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KMID : 0545120130230050689
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Journal of Microbiology and Biotechnology
2013 Volume.23 No. 5 p.689 ~ p.698
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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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Alejandra Hernandez
Olga Velasquez
Felice Leonardi
Carlos Soto
Alexander Rodriguez
Lina Lizaraso
Angela Mosquera
Jorge Bohorquez
Alejandra Coronado
Angela Espejo
Rocio Sierra
Oscar F. Sanchez
Carlos J. Almeciga-Diaz
Luis A. Barrera
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Abstract
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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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KEYWORD
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Morquio A, GALNS, Signal Peptide, Sulfatase, Recombinant enzyme, E. coli
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