Suk-Tae Kwon

Sungkyunkwan University, Sŏul, Seoul, South Korea

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Publications (55)113.55 Total impact

  • Sung Suk Cho, Mi Yu, Suk-Tae Kwon
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    ABSTRACT: Among the family B DNA polymerases, the Twa DNA polymerase from T. wiotapuensis, a hyperthermophilic archaeon, has exceedingly high fidelity. For applications in PCR, however, the enzyme is limited by its low extension rate and processivity. To resolve these weaknesses, we focused on two amino acid residues (A381 and N501) located at the palm subdomain of Twa DNA polymerase. Following replacement of these residues by site-directed mutagenesis, Twa N501R DNA polymerase showed significantly improved polymerase function compared to the wild-type enzyme in terms of processivity (3-fold), extension rate (2-fold) and PCR efficiency. Kinetic analysis using DNA as template revealed that the kcat value of the Twa N501R mutant was similar to that of wild-type, but the Km of the Twa N501R mutant was about 1.5-fold lower than that of the wild-type. These results suggest that a positive charge at residue 501 located in the forked-point does not impede catalytic activity of the polymerase domain but stabilizes interactions between the polymerase domain and the DNA template.
    Journal of biotechnology. 05/2014;
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    ABSTRACT: A family B DNA polymerase gene from the hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN4/I was highly expressed under the control of T7lac promoter of pET-28ARG in Escherichia coli BL21-CodonPlus(DE3)-RIL cells. The produced I. hospitalis (Iho) DNA polymerase was purified by heat treatment followed by HisTrap™ HP column and HiTrap™ SP column chromatographies. The molecular mass of the purified Iho DNA polymerase was 88 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH for Iho DNA polymerase activity was 7.0 and the optimal temperature was 70 °C. Iho DNA polymerase was strongly activated by the presence of magnesium ion at an optimum concentration of 3 mM. The optimal concentration of KCl for Iho DNA polymerase activity was 60 mM. The half-life of the enzyme at 94 °C was about 2 h. The optimal conditions for polymerase chain reaction (PCR) were determined. Iho DNA polymerase possesses 3' → 5' exonuclease activity, and the fidelity of the Iho DNA polymerase was similar to that of Pfu and Vent DNA polymerases. However, Iho DNA polymerase provided more enhanced efficiency of PCR amplification than Pfu and Vent DNA polymerases. Iho DNA polymerase could successfully amplify a 2-kb λ DNA target with a 10-s extension time and could amplify a DNA fragment up to 8 kb λ DNA.
    Applied biochemistry and biotechnology 04/2014; · 1.94 Impact Factor
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    ABSTRACT: Twa DNA polymerase from hyperthermophilic archaeon Thermococcus waiotapuensis has exceedingly high fidelity among family B DNA polymerases. However, Twa DNA polymerase has significant shortcomings in terms of a low extension rate and poor processivity. To resolve these weaknesses, we focused on two amino acid residues (N565 and H633) in the palm and thumb subdomains of the Twa DNA polymerase. These two residues were replaced by site-directed mutagenesis and the enzymatic properties of the mutants were analyzed. Here, Twa H633R DNA polymerase showed significantly improved polymerase function compared to wild-type Twa DNA polymerase in terms of processivity (2-fold), extension rate (1.5-fold) and PCR efficiency. Kinetic analysis using DNA as a template revealed that the kcat value of the Twa H633R mutant was similar to that of wild type, but the Km of the Twa H633R mutant was about 1.6-fold lower than that of the wild type. These result showed that the Arg residue substitution at H633 located in the thumb subdomain has a positive effect on processivity, extension rate and PCR efficiency, suggesting that the Twa H633R mutant allows a conformational change for easy access of the primer-template to the binding site of the polymerase domain.
    Enzyme and Microbial Technology 01/2014; · 2.59 Impact Factor
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    ABSTRACT: Mechanisms that allow replicative DNA polymerases to attain high processivity are often specific to a given polymerase and cannot be generalised to others. Amplification efficiency is lower in family B-type DNA polymerases than in family A-type (Taq) polymerases because of their strong 3'-5' exonuclease-activity. Here, we have red the exonuclease domain of the Thermococcus onnurineus NA1 (TNA1) DNA polymerase, especially Asn210 to Asp215 residues in Exo II motif (NXXXFD), to improve the processivity. N213D mutant protein had higher processivity and extension rate than the wild-type TNA1 DNA polymerase, retaining a lower mutation frequency than recombinant Taq DNA polymerase. Consequently, the N213D mutant could amplify target DNA up to 13.5 kb in length from human genomic DNA and 16.2 kb in length from human mitochondrial DNA while wild-type TNA1 amplified target DNA of 2.7 kb in length from human genomic DNA.
    Biotechnology Letters 12/2013; · 1.85 Impact Factor
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    ABSTRACT: The nucleotide cofactor specificity of the DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus (Hbu) was studied to investigate the evolutionary relationship of DNA ligases. The Hbu DNA ligase gene was expressed under control of the T7lac promoter of pTARG in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was purified using the IMPACT™-CN system (intein-mediated purification with an affinity chitin-binding tag) and cation-ion (Arg-tag) chromatography. The optimal temperature for Hbu DNA ligase activity was 75 °C, and the optimal pH was 8.0 in Tris-HCl. The activity was highly dependent on MgCl2 or MnCl2 with maximal activity above 5 mM MgCl2 and 2 mM MnCl2. Notably, Hbu DNA ligase can use ADP and GTP in addition to ATP. The broad nucleotide cofactor specificity of Hbu DNA ligase might exemplify an undifferentiated ancestral stage in the evolution of DNA ligases. This study provides new evidence for possible evolutionary relationships among DNA ligases.
    Extremophiles 04/2013; · 2.20 Impact Factor
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    ABSTRACT: We previously reported that Tpa-S DNA polymerase (constructed via fusion of the Sso7d DNA binding protein to the C-terminus of Thermococcus pacificus (Tpa) DNA polymerase) is more efficient in long and rapid PCR than wild-type Tpa, Taq, or Pfu DNA polymerases. However, Tpa-S DNA polymerase had a low yield of PCR products compared with commercialized Taq or Pfu DNA polymerases. To improve the yield of PCR products, mutant Tpa-S DNA polymerases were created via site-directed mutagenesis. In this study, we have targeted the N213 residue in the Exo II motif and the K501 residue in the Pol III motif. The mutant Tpa-S DNA polymerases showed enhanced PCR yields compared to that of the Tpa-S DNA polymerase. Specifically, the double mutant Tpa-S N213D/K501R DNA polymerase had an approximately three-fold increase in the yield of 8-10kb PCR products over that of the Tpa-S DNA polymerase, and catalyzed amplification of a 12kb PCR product using a lambda template with an extension time of 30s. Even though the mutation is in the Exo II motif, the error rate of the double mutant Tpa-S N213D/K501R (2.79×10(-5)) was nearly the same as that seen in the Pfu DNA polymerase (2.70×10(-5)).
    Journal of Biotechnology 02/2013; · 3.18 Impact Factor
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    ABSTRACT: We cloned and sequenced the gene encoding Thermococcus pacificus dUTPase (Tpa dUTPase). The Tpa dUTPase gene consists of 471bp and encodes a 156-amino acid protein. The deduced amino acid sequence of Tpa dUTPase has high sequence similarity with other archaeal dUTPases. The Tpa dUTPase had an 18-kDa major protein band consistent with the 17,801Da molecular mass calculated based on the amino acid sequence. The specific activity of Tpa dUTPase on dUTP at 85°C was 90,909U/mg. For Tpa dUTPase activity, we determined an optimum pH of 8.5 and temperature of 85°C. Magnesium ions strongly induced activity, with an optimum concentration of 0.75mM. The half-life of the enzyme at 94°C was about 7h. The specific activity of the Tpa dUTPase on dUTP was about 10-20-fold higher than that of Tpa dUTPase on dCTP. Tpa dUTPase enhanced the PCR amplification efficiency of long targets when Pfu and Vent DNA polymerases were used.
    Enzyme and microbial technology. 12/2012; 51(6-7):342-7.
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    ABSTRACT: The family B DNA polymerase gene from the euryarchaeon Thermococcus waiotapuensis (Twa) contains an open reading frame of 4404 bases that encodes 1467 amino acid residues. The gene is split by two intein-coding sequences that forms a continuous open reading frame with the three polymerase exteins. Twa DNA polymerase genes with (whole gene) and without (genetically intein-spliced) inteins were expressed in Escherichia coli Rosetta(DE3)pLysS. The inteins of the expressed whole gene were easily spliced during purification. The molecular mass of the purified Twa DNA polymerase was about 90kDa, as estimated by SDS-PAGE. The optimal pH for Twa DNA polymerase activity was 6.0 and the optimal temperature was 75°C. The enzyme was activated by magnesium ions. The half-life of the enzyme at 99°C was about 4h. The optimal buffer for PCR with Twa DNA polymerase was 50mM Tris-HCl (pH 8.2), 2.0mM MgCl(2), 30mM KCl, 2.0mM (NH(4))(2)SO(4), 0.01% Triton X-100, and 0.005% BSA. The PCR fidelity of Twa DNA polymerase was higher than Pfu, KOD and Vent DNA polymerases. A ratio of 15:1 Taq:Twa DNA polymerase efficiently facilitated long-range PCR.
    Enzyme and microbial technology. 12/2012; 51(6-7):334-41.
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    ABSTRACT: Polycations such as polybrene (PB) are routinely used for most retroviral vector-mediated gene transfer studies because they can increase the infectivity of retroviruses. However, it was not systematically determined if addition of the polycation is an essential prerequisite for all retroviral transductions. To test this, we measured the effects of the polycation on transduction efficiency using various combinations of target cells and pseudotyped viral envelope (Env) proteins. Here, we show polycations do not always increase retroviral transduction efficiency and that their enhancing effect depends on both the type of target cells and Env proteins. The findings presented here also suggest that high transduction rates can be achieved in primary neural stem cells in vitro and in vivo by choosing an appropriate Env protein for pseudotyping without using polycations which are potentially toxic to primary cells and may change the intrinsic characteristics of cells.
    Neurochemistry International 03/2012; 60(8):846-51. · 2.66 Impact Factor
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    ABSTRACT: The Thermococcus celericrescens (Tcel) DNA polymerase gene, which contains a 2328-bp open reading frame that encodes 775 amino acid residues, was expressed in the Escherichia coli strain Rosetta(DE3)pLysS. The expressed enzyme was purified through heat treatment, HisTrap™ HP column chromatography and then HiTrap™ SP HP column chromatography. Tcel DNA polymerase has poor thermostability and PCR efficiency compared to those of other family B DNA polymerases. To improve thermostability and PCR efficiency, mutant Tcel DNA polymerases were created via site-directed mutagenesis. Specifically, we targeted the A752 residue for enhanced thermostability and the N213 residue for improved PCR efficiency. The mutant Tcel DNA polymerases all showed enhanced PCR efficiency and thermostability compared to those of the wild-type Tcel DNA polymerase. Specifically, the double mutant TcelA752K/N213D DNA polymerase had an approximately three-fold increase in thermostability over that of the wild-type enzyme and amplified a long 10-kb PCR product in an extension time of 2min. However, there was a small change in the 3'→5' exonuclease activity compared with that of the wild-type Tcel DNA polymerase, even though the mutation is in the ExoII motif. The double mutant TcelA752K/N213D DNA polymerase had a 2.6-fold lower error rate compared to that of Taq DNA polymerase. It seems that the double mutant TcelA752K/N213D DNA polymerase can be used in LA (long and accurate) PCR.
    Journal of Biotechnology 06/2011; 155(2):156-63. · 3.18 Impact Factor
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    ABSTRACT: Notch signaling pathway enhances neural stem cell characters and regulates cell fate decisions during neural development. Interestingly, besides Notch, other γ-secretase substrates such as APP, LRP2, and ErbB4 have also proven to have biological functions in neural development. We designed a unique experimental setting, combining gain-of- (expression of Notch intracellular domain, NICD) and loss-of-function (γ-secretase inhibition) methods, and were able to examine the function of Notch alone by excluding the activity of other γ-secretase substrates. Here, we show that the frequency and size of neurospheres generated from embryonic neural stem cells (NSCs) significantly decreased by 62.7% and 37.2%, respectively, in the presence of γ-secretase inhibitor even when NICD was expressed. Under the condition of differentiation, however, the γ-secretase inhibitor treatment did not influence the promotion of astrogenesis at the expense of neurogenesis by NICD. These results indicate that other γ-secretase substrate(s) along with Notch are important in the maintenance of the stemness of NSCs, but that Notch alone can sufficiently inhibit neurogenesis without the action of the other γ-secretase substrates during differentiation.
    Biochemical and Biophysical Research Communications 01/2011; 404(1):133-8. · 2.41 Impact Factor
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    ABSTRACT: The gene encoding Thermus filiformis (Tfi) DNA polymerase was expressed under the control of the tac promoter on a high-copy plasmid, pJR, in Escherichia coli. The Tfi DNA polymerase was purified by using heat treatment and DEAE-Sephacel column chromatography. The purified enzyme had a molecular mass of 92 kDa, as estimated by SDS/PAGE. The optimum pH and temperature of the enzyme were 8.4–9.0 and 70–72.5 °C respectively. The half-life of the enzyme at 94 °C was approx. 40 min. The enzyme was activated by the bivalent cations, Mg2+ and Mn2+, and was inhibited by EDTA. The optimal Mg2+ concentration of the enzyme was 4 mM. The optimal conditions for the PCR reaction were slightly different from those for the enzyme activity except for the optimal Mg2+ concentration. Low concentrations of KCl had no effect on either the enzymic activity or the PCR amplification. The result of the PCR experiment with the enzyme indicates that Tfi DNA polymerase might be useful in DNA amplification.
    Biotechnology and Applied Biochemistry 12/2010; 30(1):19 - 25. · 1.35 Impact Factor
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    ABSTRACT: The gene encoding Psp HJ147 UDG (Psychrobacter sp. HJ147 uracil-DNA glycosylase) was cloned and sequenced. The gene consists of 735 bp for coding a protein with 244 amino acid residues. The deduced amino acid sequence of Psp HJ147 UDG showed a high similarity to that of Psychrobacter articus, Psychrobacter cryohalolentis K5 and Psychrobacter sp. PRwf-1. The PCR-amplified Psp HJ147 UDG gene was expressed under the control of the T7lac promoter on pTYB1 in Escherichia coli BL21(DE3). The expressed enzyme was purified with IMPACT™-CN (intein-mediated purification with an affinity chitin-binding tag) system. The optimum pH and temperature of the purified enzyme were 7.0–7.5 and 20–25 °C respectively. The optimum NaCl and KCl concentrations for the activity of the purified enzyme ranged from 50 to75 mM. The half-life of the enzyme at 50 °C was approx. 45 s. These heat-labile characteristics enabled Psp HJ147 UDG to control carry-over contamination in direct PCR without loss of the PCR product. Psp HJ147 UDG's contaminant control in both direct PCR and indirect PCR exhibited superiority over the UDG of the marine psychrophilic bacterium strain BMTU 3346 and that of E. coli.
    Biotechnology and Applied Biochemistry 12/2010; 52(2):167 - 175. · 1.35 Impact Factor
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    ABSTRACT: A recombinant DNA ligase from Sulfophobococcus zilligii that shows multiple cofactor specificity (ATP, ADP and GTP) was expressed in Escherichia coli and purified under reducing conditions. Crystals were obtained by the microbatch crystallization method at 295 K in a drop containing 1 µl protein solution (10 mg ml(-1)) and an equal volume of mother liquor [0.1 M HEPES pH 7.5, 10%(w/v) polyethylene glycol 10 000]. A data set was collected to 2.9 Å resolution using synchrotron radiation. The crystals belonged to space group P1, with unit-cell parameters a=63.7, b=77.1, c=77.8 Å, α=83.4, β=82.4, γ=74.6°. Assuming the presence of two molecules in the unit cell, the solvent content was estimated to be about 53.4%.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 12/2010; 66(Pt 12):1583-5. · 0.55 Impact Factor
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    ABSTRACT: Neq DNA polymerase is the first archaeal family B DNA polymerase reported to lack uracil recognition function and successfully utilize deaminated bases. We have focused on two amino acid residues (Y515, A523) in the fingers subdomain of Neq DNA polymerase, which were predicted to be located in the middle of the fingers subdomain, based on amino acid sequence alignment of the Neq DNA polymerase with structurally determined archaeal DNA polymerases. Those two residues were replaced by site-directed mutagenesis, and the enzymatic properties of the mutants were analyzed. Here, we show that the A523 residue located in the middle of the fingers subdomain affects the processivity of Neq DNA polymerase. Mutational analysis has allowed us to enhance the protein function as well as understand the function of the residues. One mutant protein, Neq A523R DNA polymerase, exhibited a roughly 3-fold enhanced processivity and extension rate compared to wild type, enabling more efficient PCR. In the presence of uracil, Neq A523R DNA polymerase outperformed Taq DNA polymerase with enhanced specificity and sensitivity. These results suggest that Neq A523R DNA polymerase could be most effectively utilized in real-time PCR using uracil-DNA glycosylase without the risk of carry-over contamination.
    Protein Engineering Design and Selection 11/2010; 23(11):835-42. · 2.59 Impact Factor
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    ABSTRACT: The family B DNA polymerase gene was amplified from Thermococcus celer genomic DNA by using the degenerate primers and DNA walking PCR. The Tce DNA polymerase gene was cloned and sequenced. The gene contains an ORF of 2,325 bp encoding 774 amino acid residues with a calculated molecular weight of 89,788.9 kDa. The Tce DNA polymerase was purified by heat treatment and heparin column chromatography. The optimal conditions for PCR were determined. Long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tce DNA polymerases (Tce plus DNA polymerase). Tce plus DNA polymerase surpassed the PCR performance of Tce, Taq and Pfu DNA polymerases in terms of yield and efficiency.
    Biotechnology Letters 10/2010; 33(2):339-46. · 1.85 Impact Factor
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    ABSTRACT: Collembolan species have been known to have beta-1,3-glucanase activity and yet the genes coding such enzymes have not been demonstrated. We report here a novel arthropod endo-beta-1,3-glucanase gene CaLam from the Antarctic springtail, Cryptopygus antarcticus. The open reading frame consists of 813bp encoding 270 amino acids with a putative signal peptide and a typical motif of glycosyl hydrolase family 16 (GHF16), E-I-D-I-T-E. The recombinant protein expressed in E. coli shows the hydrolytic activity toward laminarin (K(m) approximately 9.98mg/mL) with an optimal temperature 50 degrees C and an optimal pH 6.0. CaLam digests laminarin and laminarioligosaccharides except laminaribiose as an endo-beta-1,3-glucanase, releasing glucose, laminaribiose and laminaritriose as the major products. Analyses of molecular phylogeny of CaLam and its protein structure reveal that CaLam is closely related with bacterial beta-1,3-glucanases more than with the eukaryotic homologues. Even so, the genomic structure of the CaLam gene consisting of six exons interspersed with approximately 57 to 63bp introns confirms that it is endogenous in the genome of the Antarctic springtail. These results suggest that CaLam should have been transferred from bacteria to the lineage of the Collembolan species by horizontal gene transfer.
    Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 04/2010; 155(4):403-12. · 1.61 Impact Factor
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    ABSTRACT: The biochemical properties of the Thermococcus pacificus (Tpa) DNA polymerase were determined to evaluate its feasibility for use in polymerase chain reaction (PCR) application. The Tpa DNA polymerase gene was expressed under the control of the T7lac promoter in the pET-22b(+) plasmid in Escherichia coli BL21-CodonPlus(DE3)-RIL. The enzyme was then purified by heat treatment followed by two steps of column chromatography after which the optimum pH and temperature of the enzyme were determined to be pH 7.5 and 75 °C. The optimal PCR buffer for Tpa DNA polymerase consisted of 50 mM Tris–HCl (pH 8.4), 4 mM MgCl2, and 10 mM KCl. Tpa DNA polymerase performed significantly more efficiently in PCR amplification than Taq or Pfu DNA polymerase. By fusing the Sulfolobus solfataricus DNA binding protein Sso7d to Tpa DNA polymerase, we obtained a fusion polymerase which exhibits profound advantages over unmodified Tpa DNA polymerase in PCR applications. Tpa DNA polymerase (2.04 × 10−6) and Tpa-S DNA polymerase (2.20 × 10−6) revealed a 5-fold higher fidelity than Taq DNA polymerase (12.13 × 10−6).
    Enzyme and Microbial Technology. 01/2010;
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    ABSTRACT: The DNA polymerase gene of Thermococcus marinus (Tma) contains an intein inserted at the pol-b site that possesses a 1611-bp ORF encoding a 537-amino acid residue. The LAGLIDADG motif, often found in site-specific DNA endonucleases, was detected within the amino acid sequence of the intein. The intein endonuclease, denoted as PI-Tma, was purified as a naturally spliced product from the expression of the complete DNA polymerase gene in Escherichia coli. PI-Tma cleaved intein-less DNA sequences, leaving four-base-long, 3'-hydroxyl overhangs with 5'-phosphate. Nonpalindromic recognition sequences 19 bp long were also identified using partially complementary oligonucleotide pair sequences inserted into the plasmid pET-22b(+). Cleavage by PI-Tma was optimal when present in 50mM glycine-NaOH (pH 10.5), 150mM KCl and 12mM MgCl(2) at 70 degrees C.
    FEMS Microbiology Letters 09/2009; 297(2):180-8. · 2.05 Impact Factor
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    ABSTRACT: We developed an asexual reproductive plant, Kalanchoe pinnata, as a new bioreactor for plant-based molecular farming using a newly developed transformation method. Leaf crenate margins were pin-pricked to infect the plant with the Agrobacterium strain LBA4404 and vacuum infiltration was also applied to introduce the target gene into the plants. Subsequently, the young mother leaf produced new clones at the leaf crenate margins without the need for time- and labor-consuming tissue culture procedures. The average transformation rates were approximately 77 and 84% for pin-prickling and vacuum-infiltration methods, respectively. To functionally characterize an introduced target protein, a nucleic acid hydrolyzing recombinant 3D8 scFv was selected and the plant based 3D8 scFv proteins were purified and analyzed. Based on abzyme analysis, the purified protein expressed with this system had catalytic activity and exhibited all of properties of the protein produced in an E. coli system. This result suggested that vegetatively reproductive K. pinnata can be a novel and potent bioreactor for bio-pharmaceutical proteins.
    Plant Cell Reports 09/2009; 28(10):1593-602. · 2.94 Impact Factor