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ABSTRACT: Plant cells have been demonstrated to be an attractive heterologous expression host (using whole plants and in vitro plant cell cultures) for foreign protein production in the past 20years. In recent years in vitro liquid cultures of plant cells in a fully contained bioreactor have become promising alternatives to traditional microbial fermentation and mammalian cell cultures as a foreign protein expression platform, due to the unique features of plant cells as a production host including product safety, cost-effective biomanufacturing, and the capacity for complex protein post-translational modifications. Heterologous proteins such as therapeutics, antibodies, vaccines and enzymes for pharmaceutical and industrial applications have been successfully expressed in plant cell culture-based bioreactor systems including suspended dedifferentiated plant cells, moss, and hairy roots, etc. In this article, the current status and emerging trends of plant cell culture for in vitro production of foreign proteins will be discussed with emphasis on the technological progress that has been made in plant cell culture bioreactor systems.
Biotechnology advances 08/2011; 30(2):398-409. · 8.25 Impact Factor
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ABSTRACT: The endoglucanase (E1) from Acidothermus cellulolyticus has been used extensively in cellulase research. The goal of this work was to produce high levels of this enzyme in a system that facilitates purification. A codon-optimized synthetic gene for A. cellulolyticus E1 with a C-terminal histidine tag was cloned into the genome of Pichia pastoris. Strain KM71H expressed the most enzyme, with a yield of 550mg/L culture supernatant. The temperature optimum (80°C) and pH optimum (5.1) of the purified enzyme agree with previously determined values for the enzyme produced in other systems. Michaelis-Menten kinetic parameters were determined, using a fluorescent substrate (methylumbelliferyl-β-d-cellobioside) at various temperatures. This thermostable enzyme can be used in future cellulosic biofuels-related research.
Protein Expression and Purification 01/2011; 77(2):153-8. · 1.59 Impact Factor
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ABSTRACT: Potential epidemics of infectious diseases and the constant threat of bioterrorism demand rapid, scalable, and cost-efficient manufacturing of therapeutic proteins. Molecular farming of tobacco plants provides an alternative for the recombinant production of therapeutics. We have developed a transient production platform that uses Agrobacterium infiltration of Nicotiana benthamiana plants to express a novel anthrax receptor decoy protein (immunoadhesin), CMG2-Fc. This chimeric fusion protein, designed to protect against the deadly anthrax toxins, is composed of the von Willebrand factor A (VWA) domain of human capillary morphogenesis 2 (CMG2), an effective anthrax toxin receptor, and the Fc region of human immunoglobulin G (IgG). We evaluated, in N. benthamiana intact plants and detached leaves, the expression of CMG2-Fc under the control of the constitutive CaMV 35S promoter, and the co-expression of CMG2-Fc with nine different viral suppressors of post-transcriptional gene silencing (PTGS): p1, p10, p19, p21, p24, p25, p38, 2b, and HCPro. Overall, transient CMG2-Fc expression was higher on intact plants than detached leaves. Maximum expression was observed with p1 co-expression at 3.5 days post-infiltration (DPI), with a level of 0.56 g CMG2-Fc per kg of leaf fresh weight and 1.5% of the total soluble protein, a ten-fold increase in expression when compared to absence of suppression. Co-expression with the p25 PTGS suppressor also significantly increased the CMG2-Fc expression level after just 3.5 DPI.
International Journal of Molecular Sciences 01/2011; 12(8):4975-90. · 2.60 Impact Factor
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ABSTRACT: Plant cell culture is an alternative for the production of recombinant human therapeutic proteins because of improved product safety, lower production cost, and capability for eukaryotic post-translational modification. In this study, bioreactor production of recombinant human alpha-1-antitrypsin (rAAT) glycoprotein using a chemically inducible Cucumber mosaic virus (CMV) viral amplicon expression system in transgenic Nicotiana benthamiana cell culture is presented. Optimization of a chemically inducible plant cell culture requires evaluation of effects of timing of induction (TOI) and concentration of inducer (COI) on protein productivity and protein quality (biological functionality). To determine the optimal TOI, the oxygen uptake rate (OUR) of the plant cell culture was chosen as a physiological indicator for inducing maximum rAAT expression. Effects of COI on rAAT production were investigated using a semicontinuous culture, which enables the distinction between effects of growth rate and effects of inducer concentration. An optimized semicontinuous bioreactor operation was further proposed to maximize the recombinant protein production. The results demonstrated that the transgenic plant cells, transformed with the inducible viral amplicon expression system, maintain higher OUR and exhibit lower extracellular protease activity and lower total phenolics concentration in the optimized semicontinuous bioreactor process than in a traditional batch bioreactor operation, resulting in a 25-fold increase in extracellular functional rAAT (603 microg/L) and a higher ratio of functional rAAT to total rAAT (85-90%). Surprisingly, sustained rAAT production and steady state, long-term bioreactor operation is possible following chemical induction and establishment of the viral amplicons.
Biotechnology and Bioengineering 03/2010; 106(3):408-21. · 3.95 Impact Factor
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ABSTRACT: Two ribosome-inactivating proteins (RIPs) found in Trichosanthes kirilowii root tuber, trichosanthin (27 kDa) and TAP-29 (29 kDa), have been reported to exhibit antiviral (including HIV-1) and antitumor activities. Using SDS-PAGE and Western blotting analyses, we have studied the production of intracellular and extracellular proteins from T. kirilowii callus grown on semisolid medium, callus grown in suspension, and Agrobacterium rhizogenes A4 transformed callus grown in suspension. This transformation resulted in callus rather than hairy root growth, although the growth rate of the transformed callus on hormone-free medium was similar to that obtained for the nontransformed callus on hormone medium. Trichosanthin, identified through SDS/ PAGE and Western blotting, was detected only in root tuber and cell extracts of the transformed cell line. A 29-kDa protein was found in intracellular extracts and extracellular solutions from all of the above samples; however, the highest yield was obtained from the broth of the agrobacterium-transformed callus. Following ion-exchange purification of the shake flask broth on a strong cation-exchange column (S-Sepharose), elution fractions containing the 29-kDa protein showed a high degree of RIP activity, as evidenced by total inhibition of protein synthesis using an in vitro protein translation assay. The yield of the 29-kDa protein recovered from the broth was greater than 1.0 % (w/w) of the dry weight of the callus. For comparison, the yield of TAP-29 obtained by extraction of dried root tuber is on the order of 0.01% (w/w) of the dry weight (Lee-Huang et al., 1991); our estimate of the 29-kDa protein from our fresh root tuber is between 0.4% and 1% (w/w) on a dry weight basis.
Biotechnology Progress 09/2008; 10(4):345 - 352. · 2.34 Impact Factor
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ABSTRACT: Plant cell culture production of recombinant products offers a number of advantages over traditional eukaryotic expression systems, particularly if the product can be targeted to and purified from the cell culture broth. However, one of the main obstacles is product degradation by proteases that are produced during cell culture, and/or the loss of biological activity of secreted (extracellular) products as a result of alteration in the protein conformation. Because proteolysis activity and target protein stability can be significantly influenced by culture conditions, it is important to evaluate bioprocess conditions that minimize these effects. In this study, a bioreactor strategy using a protocol involving pH adjustment and medium exchange during plant cell culture is proposed for improving the production of functional recombinant alpha(1)-antitrypsin (rAAT), a human blood protein, produced using several alternative expression systems, including a Cauliflower mosaic virus (CaMV) 35S constitutive promoter expression system, a chemically inducible, estrogen receptor-based promoter (XVE) expression system, and a novel Cucumber mosaic virus (CMV) inducible viral amplicon (CMViva) expression system developed by our group. We have demonstrated that higher medium pH help reduce protease activity derived from cell cultures and improve the inherent stability of human AAT protein as well. This strategy resulted in a fourfold increase in the productivity of extracellular functional rAAT (100 microg/L) and a twofold increase in the ratio of functional rAAT to total rAAT (48%) in transgenic N. benthamiana cell cultures using a chemically inducible viral amplicon expression system.
Biotechnology and Bioengineering 08/2008; 102(2):508-20. · 3.95 Impact Factor
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ABSTRACT: A novel cucumber mosaic virus inducible viral amplicon (CMViva) expression system has been developed that allows for tightly regulated chemically inducible expression of heterologous genes in plant hosts. Transient production of recombinant alpha(1)-antitrypsin (rAAT), a human blood protein, was demonstrated in Nicotiana benthamiana leaves. The highest production levels were obtained by co-infiltrating leaves with Agrobacterium tumefaciens cells containing CMViva carrying the AAT gene and A. tumefaciens cells carrying a binary vector constitutively expressing the gene silencing suppressor p19. Accumulation of up to thirty-fold more rAAT was observed in leaves (24 mg per 100 g leaf tissue) when compared with the expression levels observed using the cauliflower mosaic virus (CaMV) 35S promoter. Significantly, 70% of the rAAT produced using the CMViva expression system was found to be biologically active, a 170-fold increase in functional protein compared with the CaMV 35S expression system.
Plant Biotechnology Journal 10/2006; 4(5):551-9. · 5.44 Impact Factor
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ABSTRACT: Trichosanthin (TCS) is an antiviral plant defense protein, classified as a type-I ribosome-inactivating protein, found in the root tuber and leaves of the medicinal plant Trichosanthes kirilowii. It is processed from a larger precursor protein, containing a 23 amino acid amino (N)-terminal sequence (pre sequence) and a 19 amino acid carboxy (C)-terminal extension (pro sequence). Various constructs of the TCS gene were expressed in transgenic tobacco plants to determine the effects of the amino- and carboxy-coding gene sequences on TCS expression and host toxicity in plants. The maximum TCS expression levels of 2.7% of total soluble protein (0.05% of total dry weight) were obtained in transgenic tobacco plants carrying the complete prepro-TCS gene sequence under the Cauliflower mosaic virus 35S RNA promoter. The N-terminal sequence matched the native TCS sequence indicating that the T. kirilowii signal sequence was properly processed in tobacco and the protein translation inhibitory activity of purified rTCS was similar to native TCS. One hundred-fold lower expression levels and phenotypic aberrations were evident in plants expressing the gene constructs without the C-terminal coding sequence. Transgenic tobacco plants expressing recombinant TCS exhibited delayed symptoms of systemic infection following exposure to Cucumber mosaic virus and Tobacco mosaic virus (TMV). Local lesion assays using extracts from the infected transgenic plants indicated reduced levels of TMV compared with nontransgenic controls.
Journal of Biotechnology 08/2002; 97(1):69-88. · 3.05 Impact Factor
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ABSTRACT: Development of bioreactor systems utilizing plant suspension cultures has been hindered by the lack of on-line sensors for monitoring important process variables such as biomass concentration and aggregate size. An optical technique, the focused beam reflectance method (FBRM developed by Lasentec Inc., Redmond, WA), was used to characterize several plant suspension cultures: rice (Oryza sativa), tobacco (Nicotiana benthamiana) and wild Chinese cucumber (Trichosanthes kirilowii). These cultures differ in a number of respects such as individual cell size and morphology, aggregate shape and size distribution, initial culture density, and color. For plant suspensions comprised of relatively spherical aggregates (rice and cucumber), the area under the cube-weighted FBRM chord length distribution was linearly correlated to biomass concentration (R
2>0.99) while the mean of the cube-weighted FBRM chord length distribution was nonlinearly related to aggregate size.
Biotechnology Letters 01/2001; 23(4):317-324. · 1.68 Impact Factor
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ABSTRACT: Sulfolipids are compounds found in association with the photosynthetic apparatus in most photoautotrophic organisms, although
there have been no quantitative studies on how environmental and biological factors influence the production of sulfolipids.
They are of interest due to their possible pharmaceutical use (anti-tumor and anti-HIV) and also as an interesting new class
of lipids. In this study, we are investigating the kinetics of growth and sulfolipid production using the cyanobacterium Synechocystis PCC 6803. The cyanobacteria are grown in a 2-L, fed-batch photobioreactor under well-defined conditions. We obtained a final biomass
concentration of 5 g/L after 20 days and an overall sulfolipid productivity of 0.24 mg/(L-hr) at a light intensity of 216
µE/m2/s.
12/1998: pages 459-466;
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ABSTRACT: Sulfolipids have recently emerged as promising antiHIV and antitumor therapeutics. These lipids have been found in association
with the photosynthetic apparatus in most photoautotrophic organisms. To date there have been no quantitative studies on the
effect of environmental factors on the production of sulfolipid. In this study, we present results on the effect of light
irradiance on the production of sulfolipids using the cyanobacteriumAnabaena 7120. The cyanobacteria are grown in a 2 L fedbatch photobioreactor at various external-light intensities. Total lipids are extracted
using the Folsch procedure and sulfolipids are quantified using thin-layer chromatography and scanning densitometry. We have
achieved a maximum of 14 mg sulfolipid/g dry weight of cell. Our results indicate that there are two stages in the specific
rate of production of sulfolipids, one in the declining exponential-growth phase of the cells and the other in the light-limited
stage of growth.
Applied Biochemistry and Biotechnology 06/1997; 67(1):139-152. · 1.94 Impact Factor
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ABSTRACT: This article presents a review of the recent literature describing the use of plant callus for the production of biochemicals,
as well as specific examples of work done in our laboratory to analyze the production of ribosome inactivating proteins fromTrichosanthes kirilowii callus. The article discusses research advances in the development, characterization, and improvement of plant callus cell
lines, including new cell lines and potentially useful products, influence of media composition, and environmental conditions
on growth and product distribution, cell line selection strategies, and long-term stability.
Applied Biochemistry and Biotechnology 04/1995; 54(1):93-108. · 1.94 Impact Factor
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ABSTRACT: Alfalfa (Medicago sativa L.) cells were grown in 500 ml, aerated and stirred batch bioreactors using Schenk and Hildebrant medium. For cultures in which the pH was allowed to vary, we observed two fairly distinct growth phases. Evidence is presented which indicates that the two-phase growth is most likely a result of the two nitrogen sources in the medium. The ammonium present in the medium is directly utilized during the first growth phase and ammonium resulting from intracellular nitrate reduction is utilized during the second phase. During the first growth phase, sucrose is completely hydrolyzed to glucose and fructose with some glucose and fructose consumption. In the second growth phase glucose is consumed preferentially over fructose. Attempts at maintaining the pH at 5.5 using 1N NaOH as the base titrant resulted in very little cell growth compared with cultures for which the pH was allowed to vary.
Plant Cell Reports 11/1989; 8(8):455-458. · 2.27 Impact Factor
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ABSTRACT: The need for biomanufacturing capacity for recombinant protein production to meet the expanding pharmaceutical and industrial
market demands has gained increasing importance, leading to the development of new protein expression platforms capable of
addressing requirements in terms of protein yield, product quality, and production cost. In the past few decades, molecular
farming using plant cell-based expression systems (whole plants and in vitro plant cells, organ and tissue cultures) have been investigated as an alternative for the large-scale bioproduction of recombinant
proteins. Molecular farming using bioreactor-based plant cell suspension cultures provides attractive features over recombinant
microbial fermentation and mammalian cell cultures in terms of intrinsic safety, cost-effective biomanufacturing, and the
capability for post-translation modifications. The current research and development, emerging techniques, commercialization
and future prospects of molecular farming using bioreactor-based plant cell suspension cultures for production of recombinant
proteins will be discussed in this chapter.
01/1970: pages 37-67;
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ABSTRACT: Transgenic rice suspension cultures were utilized to produce a human therapeutic protein, recombinant alpha(1)-antitrypsin (rAAT), in a cyclical, semicontinuous operation. Recombinant protein production was induced by removing the carbon source from the cell culture medium. The transgenic rice cells secreted the rAAT into the medium, and therefore medium exchanges could be performed for consecutive growth and protein expression phases. The process consisted of three cycles over a 25-28 day period, with growth phases lasting 4-6 days each and protein expression phases lasting 2.5-5 days each. Biomass and sugar concentrations, oxygen uptake rate, cell viability, culture pH, total extracellular protein, and active rAAT were measured throughout the cyclical process. The data profiles were reproducible between separate cyclical runs where, following each induction period, cell growth and viability could be reestablished once sucrose was added back to the culture. Volumetric productivities ranged from 3 to 12 mg active rAAT/(L day) for individual cycles with overall volumetric productivities of 4.5 and 7.7 mg active rAAT/(L day).
Biotechnology Progress 21(2):321-8. · 2.34 Impact Factor
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ABSTRACT: We have demonstrated that the method of chemical induction using a chemically inducible viral amplicon expression system can be optimized to increase expression of a heterologous protein in plants. A cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce a recombinant human blood protein, alpha-1-antitrypsin (AAT), by co-infiltrating intact and detached Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Infiltrated plants were induced by either topical applications or pressure injections and inducer was applied at either a single or multiple time points. Applying induction solution every 2 days via topical application resulted in increasing maximum levels of biologically functional rAAT from 0.71% to 1.3% of the total soluble protein (TSP) in detached plant leaves, a 1.8-fold improvement. Multiple applications of induction solution via pressure injection into intact leaves resulted in maximum levels of biologically functional rAAT being elevated 3-fold up to 2.4% of TSP compared to 0.8% of TSP when using the conventional method of a single topical application, and expression levels remained high 6 days post-induction. Overall production of rAAT in intact leaves was found to have a maximum level of 5.8% of TSP or 390 mg rAAT per kg leaf tissue when applying multiple injections of chemical induction solution.
Biotechnology Progress 23(6):1277-85. · 2.34 Impact Factor
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ABSTRACT: Extraction and storage of a recombinant protein produced by transient expression following agroinfiltration of lettuce were investigated. Lettuce leaves expressing beta-glucuronidase (GUS) were extracted by homogenization in several buffer combinations, and the yield and stability were assessed. The reducing agent dithiothreitol (DTT) was found to be the most important (significant) component in the extraction buffer. An extraction buffer consisting of 50 mM sodium phosphate at pH 7.0 with 10 mM DTT produced a good yield and stabilized GUS. Leaching of GUS through intact agroinfiltrated lettuce leaves was determined to be infeasible, with a maximum flux of 10 microg GUS/h/m2 and recovery of 1.7% of the GUS content in 24 h. Freeze-drying was evaluated as a method to extend the shelf life of the perishable leaf material containing GUS. First- and second-order kinetic models and the Weibull distribution were compared to describe inactivation of GUS in the freeze-dried leaves and leaf extracts. The first-order model best fit the inactivation data. An Arrhenius model fit the first-order inactivation data with respect to temperature with R2 = 1.00. Freeze-drying the lettuce leaves extended the estimated half-life of GUS to 69 days at 21 degrees C versus 11 days at 4 degrees C for fresh lettuce.
Biotechnology Progress 22(3):723-30. · 2.34 Impact Factor
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ABSTRACT: Transgenic rice cell cultures, capable of producing recombinant human alpha(1)-antitrypsin (rAAT), were scaled up from shake flasks to a 5-L bioreactor. The maximum specific growth rates (mu(max)) observed from two bioreactor runs were 0.40 day(-1) (doubling time of 1.7 days) and 0.47 day(-1) (doubling time of 1.5 days), and the maximum specific oxygen uptake rates were 0.78 and 0.84 mmol O(2)/(g dw h). Using a metabolically regulated rice alpha-amylase (RAmy3D) promoter, signal peptide, and terminator, sugar deprivation turned on rAAT expression, and rAAT was secreted into the culture medium. After 1 day of culture in sugar-free medium, there was still continued biomass growth, oxygen consumption, and viability. Extracellular concentrations of 51 and 40 mg active rAAT/L were reached 1.7 and 2.5 days, respectively, after induction in a sugar-free medium. Volumetric productivities for two batch cultures were 7.3 and 4.6 mg rAAT/(L day), and specific productivities were 3.2 and 1.6 mg rAAT/(g dw day). Several different molecular weight bands of immunoreactive rAAT were observed on immunoblots.
Biotechnology Progress 18(3):501-8. · 2.34 Impact Factor
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ABSTRACT: Transgenic plant cell cultures offer a number of advantages over alternative host expression systems, but so far relatively low product concentrations have been achieved. In this study, transgenic rice cells are used in a two-compartment membrane bioreactor (CELLine 350, Integra Biosciences) for the production of recombinant alpha-1-antitrypsin (rAAT). Expression of rAAT is controlled by the rice alpha-amylase (RAmy3D) promoter, which is induced in the absence of sugar. The extracellular product is retained in the bioreactor's relatively small cell compartment, thereby increasing product concentration. Due to the packed nature of the cell aggregates in the cell compartment, a clarified product solution can be withdrawn from the bioreactor. Active rAAT reached levels of 100-247 mg/L (4-10% of the total extracellular protein) in the cell compartment at 5-6 days postinduction, and multiple inductions of the RAmy3D promoter were demonstrated.
Biotechnology Progress 21(3):728-34. · 2.34 Impact Factor
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ABSTRACT: A review of over 15 years of research, development and commercialization of plant cell suspension culture as a bioproduction platform is presented. Plant cell suspension culture production of recombinant products offers a number of advantages over traditional microbial and/or mammalian host systems such as their intrinsic safety, cost-effective bioprocessing, and the capacity for protein post-translational modifications. Recently significant progress has been made in understanding the bottlenecks in recombinant protein expression using plant cells, including advances in plant genetic engineering for efficient transgene expression and minimizing proteolytic degradation or loss of functionality of the product in cell culture medium. In this review article, the aspects of bioreactor design engineering to enable plant cell growth and production of valuable recombinant proteins is discussed, including unique characteristics and requirements of suspended plant cells, properties of recombinant proteins in a heterologous plant expression environment, bioreactor types, design criteria, and optimization strategies that have been successfully used, and examples of industrial applications.
Biochemical Engineering Journal.
