detect recombinant proteins significantly smaller than antibodies, such as IGF-E5, a 25 kDa insulin-like growth factor derivative.     

Biotinylated-sortase self-cleavage purification (BISOP) method for cell-free produced proteins

Background  

Technology used for the purification of recombinant proteins is a key issue for the biochemical and structural analyses of proteins. In general, affinity tags, such as glutathione-S-transferase or six-histidines, are used to purify recombinant proteins. Since such affinity tags often interfere negatively with the structural and functional analyses of proteins, they are usually removed by treatment with proteases. Previously, Dr. H. Mao reported self-cleavage purification of a target protein by fusing the sortase protein to its N-terminal end, and subsequently obtained tag-free recombinant protein following expression in Escherichia coli. This method, however, is yet to be applied to the cell-free based protein production.     

Expression of an endotoxin-free S-layer/allergen fusion protein in gram-positive <Emphasis Type="Italic">Bacillus subtilis</Emphasis> 1012 for the potential application as vaccines for immunotherapy of atopic allergy

Background  

Genetic fusion of the major birch pollen allergen (Bet v1) to bacterial surface-(S)-layer proteins resulted in recombinant proteins exhibiting reduced allergenicity as well as immunomodulatory capacity. Thus, S-layer/allergen fusion proteins were considered as suitable carriers for new immunotherapeutical vaccines for treatment of Type I hypersensitivity. Up to now, endotoxin contamination of the fusion protein which occurred after isolation from the gram-negative expression host E. coli had to be removed by an expensive and time consuming procedure. In the present study, in order to achieve expression of pyrogen-free, recombinant S-layer/allergen fusion protein and to study the secretion of a protein capable to self-assemble, the S-layer/allergen fusion protein rSbpA/Bet v1 was produced in the gram-positive organism Bacillus subtilis 1012.     

Goat uromodulin promoter directs kidney-specific expression of GFP gene in transgenic mice

Background  

Uromodulin is the most abundant protein found in the urine of mammals. In an effort to utilize the uromodulin promoter in order to target recombinant proteins in the urine of transgenic animals we have cloned a goat uromodulin gene promoter fragment (GUM promoter) and used it to drive expression of GFP in the kidney of transgenic mice.     

Induction of protein body formation in plant leaves by elastin-like polypeptide fusions

Background  

Elastin-like polypeptides are synthetic biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the simple non-chromatographic purification of recombinant proteins. In addition, elastin-like polypeptide fusions have been shown to enhance the accumulation of a range of different recombinant proteins in plants, thus addressing the major limitation of plant-based expression systems, which is a low production yield. This study's main objectives were to determine the general utility of elastin-like polypeptide protein fusions in various intracellular compartments and to elucidate elastin-like polypeptide's mechanism of action for increasing recombinant protein accumulation in the endoplasmic reticulum of plants.