Production Of Foreign Proteins Research Articles

Enhancement of heterologous protein expression in Escherichia coli by co-expression of nonspecific DNA-binding stress protein, Dps

The over-expression of foreign proteins imposes metabolic burden on host strains that may lead to reduced cell growth and even yield of target heterologous protein. We investigated the effect of co-expression of nonspecific DNA-binding protein, Dps, one of the many stress proteins against oxidative damage and nutrient starvation, on the over-expression of heterologous protein in Escherichia coli expression system. It was observed that the co-expression of recombinant Dps reduced the growth rate in minimal M9 medium. On the contrary, Dps had a positive effect on cell growth in rich LB medium. It was also observed that Dps was capable of enhancing the specific production of insoluble target foreign protein, baculoviral polyhedrin (Polh) and green fluorescent protein (GFP) fusion in both media, demonstrating that the co-expression of Dps has general positive effects on foreign protein production regardless of medium types. Even though the mechanism of Dps on foreign protein production remains unclear, the ability for significant enhancement of target protein production (about 46% of target protein fraction in total cellular proteins and about 2.5-fold increase in product yield) may be successfully applied in practical culture process.

Modeling of the induced expression for high-level production of a foreign protein by recombinant E. coli under the control of the T7 phage promoter

A structured four-compartment mathematical model is proposed for describing the behaviour of three expression systems. In system [R], Taq I endonuclease gene was expressed under the control of T7 RNA polymerase promoter in recombinant Escherichia coli cells. Methylase protection was provided either by cloning the Taq I methylase gene on to a second plasmid (system [R, M]) or by constructing a recombinant plasmid harbouring both genes (system [R+M]). Considering the promoter strength, the inducer effect is included in the rate equation for foreign protein production. A separate set of parameters was estimated for simulations of the uninduced and induced phases (dual-parameter set) of systems [R] and [R, M]. A single parameter set describing both phases was also determined. Simulation results indicate that the dual-parameter approach is more successful in predicting the experimental observations. Model validation conducted on system [R+M] using parameter sets of system [R, M] revealed good agreement.

Effect of hyperosmolarity on recombinant protein productivity in baculovirus expression system

A lot of strategies were applied to improve recombinant protein productivity in the baculovirus expression system. In this study we propose for foreign protein production fed-batch cultivation method at hyperosmotic environment induced by increased NaCl content. Obtained results suggested relatively high tolerance and adaptation abilities of Tn-5 insect cells exposed to hyperosmotic stress. The cells under hyperosmotic conditions increased the specific rate of glucose consumption and lactate production. The release of additional energy and precursors as a result of increased metabolism by osmotically stressed culture was involved in recombinant protein synthesis. Recombinant nucleoprotein productivity in nutritional feeding cultures exposed to hyperosmolarity was about 72% higher than that obtained in batch culture at physiological osmolarity, but 31% was a result of feeding and the rest 41% was a result of hyperosmolarity and increasing Na + concentration.

Proteome analysis of antibody-expressing CHO cells in response to hyperosmotic pressure.

To better understand intracellular responses to hyperosmotic pressure of recombinant Chinese hamster ovary (rCHO) cells expressing an antibody, we have taken a proteomics approach. Using two-dimensional electrophoresis and mass spectrometry, a proteome profile of rCHO cells comprising 23 identified proteins was established. On the basis of this proteome profile, we found three proteins of which expression levels were significantly changed at 450 mOsm/kg. Compared to the results at 300 mOsm/kg, two glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase, were found to be up-regulated, probably leading to an increased metabolic energy for antibody synthesis. The elevation of specific glucose consumption rate at 450 mOsm/kg agreed with the up-regulation of these glycolytic enzymes. On the other hand, tubulin expression was down-regulated, reflecting a depressed cell growth rate at 450 mOsm/kg. Taken together, this study shows the potential of the proteomics approach in understanding intracellular and physiological changes in cells and seeking a better insight into possible environmental or genetic manipulation approaches for increasing foreign protein production in rCHO cells.

Construction of engineered CHO strains for high-level production of recombinant proteins.

We constructed engineered CHO strains that can be used for high-level production of foreign proteins by gene-targeting. After transfecting dihydroforate reductase (DHFR)-deficient CHO cells with a plasmid carrying a loxP-green fluorescent protein (GFP) fusion gene and a DHFR gene, we screened colonies by fluorescent intensity. We selected 16 clones that expressed high levels of GFP and carried one copy of the plasmid in their chromosomes and treated them with methotrexate (MTX) to examine their ability for DHFR-mediated gene amplification. Two clones, MK1 and MK2, showed increased GFP expression upon gene amplification. In those clones, the loxP-GFP gene was integrated at a transcription-active, DHFR-mediated, gene-amplifiable locus in the chromosomes. A gene-targeting vector, carrying a loxP-fused hygromycin-resistance gene, was constructed to target desired genes in chromosomal loxP by Cre recombinase-mediated site-specific recombination. Using this cell-vector system, we could reproducibly obtain high producers of recombinant proteins by gene-targeting and gene amplification. In human monoclonal antibody production, after gene-targeting of loxP in MK2 and gene amplification with MTX, the MTX-resistant colonies showed high levels of antibody production. The most productive clone was able to produce 160 mg/l in 7 days in a low-protein medium in a spinner-flask.

Increased production and recovery of secreted foreign proteins from plant cell cultures using an affinity chromatography bioreactor

Although plant cell cultures can be used to produce a variety of proteins, the instability and degradation of secreted proteins hinders product recovery from culture medium. To solve this problem, an affinity chromatography bioreactor (ACBR) scheme was developed for the simultaneous production and purification of foreign proteins from genetically modified plant suspension cultures. Two model products, the heavy chain of a mouse monoclonal antibody (HC MAb) and 6-his tagged human granulocyte macrophage colony-stimulating factor (GM-CSF), were produced in a modified shake flask and separated using protein G and iminodiacetic acid metal affinity resins, respectively. Results showed that the ACBR scheme increased the production of HC MAb by as much as eight-fold and GM-CSF by more than two-fold compared with batch yields. Product recovery was optimized by varying the aeration method, flow rate, pH, and resin type for the experimental system. These results suggest that in situ protein removal from plant cell media using the ACBR strategy can isolate the product from degrading influences and remove the protein from product inhibition pathways.

Assessment of foreign protein production by recombinant Heliothis (Helicoverpa) armigera entomopoxviruses in Spodoptera frugiperda cells.

This report describes the first production of recombinant forms of Heliothis (Helicoverpa) armigera entomopoxvirus (HaEPV). These HaEPVs are engineered at either the spheroidin or fusolin locus, to produce the green fluorescent marker protein (GFP). The growth properties of these recombinant HaEPVs, in comparison to the parental HaEPV, were assessed in cultured Spodoptera frugiperda Sf9 cells. Additionally, GFP production by these recombinant HaEPVs was compared to that of a GFP-expressing recombinant of the baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) in the same in vitro system, at various multiplicities of infection. Expression of GFP from the HaEPV spheroidin locus produced up to 60% of that generated from the AcNPV polyhedrin locus, albeit over a longer period of infection. A considerably lower yield was recorded from the HaEPV fusolin locus, a result that contrasted markedly with the apparent activity of this promoter in caterpillar infections in vivo. The potential applications for further development of HaEPV expression systems are discussed.

Response of recombinant Chinese hamster ovary cells to hyperosmotic pressure: effect of Bcl-2 overexpression

In an attempt to use the hyperosmotic pressure for improved foreign protein production in recombinant Chinese hamster ovary (rCHO) cells, the response of rCHO cells producing a humanized antibody (SH2-0.32-Δbcl-2 cells) to hyperosmotic pressure was determined in regard to cell growth and death, and antibody production. Further, the feasibility of Bcl-2 overexpression in improving rCHO cell viability under hyperosmotic pressure was also determined by comparing control cells (SH2-0.32-Δbcl-2) with Bcl-2 overexpressing cells (14C6-bcl-2). After 3 days of cultivation in the standard medium (294 mOsm kg −1), the spent medium was exchanged with the fresh media with various osmolalities (294–640 mOsm kg −1). The results obtained show that hyperosmotic pressure inhibited cell growth in a dose-dependent manner, though 14C6-bcl-2 cells were less susceptible to hyperosmotic pressure than SH2-0.32-Δbcl-2 cells. At 522 mOsm kg −1, SH2-0.32-Δbcl-2 cells underwent a gradual cell death mainly through apoptosis due to the cytotoxic effect of hyperosmotic pressure. In contrast, Bcl-2 overexpression in 14C6-bcl-2 cells could delay the apoptosis induced by 522 mOsm kg −1 by inhibiting caspase-3 activation. Bcl-2 overexpression could also improve the cellular membrane integrity of 14C6-bcl-2 cells. When subjected to hyperosmotic pressure, the specific antibody productivity of SH2-0.32-Δbcl-2 cells and 14C6-bcl-2 cells was increased in a similar extent. As a result, the final antibody concentration achieved in 14C6-bcl-2 cells at 522 mOsm kg −1 was 2.5-fold higher than that at 294 mOsm kg −1. At 580 mOsm kg −1, acute hyperosmotic pressure induced the rapid loss of viability in both SH2-0.32-Δbcl-2 and 14C6-bcl-2 cells through necrosis rather than through apoptosis. Taken together, Bcl-2 overexpression and optimized hyperosmotic pressure could improve the antibody production of rCHO cells.

Large-scale extraction of proteins.

The production of foreign proteins using selected host with the necessary posttranslational modifications is one of the key successes in modern biotechnology. This methodology allows the industrial production of proteins that otherwise are produced in small quantities. However, the separation and purification of these proteins from the fermentation media constitutes a major bottleneck for the widespread commercialization of recombinant proteins. The major production costs (50-90%) for typical biological product resides in the purification strategy. There is a need for efficient, effective, and economic large-scale bioseparation techniques, to achieve high purity and high recovery, while maintaining the biological activity of the molecule. Aqueous two-phase systems (ATPS) allow process integration as simultaneously separation and concentration of the target protein is achieved, with posterior removal and recycle of the polymer. The ease of scale-up combined with the high partition coefficients obtained allow its potential application in large-scale downstream processing of proteins produced by fermentation. The equipment and the methodology for aqueous two-phase extraction of proteins on a large scale using mixer-settlerand column contractors are described. The operation of the columns, either stagewise or differential, are summarized. A brief description of the methods used to account for mass transfer coefficients, hydrodynamics parameters of hold-up, drop size, and velocity, back mixing in the phases, and flooding performance, required for column design, is also provided.

Development of an edible subunit vaccine in corn against enterotoxigenic strains of escherichia coli

Advances in the development of subunit vaccines and in the production of foreign proteins in plants together offer the prospect of stable and inexpensive vaccine delivery systems. Various bacterial and viral proteins stably produced in plants have been shown to elicit immune responses in feeding trials. We have extended this approach by using Zea mays as the plant production system. Corn has several advantages as a vaccine delivery vehicle, most notably established technologies to generate transgenic plants, to optimize traits through breeding and to process the seed into a palatable form. Here we report on the production in corn seed of the GM1 receptor binding (B) subunit of the heat-labile toxin (Lt) from enterotoxigenic strains of Escherichia coli. Versions of the Lt-B gene were synthesized to give optimum codon usage for corn and to target the protein to either the cell surface or the cytoplasm. These synthetic genes were fused to a strong promoter and transformed into corn. Lt-B was highly expressed in corn seed at up to 1.8% of the total soluble protein and this was further increased approximately five-fold through plant breeding. As in E. coli. Lt-B produced in corn forms a functional pentamer that can bind to the GM1 receptor. Furthermore, Lt-B pentamer stored in corn seed is much more resistant to heat than is the pure protein, allowing the transgenic corn to be readily processed into an edible form. This work demonstrates the potential of using products derived from transgenic corn seed as delivery vehicles for subunit vaccines.

Strategies for enhancing monoclonal antibody accumulation in plant cell and organ cultures.

Various strategies aimed at improving IgG(1) antibody accumulation in transgenic tobacco cell and organ cultures were tested. The form of tissue had a significant effect on antibody levels; shooty teratomas were less productive than hairy roots or suspended cells. Although there were several disadvantages associated with hairy roots compared with suspensions, such as slower growth, slower antibody production, and formation of a greater number of antibody fragments, the roots exhibited superior long-term culture stability. Antibody accumulation in hairy root cultures was improved by increasing the dissolved oxygen tension to 150% air saturation, indicating the need for effective oxygen transfer in root reactors used for antibody production. Preventing N-linked glycosylation using tunicamycin or inhibition of subsequent glycan processing by castanospermine reduced antibody accumulation in the biomass and/or medium in cell suspensions. Loss of antibody from the cultures after its secretion and release into the medium was identified as a major problem. This effect was minimized by inhibiting protein transport in the secretory pathway using Brefeldin A, resulting in antibody accumulation levels up to 2.7 times those in untreated cells. Strategies for protecting secreted antibody, such as addition of poly(vinylpyrrolidone) and periodic harvesting from the medium using hydroxyapatite resin, also increased antibody titers. The mechanisms responsible for the disappearance of antibody from plant culture media were not clearly identified; degradation by proteases and conformational modification of the antibody, such as formation of aggregates, provided an explanation for some but not all the phenomena observed. This work demonstrates that the manipulation and control of culture conditions and metabolic processes in plant tissue cultures can be used to improve the production of foreign proteins. However, loss of secreted antibody from plant culture medium is a significant problem that may limit the feasibility of using product recovery from the medium to reduce downstream processing costs relative to agricultural systems.

Characterization of a Saccharomyces cerevisiae mutant with oversecretion phenotype.

An oversecreting mutant of Saccharomyces cerevisiae was obtained from about 400 meiotic segregants derived from thediploid cells made by crossing the HBsAg-induced mutant NI-C with the wild-type strain Sey6211. When transformed with a plasmid containing mouse alpha-amylase cDNA, the mutant (NI-C-D4) exhibited an increased capacity (up to 13-fold) for the secretion of mouse alpha-amylase, higher than the parental strains and other standard wild-type strains. It was also shown that alpha-amylase secreted by the oversecreting mutant had a higher activity and contained more of the non-glycosylated form than the glycosylated form. This isolated oversecreting, low-glycosylation mutant may prove to be a potential S. cerevisiae host for the production of foreign proteins. Further genetic analysis suggested that the mutation responsible for the mutant's oversecretion was partially dominant and that both the oversecretion and low-glycosylation phenotypes were governed by a single chromosome mutation. These pleiotrophic phenotypes may be attributed to a defect in the synthesis of an ER-resident chaperone.

The production of foreign proteins from genetically modified plant cells.

While traditionally used to produce natural products, plant suspension cultures can also be utilized for the production of foreign proteins. Production of these high-value products in plant cells is an economically viable alternative to other systems, particularly in cases where the protein must be biologically active. There are several advantages to using plant cells for the large-scale production of secreted proteins. Plant cell media are composed of simple sugars and salts and are therefore less expensive and complex than mammalian media. Consequently, purification of secreted protein is simpler and more economical. Additionally, plant cell derived proteins are likely to be safer than those derived from other systems, since plant cell pathogens are not harmful to humans. In this chapter, we will review foreign protein production from plant cells. To begin, we will discuss the behavior of plant cell cultures, products produced by plant cells, protein secretion and its relationship to purification, and the performance of plant cells as compared to whole plants and other alternative hosts. After a brief discussion of gene transfer techniques, we will present strategies to overcome the limitations of protein production, including protein stabilization, novel production schemes, modeling, and scale-up considerations. To conclude, we will discuss implications for future development of this technology.

Regulation of the amylolytic and (hemi-)cellulolytic genes in aspergilli.

Filamentous fungi produce high levels of polysaccharide-degrading enzymes and are frequently used for the production of industrial enzymes. Because of the high secretory capacity for enzymes, filamentous fungi are effective hosts for the production of foreign proteins. Genetic studies with Aspergillus nidulans have shown pathway-specific regulatory systems that control a set of genes that must be expressed to catabolize particular substrates. Besides the pathway-specific regulation, wide domain regulatory systems exist that affect a great many individual genes in different pathways. A molecular analysis of various regulated systems has confirmed the formal models derived from purely genetic data. In general, many genes are subject to more than one regulatory system. In this article, we describe two transcriptional activators, AmyR and XlnR, and an enhancer, Hap complex, in view of their regulatory roles in the expression of the amylolytic and (hemi-)cellulolytic genes mainly in aspergilli. The amyR gene has been isolated as a transcriptional activator involved in the expression of amylolytic genes from A. oryzae, A. niger, and A. nidulans, and the xlnR gene, which has been isolated from A. niger and A. oryzae, activates the expression of xylanolytic genes as well as some cellulolytic genes in aspergilli. Both AmyR and XlnR have a typical zinc binuclear cluster DNA-binding domain at their N-terminal regions. Hap complex, a CCAAT-binding complex, enhances the overall promoter activity and increases the expression levels of many fungal genes, including the Taka-amylase A gene. Hap complex comprises three subunits, HapB, HapC, and HapE, in A. nidulans and A. oryzae as well as higher eukaryotes, whereas HAP complex in Saccharomyces cerevisiae and Kluyveromyces lactis has the additional subunit, Hap4p, which is responsible for the transcriptional activation. Hap complex is suggested to enhance transcription by remodeling the chromatin structure. The regulation of gene expression in filamentous fungi of industrial interest could follow basically the same general principles as those discovered in A. nidulans. The knowledge of regulation of gene expression in combination with traditional genetic techniques is expected to be increasingly utilized for strain breeding. Furthermore, this knowledge provides a basis for the rational application of transcriptional regulators for biotechnological processes in filamentous fungi.

Construction of spore mutants of Bacillus subtilis for the development as a host for foreign protein production

For the development of Bacillus subtilis as a host for foreign protein synthesis, three types of sigma factor deleted mutants (spoIIAC, spoIIIG and spoIIIC) were constructed by antibiotic marker insertion using plasmid vector-mediated method or LFH (Long Flanking Homology)-PCR. Mother cell specific sigma factor mutants of B. subtilis (σK), B. subtilis DB104 ΔspoIIIC (km r)::pMK101, had two to three times higher subtilisin activity than the wild type DB104::pMK101. Subtilisin expression by the other two mutants, B. subtilis DB104 ΔspoIIAC (km r)::pMK101 and DB104 ΔspoIIIG (km r)::pMK101, which are pre-spore specific sigma factor (σF and σG) deleted strains, was similar to, or less than that of the wild type.

Osmoprotective Effect of Glycine Betaine on Thrombopoietin Production in Hyperosmotic Chinese Hamster Ovary Cell Culture: Clonal Variations

When 23 recombinant Chinese hamster ovary (rCHO) cell clones were cultivated in hyperosmolar medium resulting from NaCl addition (533 mOsm/kg), their specific thrombopoietin (TPO) productivity (q(TPO)) was increased. However, due to depressed cell growth at elevated osmolality, no enhancement in the maximum TPO titer was made in batch cultures of all 23 clones. To test the feasibility of using glycine betaine, known as a strong osmoprotective compound, for improved TPO production in hyperosmotic rCHO cell cultures, hyperosmotic batch cultures of 23 clones were performed in the presence of 15 mM glycine betaine. Glycine betaine was found to have a strong osmoprotective effect on all 23 clones. Inclusion of 15 mM glycine betaine in hyperosmolar medium enabled 22 clones to grow at 542 mOsm/kg, where most clones could not grow in the absence of glycine betaine, but at a cost of reduced q(TPO). However, the relative decrease in q(TPO) varied significantly among clones. Thus, efficacy of the simultaneous use of hyperosmotic pressure and glycine betaine as a means to improve foreign protein production was variable among clones. Six out of 23 clones displayed more than a 40% increase in the maximum TPO titer in the hyperosmolar medium containing glycine betaine, compared with that in the standard medium with a physiological osmolality. Taken together, the results obtained here emphasize the importance of selection of clones for the successful use of hyperosmotic pressure and glycine betaine as an economical means to improve TPO production.

Effect of the truncation of the C-terminal region of Kex2 endoprotease on processing of the recombinant Rhizopus oryzae lipase precursor in the co-expression system in yeast

The Saccharomyces cerevisiae Kex2p is a membrane-bound endoprotease and cleaves at a C-terminal site of Lys–Arg of the α-factor precursor in late Golgi compartment. The complete cleavage of a secreted recombinant Rhizopus oryzae lipase (ROL) precursor (rProROL) having an internal Lys–Arg has been examined by the co-expression of the KEX2 gene. The cleavage of rProROL was not sufficient. The co-expression system of the truncated gene encoding the soluble form of Kex2p (sKex2p) lacking C-terminal 201 amino acids containing the transmembrane domain (TMD) was constructed. sKex2p co-expressed was detected in the culture medium and the carboxyl site of Lys(-30)–Arg(-29) in the prosequence of rProROL was completely cleaved. These results revealed that on the process of secretion, including in the Golgi apparatus and secretion vesicles, attack of sKex2p to the produced protein occurred more efficiently than that of Kex2p. The cells having the co-expression system of the gene encoding sKex2p will be useful for production of foreign proteins designed to cleave the internal Lys–Arg site for their activation or for the application of the prepro-α-factor leader sequence.

A pH-based fed-batch process for the production of a chimeric recombinant infectious bursal disease virus (IBDV) structural protein (rVP2H) in insect cells

For the production of a rVP2H vaccine, to protect young chickens from infection by an infectious bursal disease virus (IBDV), Trichoplusia ni (Hi-5) cells were cultured in a 500-ml spinner flask to evaluate the productivity of the rVP2H protein. The spinner flask, with a working volume of 500 ml, was equipped with a sparger to bubble the air at a flow rate of 0.134 vvm to provide adequate oxygen transfer. The oxygen transfer rate coefficient, k L a, was 24/h under these conditions. Hi-5 cells were grown and then infected with a recombinant rVP2H expressing baculovirus in spinner flasks. The Hi-5 cells generated a higher rVP2H yield at a low cell density (1×10 6 cells/ml) than at higher cell density (2–3×10 6 cells/ml). Nutrient depletion, especially glucose and glutamine, caused a decrease in specific productivity (i.e. production per cell) with increase in cell density at infection. A pH-based fed-batch culture method, in which the increase in pH, signalled by a ‘metabolic switch’, was used as an indicator for initiating supplemental glucose and glutamine application. By applying this method, the production of rVP2H was increased from 25 to 54 mg/l when the Hi-5 cells were infected at a cell density of 2×10 6 cells/ml. This feeding strategy was carried out with simple instruments and could facilitate a significant increased-yield of rVP2H protein in spinner flasks. This technique should be beneficial in research laboratories employing the Hi-5 cells/baculovirus expression system as a rapid and efficient system for the production of foreign proteins.

Osmoprotective effect of glycine betaine on foreign protein production in hyperosmotic recombinant Chinese hamster ovary cell cultures differs among cell lines

When three recombinant Chinese hamster ovary (rCHO) cell lines, CHO/dhfr-B22-4, CS13-1.00*, and CS13-0.02*, were cultivated in hyperosmolar media resulting from NaCl addition, their specific foreign protein productivity increased with medium osmolality. However, due to a simultaneous suppression of cell growth at elevated osmolality, no enhancement in the maximum foreign protein titer was made in batch cultures. To test the feasibility of using glycine betaine, known as a strong osmoprotective compound, for improved foreign protein production in hyperosmotic rCHO cell cultures, hyperosmotic batch cultures were carried out in the presence of 15 mM glycine betaine. Glycine betaine was found to have a strong osmoprotective effect on all three rCHO cell lines. Inclusion of 15 mM glycine betaine in hyperosmolar medium enabled rCHO cell lines to grow at 557 to 573 mOsm/kg, whereas they could not grow in the absence of glycine betaine. However, effect of glycine betaine inclusion in hyperosmolar medium on foreign protein production differed among rCHO cell lines. CHO/dhfr-B22-4 cells retained enhanced specific human thrombopoietin (hTPO) productivity in the presence of glycine betaine, and thereby the maximum hTPO titer obtained at 573 mOsm/kg was increased by 72% over that obtained in the control culture with physiological osmolality (292 mOsm/kg). On the other hand, enhanced specific antibody productivity of CS13-1.00* and CS13-0.02* at elevated osmolality was decreased significantly in the presence of glycine betaine. As a result, the maximum antibody titer at 557 mOsm/kg was similar to that obtained in the control culture with physiological osmolality. The mRNA contents per cell determined by northern blot hybridization correlated with q in all three rCHO cell lines, indicating that transcriptional regulation is responsible in part for q enhancement at hyperosmolality in the absence as well as the presence of glycine betaine. Taken together, efficacy of the simultaneous use of hyperosmotic pressure and glycine betaine as a means to improve foreign protein production was variable among different rCHO cell lines.

In planta expression of HIV-1 p24 protein using an RNA plant virus-based expression vector.

Plant viruses show significant potential as expression vectors for the production of foreign proteins (e.g., antigens) in plants. The HIV-1 p24 nucleocapsid protein is an important early marker of HIV infection and has been used as an antigen in the development of HIV vaccines. Toward developing a plant-based expression system for the production of p24, we have investigated the use of a (positive)-strand RNA plant virus, tomato bushy stunt virus (TBSV), as an expression vector. The HIV p24 open reading frame (ORF) was introduced into a cloned cDNA copy of the TBSV genome as an in-frame fusion with a 5'-terminal portion of the TBSV coat protein ORF. In vitro-generated RNA transcripts corresponding to the engineered virus vector were infectious when inoculated into plant protoplasts; Northern and Western blot analyses verified the accumulation of a predicted p24-encoding viral subgenomic mRNA and the production of p24 fusion product. Whole-plant infections with the viral vector led to the accumulation of p24 fusion protein in inoculated leaves, which cross-reacted with p24-specific antibodies, thus confirming the maintenance of key antigenic determinants. This study is the first to demonstrate that TBSV can be engineered to express a complete foreign protein of clinical importance. Strategies for optimizing protein yield from this viral vector are discussed.