title: Effect of culture conditions and signal peptide on production of human recombinant N-acetylgalactosamine-6-sulfate sulfatase in Escherichia coli BL21.
abstract: 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.
authors: Department of Chemical Engineering, Universidad de Los Andes, Bogota, Colombia.
authors: Alejandra
authors: A
authors: Hernández
authors: Olga
authors: O
authors: Velásquez
authors: Felice
authors: F
authors: Leonardi
authors: Carlos
authors: C
authors: Soto
authors: Alexander
authors: A
authors: Rodríguez
authors: Lina
authors: L
authors: Lizaraso
authors: Ángela
authors: Á
authors: Mosquera
authors: Jorge
authors: J
authors: Bohórquez
authors: Alejandra
authors: A
authors: Coronado
authors: Ángela
authors: Á
authors: Espejo
authors: Rocio
authors: R
authors: Sierra
authors: Oscar F
authors: OF
authors: Sánchez
authors: Carlos J
authors: CJ
authors: Alméciga-Díaz
authors: Luis A
authors: LA
authors: Barrera
mesh_terms: D002811:Chondroitinsulfatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015202:Protein Engineering; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D003470:Culture Media; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D002811:Chondroitinsulfatases; C102295:GALNS protein, human