The Experts below are selected from a list of 303 Experts worldwide ranked by ideXlab platform
Martha Guerrero-olazarán - One of the best experts on this subject based on the ideXlab platform.
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Optimization of five environmental factors to increase beta‐propeller phytase production in Pichia pastoris and impact on the physiological response of the host
Biotechnology progress, 2013Co-Authors: José M. Viader-salvadó, Miguel Castillo-galván, José Antonio Fuentes-garibay, María M. Iracheta-cárdenas, Martha Guerrero-olazaránAbstract:Recently, we engineered Pichia pastoris Mut(s) strains to produce several beta-propeller phytases, one from Bacillus subtilis and the others designed by a structure-guided consensus approach. Furthermore, we demonstrated the ability of P. pastoris to produce and secrete these phytases in an active form in shake-flask cultures. In the present work, we used a design of experiments strategy (Simplex optimization method) to optimize five environmental factors that define the culture conditions in the Induction Step to increase beta-propeller phytase production in P. pastoris bioreactor cultures. With the optimization process, up to 347,682 U (82,814 U/L or 6.4 g/L culture medium) of phytase at 68 h of Induction was achieved. In addition, the impact of the optimization process on the physiological response of the host was evaluated. The results indicate that the increase in extracellular phytase production through the optimization process was correlated with an increase in metabolic activity of P. pastoris, shown by an increase in oxygen demand and methanol consumption, that increase the specific growth rate. The increase in extracellular phytase production also occurred with a decrease in extracellular protease activity. Moreover, the optimized culture conditions increased the recombinant protein secretion by up to 88%, along with the extracellular phytase production efficiency per cell.
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Shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris bioreactor cultures.
Biotechnology progress, 2012Co-Authors: José M. Viader-salvadó, José Antonio Fuentes-garibay, María M. Iracheta-cárdenas, Mauricio Castillo-galván, Luis J. Galán-wong, Martha Guerrero-olazaránAbstract:Recently, we engineered a Pichia pastoris Mut+ strain to produce and secrete recombinant Litopenaeus vannamei trypsinogen. Despite the observed toxicity of the recombinant shrimp trypsinogen to the P. pastoris cell host, when high density cell cultures in shake flasks with alanine in the Induction medium were used recombinant shrimp trypsinogen could be produced. To further improve the product yield, in this work, we evaluated L. vannamei trypsinogen production in P. pastoris using a bioreactor and two recombinant P. pastoris strains with different methanol utilization (Mut) phenotypes. The effect of pH and temperature during the Induction Step on the trypsinogen production was also evaluated. The results indicate that temperature, pH, and Mut phenotypes influence the production of the recombinant protein, with almost no observed effect on cell growth. All cultures with the Mut+ strain had significant operational difficulties, such as in lowering the Induction temperature, maintaining dissolved oxygen (DO) above 20%, and maintaining the methanol concentration at a constant value, and showed a decrease in metabolic activity due to trypsinogen toxicity to the cell host. In the culture with the Muts strain, however, the temperature, methanol concentration, and DO could be more easily controlled, the temperature could be easily decreased, and the trypsinogen caused the lowest toxicity to the host cells. After 96 h of Muts strain Induction (pH 6 and 25°C), about 250 mg/L recombinant trypsinogen was detected in the culture medium. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013
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Recombinant shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris.
Biotechnology progress, 2009Co-Authors: Martha Guerrero-olazarán, Mauricio Castillo-galván, Luis L. Escamilla-treviño, Juan A Gallegos-lópez, José M. Viader-salvadóAbstract:Shrimp (Litopenaeus vannamei) trypsinogen has never been isolated from its natural source. To assess the production of L. vannamei trypsinogen, we engineered Pichia pastoris strains and evaluated two culture approaches with three Induction culture media, to produce recombinant shrimp trypsinogen for the first time. The trypsinogen II cDNA was fused to the signal sequence of the Saccharomyces cerevisiae alpha mating factor, placed under the control of the P. pastoris AOX1 promoter, and integrated into the genome of P. pastoris host strain GS115. Using standard culture conditions for heterologous gene Induction of a GS115 strain in shake flasks, recombinant shrimp trypsinogen was not detected by SDS-PAGE and Western blot analysis. Growth kinetics revealed a toxicity of recombinant shrimp trypsinogen or its activated form over the cell host. Thus, a different culture approach was tested for the Induction Step, involving the use of high cell density cultures, a higher frequency of methanol feeding (every 12 h), and a buffered minimal methanol medium supplemented with sorbitol or alanine; alanine supplemented medium was found to be more efficient. After 96 h of Induction with alanine supplemented medium, a 29-kDa band from the cell-free culture medium was clearly observed by SDS-PAGE, and confirmed by Western blot to be shrimp trypsinogen, at a concentration of 14 microg/mL. Our results demonstrate that high density cell cultures with alanine in the Induction medium allow the production of recombinant shrimp trypsinogen using the P. pastoris expression system, because of improved cell viability and greater stability of the recombinant trypsinogen.
José M. Viader-salvadó - One of the best experts on this subject based on the ideXlab platform.
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Optimization of five environmental factors to increase beta‐propeller phytase production in Pichia pastoris and impact on the physiological response of the host
Biotechnology progress, 2013Co-Authors: José M. Viader-salvadó, Miguel Castillo-galván, José Antonio Fuentes-garibay, María M. Iracheta-cárdenas, Martha Guerrero-olazaránAbstract:Recently, we engineered Pichia pastoris Mut(s) strains to produce several beta-propeller phytases, one from Bacillus subtilis and the others designed by a structure-guided consensus approach. Furthermore, we demonstrated the ability of P. pastoris to produce and secrete these phytases in an active form in shake-flask cultures. In the present work, we used a design of experiments strategy (Simplex optimization method) to optimize five environmental factors that define the culture conditions in the Induction Step to increase beta-propeller phytase production in P. pastoris bioreactor cultures. With the optimization process, up to 347,682 U (82,814 U/L or 6.4 g/L culture medium) of phytase at 68 h of Induction was achieved. In addition, the impact of the optimization process on the physiological response of the host was evaluated. The results indicate that the increase in extracellular phytase production through the optimization process was correlated with an increase in metabolic activity of P. pastoris, shown by an increase in oxygen demand and methanol consumption, that increase the specific growth rate. The increase in extracellular phytase production also occurred with a decrease in extracellular protease activity. Moreover, the optimized culture conditions increased the recombinant protein secretion by up to 88%, along with the extracellular phytase production efficiency per cell.
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Shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris bioreactor cultures.
Biotechnology progress, 2012Co-Authors: José M. Viader-salvadó, José Antonio Fuentes-garibay, María M. Iracheta-cárdenas, Mauricio Castillo-galván, Luis J. Galán-wong, Martha Guerrero-olazaránAbstract:Recently, we engineered a Pichia pastoris Mut+ strain to produce and secrete recombinant Litopenaeus vannamei trypsinogen. Despite the observed toxicity of the recombinant shrimp trypsinogen to the P. pastoris cell host, when high density cell cultures in shake flasks with alanine in the Induction medium were used recombinant shrimp trypsinogen could be produced. To further improve the product yield, in this work, we evaluated L. vannamei trypsinogen production in P. pastoris using a bioreactor and two recombinant P. pastoris strains with different methanol utilization (Mut) phenotypes. The effect of pH and temperature during the Induction Step on the trypsinogen production was also evaluated. The results indicate that temperature, pH, and Mut phenotypes influence the production of the recombinant protein, with almost no observed effect on cell growth. All cultures with the Mut+ strain had significant operational difficulties, such as in lowering the Induction temperature, maintaining dissolved oxygen (DO) above 20%, and maintaining the methanol concentration at a constant value, and showed a decrease in metabolic activity due to trypsinogen toxicity to the cell host. In the culture with the Muts strain, however, the temperature, methanol concentration, and DO could be more easily controlled, the temperature could be easily decreased, and the trypsinogen caused the lowest toxicity to the host cells. After 96 h of Muts strain Induction (pH 6 and 25°C), about 250 mg/L recombinant trypsinogen was detected in the culture medium. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013
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Recombinant shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris.
Biotechnology progress, 2009Co-Authors: Martha Guerrero-olazarán, Mauricio Castillo-galván, Luis L. Escamilla-treviño, Juan A Gallegos-lópez, José M. Viader-salvadóAbstract:Shrimp (Litopenaeus vannamei) trypsinogen has never been isolated from its natural source. To assess the production of L. vannamei trypsinogen, we engineered Pichia pastoris strains and evaluated two culture approaches with three Induction culture media, to produce recombinant shrimp trypsinogen for the first time. The trypsinogen II cDNA was fused to the signal sequence of the Saccharomyces cerevisiae alpha mating factor, placed under the control of the P. pastoris AOX1 promoter, and integrated into the genome of P. pastoris host strain GS115. Using standard culture conditions for heterologous gene Induction of a GS115 strain in shake flasks, recombinant shrimp trypsinogen was not detected by SDS-PAGE and Western blot analysis. Growth kinetics revealed a toxicity of recombinant shrimp trypsinogen or its activated form over the cell host. Thus, a different culture approach was tested for the Induction Step, involving the use of high cell density cultures, a higher frequency of methanol feeding (every 12 h), and a buffered minimal methanol medium supplemented with sorbitol or alanine; alanine supplemented medium was found to be more efficient. After 96 h of Induction with alanine supplemented medium, a 29-kDa band from the cell-free culture medium was clearly observed by SDS-PAGE, and confirmed by Western blot to be shrimp trypsinogen, at a concentration of 14 microg/mL. Our results demonstrate that high density cell cultures with alanine in the Induction medium allow the production of recombinant shrimp trypsinogen using the P. pastoris expression system, because of improved cell viability and greater stability of the recombinant trypsinogen.
Edward R Dougherty - One of the best experts on this subject based on the ideXlab platform.
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A CoD-based stationary control policy for intervening in large gene regulatory networks.
BMC bioinformatics, 2011Co-Authors: Noushin Ghaffari, Ivan Ivanov, Xiaoning Qian, Edward R DoughertyAbstract:One of the most important goals of the mathematical modeling of gene regulatory networks is to alter their behavior toward desirable phenotypes. Therapeutic techniques are derived for intervention in terms of stationary control policies. In large networks, it becomes computationally burdensome to derive an optimal control policy. To overcome this problem, greedy intervention approaches based on the concept of the Mean First Passage Time or the steady-state probability mass of the network states were previously proposed. Another possible approach is to use reduction mappings to compress the network and develop control policies on its reduced version. However, such mappings lead to loss of information and require an Induction Step when designing the control policy for the original network. In this paper, we propose a novel solution, CoD-CP, for designing intervention policies for large Boolean networks. The new method utilizes the Coefficient of Determination (CoD) and the Steady-State Distribution (SSD) of the model. The main advantage of CoD-CP in comparison with the previously proposed methods is that it does not require any compression of the original model, and thus can be directly designed on large networks. The simulation studies on small synthetic networks shows that CoD-CP performs comparable to previously proposed greedy policies that were induced from the compressed versions of the networks. Furthermore, on a large 17-gene gastrointestinal cancer network, CoD-CP outperforms other two available greedy techniques, which is precisely the kind of case for which CoD-CP has been developed. Finally, our experiments show that CoD-CP is robust with respect to the attractor structure of the model. The newly proposed CoD-CP provides an attractive alternative for intervening large networks where other available greedy methods require size reduction on the network and an extra Induction Step before designing a control policy.
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A CoD-based stationary control policy for intervening in large gene regulatory networks
BMC Bioinformatics, 2011Co-Authors: Noushin Ghaffari, Ivan Ivanov, Xiaoning Qian, Edward R DoughertyAbstract:Abstract Background One of the most important goals of the mathematical modeling of gene regulatory networks is to alter their behavior toward desirable phenotypes. Therapeutic techniques are derived for intervention in terms of stationary control policies. In large networks, it becomes computationally burdensome to derive an optimal control policy. To overcome this problem, greedy intervention approaches based on the concept of the Mean First Passage Time or the steady-state probability mass of the network states were previously proposed. Another possible approach is to use reduction mappings to compress the network and develop control policies on its reduced version. However, such mappings lead to loss of information and require an Induction Step when designing the control policy for the original network. Results In this paper, we propose a novel solution, CoD-CP, for designing intervention policies for large Boolean networks. The new method utilizes the Coefficient of Determination (CoD) and the Steady-State Distribution (SSD) of the model. The main advantage of CoD-CP in comparison with the previously proposed methods is that it does not require any compression of the original model, and thus can be directly designed on large networks. The simulation studies on small synthetic networks shows that CoD-CP performs comparable to previously proposed greedy policies that were induced from the compressed versions of the networks. Furthermore, on a large 17-gene gastrointestinal cancer network, CoD-CP outperforms other two available greedy techniques, which is precisely the kind of case for which CoD-CP has been developed. Finally, our experiments show that CoD-CP is robust with respect to the attractor structure of the model. Conclusions The newly proposed CoD-CP provides an attractive alternative for intervening large networks where other available greedy methods require size reduction on the network and an extra Induction Step before designing a control policy.
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A CoD-based stationary control policy for intervening in large gene regulatory networks
BMC Bioinformatics, 2011Co-Authors: Noushin Ghaffari, Ivan Ivanov, Xiaoning Qian, Edward R DoughertyAbstract:Background One of the most important goals of the mathematical modeling of gene regulatory networks is to alter their behavior toward desirable phenotypes. Therapeutic techniques are derived for intervention in terms of stationary control policies. In large networks, it becomes computationally burdensome to derive an optimal control policy. To overcome this problem, greedy intervention approaches based on the concept of the Mean First Passage Time or the steady-state probability mass of the network states were previously proposed. Another possible approach is to use reduction mappings to compress the network and develop control policies on its reduced version. However, such mappings lead to loss of information and require an Induction Step when designing the control policy for the original network.
Mauricio Castillo-galván - One of the best experts on this subject based on the ideXlab platform.
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Shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris bioreactor cultures.
Biotechnology progress, 2012Co-Authors: José M. Viader-salvadó, José Antonio Fuentes-garibay, María M. Iracheta-cárdenas, Mauricio Castillo-galván, Luis J. Galán-wong, Martha Guerrero-olazaránAbstract:Recently, we engineered a Pichia pastoris Mut+ strain to produce and secrete recombinant Litopenaeus vannamei trypsinogen. Despite the observed toxicity of the recombinant shrimp trypsinogen to the P. pastoris cell host, when high density cell cultures in shake flasks with alanine in the Induction medium were used recombinant shrimp trypsinogen could be produced. To further improve the product yield, in this work, we evaluated L. vannamei trypsinogen production in P. pastoris using a bioreactor and two recombinant P. pastoris strains with different methanol utilization (Mut) phenotypes. The effect of pH and temperature during the Induction Step on the trypsinogen production was also evaluated. The results indicate that temperature, pH, and Mut phenotypes influence the production of the recombinant protein, with almost no observed effect on cell growth. All cultures with the Mut+ strain had significant operational difficulties, such as in lowering the Induction temperature, maintaining dissolved oxygen (DO) above 20%, and maintaining the methanol concentration at a constant value, and showed a decrease in metabolic activity due to trypsinogen toxicity to the cell host. In the culture with the Muts strain, however, the temperature, methanol concentration, and DO could be more easily controlled, the temperature could be easily decreased, and the trypsinogen caused the lowest toxicity to the host cells. After 96 h of Muts strain Induction (pH 6 and 25°C), about 250 mg/L recombinant trypsinogen was detected in the culture medium. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013
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Recombinant shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris.
Biotechnology progress, 2009Co-Authors: Martha Guerrero-olazarán, Mauricio Castillo-galván, Luis L. Escamilla-treviño, Juan A Gallegos-lópez, José M. Viader-salvadóAbstract:Shrimp (Litopenaeus vannamei) trypsinogen has never been isolated from its natural source. To assess the production of L. vannamei trypsinogen, we engineered Pichia pastoris strains and evaluated two culture approaches with three Induction culture media, to produce recombinant shrimp trypsinogen for the first time. The trypsinogen II cDNA was fused to the signal sequence of the Saccharomyces cerevisiae alpha mating factor, placed under the control of the P. pastoris AOX1 promoter, and integrated into the genome of P. pastoris host strain GS115. Using standard culture conditions for heterologous gene Induction of a GS115 strain in shake flasks, recombinant shrimp trypsinogen was not detected by SDS-PAGE and Western blot analysis. Growth kinetics revealed a toxicity of recombinant shrimp trypsinogen or its activated form over the cell host. Thus, a different culture approach was tested for the Induction Step, involving the use of high cell density cultures, a higher frequency of methanol feeding (every 12 h), and a buffered minimal methanol medium supplemented with sorbitol or alanine; alanine supplemented medium was found to be more efficient. After 96 h of Induction with alanine supplemented medium, a 29-kDa band from the cell-free culture medium was clearly observed by SDS-PAGE, and confirmed by Western blot to be shrimp trypsinogen, at a concentration of 14 microg/mL. Our results demonstrate that high density cell cultures with alanine in the Induction medium allow the production of recombinant shrimp trypsinogen using the P. pastoris expression system, because of improved cell viability and greater stability of the recombinant trypsinogen.
Noushin Ghaffari - One of the best experts on this subject based on the ideXlab platform.
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A CoD-based stationary control policy for intervening in large gene regulatory networks.
BMC bioinformatics, 2011Co-Authors: Noushin Ghaffari, Ivan Ivanov, Xiaoning Qian, Edward R DoughertyAbstract:One of the most important goals of the mathematical modeling of gene regulatory networks is to alter their behavior toward desirable phenotypes. Therapeutic techniques are derived for intervention in terms of stationary control policies. In large networks, it becomes computationally burdensome to derive an optimal control policy. To overcome this problem, greedy intervention approaches based on the concept of the Mean First Passage Time or the steady-state probability mass of the network states were previously proposed. Another possible approach is to use reduction mappings to compress the network and develop control policies on its reduced version. However, such mappings lead to loss of information and require an Induction Step when designing the control policy for the original network. In this paper, we propose a novel solution, CoD-CP, for designing intervention policies for large Boolean networks. The new method utilizes the Coefficient of Determination (CoD) and the Steady-State Distribution (SSD) of the model. The main advantage of CoD-CP in comparison with the previously proposed methods is that it does not require any compression of the original model, and thus can be directly designed on large networks. The simulation studies on small synthetic networks shows that CoD-CP performs comparable to previously proposed greedy policies that were induced from the compressed versions of the networks. Furthermore, on a large 17-gene gastrointestinal cancer network, CoD-CP outperforms other two available greedy techniques, which is precisely the kind of case for which CoD-CP has been developed. Finally, our experiments show that CoD-CP is robust with respect to the attractor structure of the model. The newly proposed CoD-CP provides an attractive alternative for intervening large networks where other available greedy methods require size reduction on the network and an extra Induction Step before designing a control policy.
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A CoD-based stationary control policy for intervening in large gene regulatory networks
BMC Bioinformatics, 2011Co-Authors: Noushin Ghaffari, Ivan Ivanov, Xiaoning Qian, Edward R DoughertyAbstract:Abstract Background One of the most important goals of the mathematical modeling of gene regulatory networks is to alter their behavior toward desirable phenotypes. Therapeutic techniques are derived for intervention in terms of stationary control policies. In large networks, it becomes computationally burdensome to derive an optimal control policy. To overcome this problem, greedy intervention approaches based on the concept of the Mean First Passage Time or the steady-state probability mass of the network states were previously proposed. Another possible approach is to use reduction mappings to compress the network and develop control policies on its reduced version. However, such mappings lead to loss of information and require an Induction Step when designing the control policy for the original network. Results In this paper, we propose a novel solution, CoD-CP, for designing intervention policies for large Boolean networks. The new method utilizes the Coefficient of Determination (CoD) and the Steady-State Distribution (SSD) of the model. The main advantage of CoD-CP in comparison with the previously proposed methods is that it does not require any compression of the original model, and thus can be directly designed on large networks. The simulation studies on small synthetic networks shows that CoD-CP performs comparable to previously proposed greedy policies that were induced from the compressed versions of the networks. Furthermore, on a large 17-gene gastrointestinal cancer network, CoD-CP outperforms other two available greedy techniques, which is precisely the kind of case for which CoD-CP has been developed. Finally, our experiments show that CoD-CP is robust with respect to the attractor structure of the model. Conclusions The newly proposed CoD-CP provides an attractive alternative for intervening large networks where other available greedy methods require size reduction on the network and an extra Induction Step before designing a control policy.
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A CoD-based stationary control policy for intervening in large gene regulatory networks
BMC Bioinformatics, 2011Co-Authors: Noushin Ghaffari, Ivan Ivanov, Xiaoning Qian, Edward R DoughertyAbstract:Background One of the most important goals of the mathematical modeling of gene regulatory networks is to alter their behavior toward desirable phenotypes. Therapeutic techniques are derived for intervention in terms of stationary control policies. In large networks, it becomes computationally burdensome to derive an optimal control policy. To overcome this problem, greedy intervention approaches based on the concept of the Mean First Passage Time or the steady-state probability mass of the network states were previously proposed. Another possible approach is to use reduction mappings to compress the network and develop control policies on its reduced version. However, such mappings lead to loss of information and require an Induction Step when designing the control policy for the original network.