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Hyung Joon Cha - One of the best experts on this subject based on the ideXlab platform.

  • solubility dependency of co expression effects of stress induced Protein dps on Foreign Protein expression in escherichia coli
    Enzyme and Microbial Technology, 2006
    Co-Authors: Youngsoo Kim, Hyung Joon Cha
    Abstract:

    Abstract The over-expression of Foreign Proteins imposes metabolic burden on host strains that may lead to reduced cell growth and even yield of target Protein. We investigated dependency of co-expression effects of stress-induced non-specific DNA-binding Protein, Dps, on cell growth and Foreign Protein expression according to solubility in Escherichia coli . Dps co-expression showed clear distinct effects according to solubility of target Proteins. Under co-expression of recombinant Dps, cell growth for the strain expressing baculoviral polyhedrin (Polh)-green fluorescent Protein (GFP) fusion Protein or human interleukin-2 (hIL-2) that were expressed as insoluble inclusion body had tendency to decline slightly compared to each Dps non-expressing strain. However, cell growth for the strain expressing soluble GFP or mussel adhesive Protein type 5 (Mgfp-5) was somewhat increased. While Dps co-expression had somewhat negative effects on expression of soluble Protein, it showed huge impacts on product yield of insoluble Proteins (1.6–1.8-fold for Polh-GFP and 4–5-fold for hIL-2). Therefore, it was obvious that co-expression of Dps has different effects on Foreign Protein production according to solubility. Proteomic analyses revealed that Dps co-expression induced significantly different global patterns through interaction with target Foreign Protein according to target solubility. These global pattern alterations might be favorable for production of insoluble Foreign Proteins indirectly.

  • functional display of Foreign Protein on surface of escherichia coli using n terminal domain of ice nucleation Protein
    Biotechnology and Bioengineering, 2004
    Co-Authors: Dong Gyun Kang, Hyung Joon Cha
    Abstract:

    We investigated the ability of the N-terminal domain of InaK, an ice nucleation Protein from Pseudomonas syringae KCTC1832, to act as an anchoring motif for the display of Foreign Proteins on the Escherichia coli cell surface. Total expression level and surface display efficiency of green fluorescent Protein (GFP) was compared following their fusion with either the N-terminal domain of InaK (InaK-N), or with the known truncated InaK containing both N- and C-terminal domains (InaK-NC). We report that the InaK-N/GFP fusion Protein showed a similar cell surface display efficiency (∼50%) as InaK-NC/GFP, demonstrating that the InaK N-terminal region alone can direct translocation of Foreign Proteins to the cell surface and can be employed as a potential cell surface display motif. Moreover, InaK-N/GFP showed the highest levels of total expression and surface display based on unit cell density. InaK-N was also successful in directing cell surface display of organophosphorus hydrolase (OPH), confirming its ability to act as a display motif. © 2003 Wiley Periodicals, Inc.

  • functional display of Foreign Protein on surface of escherichia coli using n terminal domain of ice nucleation Protein
    Biotechnology and Bioengineering, 2004
    Co-Authors: Dong Gyun Kang, Hyung Joon Cha
    Abstract:

    We investigated the ability of the N-terminal domain of InaK, an ice nucleation Protein from Pseudomonas syringae KCTC1832, to act as an anchoring motif for the display of Foreign Proteins on the Escherichia coli cell surface. Total expression level and surface display efficiency of green fluorescent Protein (GFP) was compared following their fusion with either the N-terminal domain of InaK (InaK-N), or with the known truncated InaK containing both N- and C-terminal domains (InaK-NC). We report that the InaK-N/GFP fusion Protein showed a similar cell surface display efficiency ( approximately 50%) as InaK-NC/GFP, demonstrating that the InaK N-terminal region alone can direct translocation of Foreign Proteins to the cell surface and can be employed as a potential cell surface display motif. Moreover, InaK-N/GFP showed the highest levels of total expression and surface display based on unit cell density. InaK-N was also successful in directing cell surface display of organophosphorus hydrolase (OPH), confirming its ability to act as a display motif.

  • down regulation of acetate pathway through antisense strategy in escherichia coli improved Foreign Protein production
    Biotechnology and Bioengineering, 2003
    Co-Authors: Jaoon Y H Kim, Hyung Joon Cha
    Abstract:

    A problem with the use of Escherichia coli to produce Foreign Proteins is that although endogenously produced acetate is physiologically indispensable, it inhibits Protein expression. Here we firstly employed an antisense RNA strategy as an elaborate metabolic engineering tool to partially block biosynthesis of two major acetate pathway enzymes, phosphotransacetylase (PTA) and acetate kinase (ACK). Three recombinant plasmids containing antisense genes targeting either or both of pta and ackA were constructed, and their effects on the acetate pathway and Foreign Protein productivity compared to control plasmid without any antisense genes were determined in E. coli BL21. Green fluorescent Protein (GFP) was employed as a model Foreign Protein, and timing of antisense expression was controlled by using the intrinsic ackA promoter. We found that the antisense method partially reduced mRNA levels of target enzyme genes and, over time, lowered the concentration of acetate in culture media in all antisense-regulated strains. Notably, total production of GFP was enhanced 1.6- to 2.1-fold in antisense-regulated strains, even though the degree of acetate reduction was not significantly large. It was revealed that the acetate pathway has more critical roles in cellular physiology than expected in the previous reports. When the scale of culture was increased, enhancement of Protein production became larger, demonstrating that this antisense strategy can be successfully applied to practical large-scale Protein production processes.

  • enhancement of heterologous Protein expression in escherichia coli by co expression of nonspecific dna binding stress Protein dps
    Enzyme and Microbial Technology, 2003
    Co-Authors: Youngsoo Kim, Jeong Hyun Seo, Hyung Joon Cha
    Abstract:

    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.

Fred W Ramirez - One of the best experts on this subject based on the ideXlab platform.

  • dynamic hybrid neural network model of an industrial fed batch fermentation process to produce Foreign Protein
    Computers & Chemical Engineering, 2007
    Co-Authors: Siris Laursen, Daniel Webb, Fred W Ramirez
    Abstract:

    An industrial pharmaceutical company has provided industrial pilot scale fed-batch data from a biological process used to produce a Foreign Protein from fed-batch fermentation. This process had proven difficult to control due to the complex behavior of the bacteria after induction. Because of the difficulty of modeling the process fundamentally, neural networks are an attractive alternative. To capture dynamic systems a gray box model approach of parameter function neural networks was used. The parameter function neural network approach has been able to capture well this pilot scale fed-batch fermentation process. In order to obtain accurate training data, the data sets were fit and smoothed using smoothing cubic spline functions. Neural networks were found for the five critical parameter functions of growth rate, glucose consumption rate, oxygen consumption rate, acetate production rate, and Protein production rate. Relatively simple networks were used in order to capture process behavior and not the significant noise in the industrial scale pilot data. Simulations using the neural network parameters predicted dynamic response data well.

  • neural network modeling and optimization of induced Foreign Protein production
    Aiche Journal, 1999
    Co-Authors: Arun Tholudur, Fred W Ramirez
    Abstract:

    An experimental verification and validation of the neural network parameter function approach to modeling dynamic systems is provided. The neural-network parameter-function modeling scheme utilizes some a priori process knowledge (usually material balances) and experimental data to develop a dynamic neural-network model. Other models based on fundamental principles are also developed. The experimental system under consideration is the host-vector system Escherichia coli D1210 and plasmid pSD8, which produces the Foreign Protein β-galactosidase under the effect of the inducer IPTG. Optimal operational conditions are derived and the neural-network-based model is shown to better predict the dynamics and optimum for Protein production than the proposed fundamental kinetic models.

  • optimal fed batch control of induced Foreign Protein production by recombinant bacteria
    Aiche Journal, 1994
    Co-Authors: Jongdae Lee, Fred W Ramirez
    Abstract:

    Optimal control strategies for maximizing the production of induced Foreign Protein by recombinant bacteria were sought by the optimal control theory. Nutrient and inducer feeding rates were selected as key control variables. Since the problem is linear in the control variables, the optimal control is bang-bang or singular. Singular solutions are shown to exist. The optimal control theory showed that the specific growth rate with respect to nutrient concentration must be kept in its maximum phase and that there exist both a cell growth period and a Protein production periods. The optimal control theory calculates exactly the growth and production periods. The glucose concentration is controlled along a singular are to give a maximum specific growth rate. The inducer level is controlled along a separate singular arc.

  • mathematical modeling of induced Foreign Protein production by recombinant bacteria
    Biotechnology and Bioengineering, 1992
    Co-Authors: Jongdae Lee, Fred W Ramirez
    Abstract:

    A new generalized mathematical model for recombinant bacteria which includes inducer effects on cell growth and Foreign Protein production is developed. The model equation set was applied to a host-vector system, Escherichi coli D1210 and plasmid pSD8. Batch experiments were designed and performed in shake flasks to verify the model. A parameter estimation method was developed and proven to be efficient. Although simple, the model can effectively describe the dynamics of the production of Foreign Protein in recombinant bacteria and can be used for optimization and control studies to maximize Foreign Protein production.

Yeon-gu Kim - One of the best experts on this subject based on the ideXlab platform.

  • Co-amplification of EBNA-1 and PyLT through dhfr-mediated gene amplification for improving Foreign Protein production in transient gene expression in CHO cells
    Applied Microbiology and Biotechnology, 2018
    Co-Authors: Joo-hyoung Lee, Sun-hye Park, Jee Yon Kim, Jeong-ki Min, Gyun Min Lee, Sun Hong Kim, Jong Ho Park, Yeon-gu Kim
    Abstract:

    Despite the relatively low transfection efficiency and low specific Foreign Protein productivity (qp) of Chinese hamster ovary (CHO) cell-based transient gene expression (TGE) systems, TGE-based recombinant Protein production technology predominantly employs CHO cells for pre-clinical research and development purposes. To improve TGE in CHO cells, Epstein-Barr virus nuclear antigen-1 (EBNA-1)/polyoma virus large T antigen (PyLT)-co-amplified recombinant CHO (rCHO) cells stably expressing EBNA-1 and PyLT were established using dihydrofolate reductase/methotrexate-mediated gene amplification. The level of transiently expressed Fc-fusion Protein was significantly higher in the EBNA-1/PyLT-co-amplified pools compared to control cultures. Increased Fc-fusion Protein production by EBNA-1/PyLT-co-amplification resulted from a higher qp attributable to EBNA-1 but not PyLT expression. The qp for TGE-based production with EBNA-1/PyLT-co-amplified rCHO cells (EP-amp-20) was approximately 22.9-fold that of the control culture with CHO-DG44 cells. Rather than improved transfection efficiency, this cell line demonstrated increased levels of mRNA expression and replicated DNA, contributing to an increased qp. Furthermore, there was no significant difference in N-glycan profiles in Fc-fusion Proteins produced in the TGE system. Taken together, these results showed that the use of rCHO cells with co-amplified expression of the viral elements EBNA-1 and PyLT improves TGE-based therapeutic Protein production dramatically. Therefore, EBNA-1/PyLT-co-amplified rCHO cells will likely be useful as host cells in CHO cell-based TGE systems.

  • investigation of relationship between ebna 1 expression level and specific Foreign Protein productivity in transient gene expression of hek293 cells
    Process Biochemistry, 2017
    Co-Authors: Joo-hyoung Lee, Sun-hye Park, Jeong-ki Min, Gyun Min Lee, Sungmin Lim, Yeon-gu Kim
    Abstract:

    Abstract In an attempt to determine the relationship between the Epstein–Barr virus nuclear antigen-1 (EBNA-1) expression level and specific Foreign Protein productivity (qp), EBNA-1-amplifed HEK293 cells, which achieved a higher EBNA-1 expression level than that achieved by HEK293E cells, were established using dihydrofolate reductase (dhfr)-mediated gene amplification. Compared with a control culture in a null pool, Fc-fusion Protein production by transient transfection in the EBNA-1-amplified pool showed a significant improvement. qp was linearly correlated with the EBNA-1 expression level in the transient transfection of EBNA-1-amplified clones, as indicated by the correlation coefficient (R2 = 0.7407). The Fc-fusion Protein production and qp in a transient gene expression-based culture with EBNA-1-amplified HEK293 cells, E-amp-68, were approximately 2.0 and 3.2 times, respectively, higher than those in a culture with HEK293E cells. The increase in qp by EBNA-1 amplification mainly resulted from an enhancement in the amount of replicated DNA and level of mRNA expression but not an improved transfection efficiency. Taken together, it was found that EBNA-1 amplification could improve the therapeutic Protein production in an HEK293 cell-based transient gene expression system.

Gyun Min Lee - One of the best experts on this subject based on the ideXlab platform.

  • Co-amplification of EBNA-1 and PyLT through dhfr-mediated gene amplification for improving Foreign Protein production in transient gene expression in CHO cells
    Applied Microbiology and Biotechnology, 2018
    Co-Authors: Joo-hyoung Lee, Sun-hye Park, Jee Yon Kim, Jeong-ki Min, Gyun Min Lee, Sun Hong Kim, Jong Ho Park, Yeon-gu Kim
    Abstract:

    Despite the relatively low transfection efficiency and low specific Foreign Protein productivity (qp) of Chinese hamster ovary (CHO) cell-based transient gene expression (TGE) systems, TGE-based recombinant Protein production technology predominantly employs CHO cells for pre-clinical research and development purposes. To improve TGE in CHO cells, Epstein-Barr virus nuclear antigen-1 (EBNA-1)/polyoma virus large T antigen (PyLT)-co-amplified recombinant CHO (rCHO) cells stably expressing EBNA-1 and PyLT were established using dihydrofolate reductase/methotrexate-mediated gene amplification. The level of transiently expressed Fc-fusion Protein was significantly higher in the EBNA-1/PyLT-co-amplified pools compared to control cultures. Increased Fc-fusion Protein production by EBNA-1/PyLT-co-amplification resulted from a higher qp attributable to EBNA-1 but not PyLT expression. The qp for TGE-based production with EBNA-1/PyLT-co-amplified rCHO cells (EP-amp-20) was approximately 22.9-fold that of the control culture with CHO-DG44 cells. Rather than improved transfection efficiency, this cell line demonstrated increased levels of mRNA expression and replicated DNA, contributing to an increased qp. Furthermore, there was no significant difference in N-glycan profiles in Fc-fusion Proteins produced in the TGE system. Taken together, these results showed that the use of rCHO cells with co-amplified expression of the viral elements EBNA-1 and PyLT improves TGE-based therapeutic Protein production dramatically. Therefore, EBNA-1/PyLT-co-amplified rCHO cells will likely be useful as host cells in CHO cell-based TGE systems.

  • investigation of relationship between ebna 1 expression level and specific Foreign Protein productivity in transient gene expression of hek293 cells
    Process Biochemistry, 2017
    Co-Authors: Joo-hyoung Lee, Sun-hye Park, Jeong-ki Min, Gyun Min Lee, Sungmin Lim, Yeon-gu Kim
    Abstract:

    Abstract In an attempt to determine the relationship between the Epstein–Barr virus nuclear antigen-1 (EBNA-1) expression level and specific Foreign Protein productivity (qp), EBNA-1-amplifed HEK293 cells, which achieved a higher EBNA-1 expression level than that achieved by HEK293E cells, were established using dihydrofolate reductase (dhfr)-mediated gene amplification. Compared with a control culture in a null pool, Fc-fusion Protein production by transient transfection in the EBNA-1-amplified pool showed a significant improvement. qp was linearly correlated with the EBNA-1 expression level in the transient transfection of EBNA-1-amplified clones, as indicated by the correlation coefficient (R2 = 0.7407). The Fc-fusion Protein production and qp in a transient gene expression-based culture with EBNA-1-amplified HEK293 cells, E-amp-68, were approximately 2.0 and 3.2 times, respectively, higher than those in a culture with HEK293E cells. The increase in qp by EBNA-1 amplification mainly resulted from an enhancement in the amount of replicated DNA and level of mRNA expression but not an improved transfection efficiency. Taken together, it was found that EBNA-1 amplification could improve the therapeutic Protein production in an HEK293 cell-based transient gene expression system.

  • osmoprotective effect of glycine betaine on Foreign Protein production in hyperosmotic recombinant chinese hamster ovary cell cultures differs among cell lines
    Biotechnology and Bioengineering, 2000
    Co-Authors: Joon Soo Ryu, Tae Kyung Kim, Joo Young Chung, Gyun Min Lee
    Abstract:

    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.

Valeri Barsegov - One of the best experts on this subject based on the ideXlab platform.

  • Assembly and mechanical properties of the cargo-free and cargo-loaded bacterial nanocompartment encapsulin
    Biomacromolecules, 2016
    Co-Authors: Joost Snijder, Ioana M. Barbu, W. Frederik Rurup, Rebecca J. Burnley, Charlotte Uetrecht, Wouter H Roos, Olga Kononova, Melissa S. T. Koay, Jeroen J L M Cornelissen, Valeri Barsegov
    Abstract:

    Prokaryotes mostly lack membranous compartments that are typical of eukaryotic cells, but instead, they have various Protein-based organelles. These include bacterial microcompartments like the carboxysome and the virus-like nanocompartment encapsulin. Encapsulins have an adaptable mechanism for enzyme packaging, which makes it an attractive platform to carry a Foreign Protein cargo. Here we investigate the assembly pathways and mechanical properties of the cargo-free and cargo-loaded nanocompartments, using a combination of native mass spectrometry, atomic force microscopy and multiscale computational molecular modeling. We show that encapsulin dimers assemble into rigid single-enzyme bacterial containers. Moreover, we demonstrate that cargo encapsulation has a mechanical impact on the shell. The structural similarity of encapsulins to virus capsids is reflected in their mechanical properties. With these robust mechanical properties encapsulins provide a suitable platform for the development of nanotechn...

  • Assembly and Mechanical Properties of the Cargo-Free and Cargo-Loaded Bacterial Nanocompartment Encapsulin
    2016
    Co-Authors: Joost Snijder, Ioana M. Barbu, Rebecca J. Burnley, Charlotte Uetrecht, Wouter H Roos, Olga Kononova, Jeroen J L M Cornelissen, Frederik W. Rurup, Melissa S. T. Koay, Valeri Barsegov
    Abstract:

    Prokaryotes mostly lack membranous compartments that are typical of eukaryotic cells, but instead, they have various Protein-based organelles. These include bacterial microcompartments like the carboxysome and the virus-like nanocompartment encapsulin. Encapsulins have an adaptable mechanism for enzyme packaging, which makes it an attractive platform to carry a Foreign Protein cargo. Here we investigate the assembly pathways and mechanical properties of the cargo-free and cargo-loaded nanocompartments, using a combination of native mass spectrometry, atomic force microscopy and multiscale computational molecular modeling. We show that encapsulin dimers assemble into rigid single-enzyme bacterial containers. Moreover, we demonstrate that cargo encapsulation has a mechanical impact on the shell. The structural similarity of encapsulins to virus capsids is reflected in their mechanical properties. With these robust mechanical properties encapsulins provide a suitable platform for the development of nanotechnological applications