Effect of Culture Conditions and Signal Peptide on Production of Human Recombinant N-Acetylgalactosamine-6-Sulfate Sulfatase in Escherichia coli BL21

The production and characterization of an active recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) in Escherichia coli BL21(DE3) has been previously reported. In this study, the effect of the signal peptide (SP), inducer concentration, process scale, and operational mode (batch and semi-...

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Veröffentlicht in:Journal of microbiology and biotechnology 2013-05, Vol.23 (5), p.689-698
Hauptverfasser: Hernandez, Alejandra, Velasquez, Olga, Leonardi, Felice, Soto, Carlos, Rodriguez, Alexander, Lizaraso, Lina, Mosquera, Angela, Bohorquez, Jorge, Coronado, Alejandra, Espejo, Angela, Sierra, Rocio, Sanchez, Oscar F, Almeciga-Diaz, Carlos J, Barrera, Luis A
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container_issue 5
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container_title Journal of microbiology and biotechnology
container_volume 23
creator Hernandez, Alejandra
Velasquez, Olga
Leonardi, Felice
Soto, Carlos
Rodriguez, Alexander
Lizaraso, Lina
Mosquera, Angela
Bohorquez, Jorge
Coronado, Alejandra
Espejo, Angela
Sierra, Rocio
Sanchez, Oscar F
Almeciga-Diaz, Carlos J
Barrera, Luis A
description The production and characterization of an active recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) in Escherichia coli BL21(DE3) has been previously reported. In this study, the effect of the signal peptide (SP), inducer concentration, process scale, and operational mode (batch and semi-continuous) on GALNS production were evaluated. When native SP was presented, higher enzyme activity levels were observed in both soluble and inclusion bodies fractions, and its removal had a significant impact on enzyme activation. At shake scale, the optimal IPTG concentrations were 0.5 and 1.5 mM for the strains with and without SP, respectively, whereas at bench scale, the highest enzyme activities were observed with 1.5 mM IPTG for both strains. Noteworthy, enzyme activity in the culture media was only detected when SP was presented and the culture was carried out under semi-continuous mode. We showed for the first time that the mechanism that in prokaryotes recognizes the SP to mediate sulfatase activation can also recognize a eukaryotic SP, favoring the activation of the enzyme, and could also favor the secretion of the recombinant protein. These results offer significant information for scaling-up the production of human sulfatases in E. coli.
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subjects coli
E
GALNS
Morquio A
recombinant enzyme
signal peptide
sulfatase
title Effect of Culture Conditions and Signal Peptide on Production of Human Recombinant N-Acetylgalactosamine-6-Sulfate Sulfatase in Escherichia coli BL21
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