Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12

Edwin David Morales-Álvarez; Claudia Marcela Rivera-Hoyos; Angélica María Baena-Moncada; Patricia Landázuri; Raúl A. Poutou-Piñales; Homero Sáenz-Suárez; Luis A. Barrera; Olga Y. Echeverri-Peña

doi:10.1007/s12275-013-2416-2

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Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12: Edwin David Morales-Álvarez ^1,2, Claudia Marcela Rivera-Hoyos ^1,3, Angélica María Baena-Moncada ¹, Patricia Landázuri ¹, Raúl A. Poutou-Piñales ³, Homero Sáenz-Suárez ⁴, Luis A. Barrera ⁵, Olga Y. Echeverri-Peña ⁵; Journal of Microbiology 2013;51(2):213-221.
DOI: https://doi.org/10.1007/s12275-013-2416-2
Published online: April 27, 2013

¹Grupo de Investigación en Enfermedades Cardiovasculares y Metabólicas (GECAVYME), Facultad de Ciencias de la Salud, Universidad del Quindío, Armenia-Quindío, Colombia, ²Departamento de Química, Grupo de Investigación en Genética, Biodiversidad y Manejo de Ecosistemas (GEBIOME), Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales-Caldas, Colombia, ³Laboratorio de Biotecnología Molecular. Grupo de Biotecnología Ambiental e Industrial (GBAI), Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, D.C. Colombia, ⁴Unidad de Biología Celular y Microscopía, Decanato de Ciencias de la Salud, Universidad Centroccidental Lisandro Alvarado, Barquisimeto, Venezuela, ⁵Instituto de Errores Innatos del Metabolismo (IEIM), Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, D.C. Colombia

Corresponding author: Patricia Landázuri , Tel: (57-6) 746 01 58,

Received: 8 August 2012 • Accepted: 31 October 2012

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Abstract

The sulfatase family involves a group of enzymes with a large degree of similarity. Until now, sixteen human sulfatases have been identified, most of them found in lysosomes. Human deficiency of sulfatases generates various genetic disorders characterized by abnormal accumulation of sulfated intermediate compounds. Mucopolysaccharidosis type II is characterized by the deficiency of iduronate 2-sulfate sulfatase (IDS), causing the lysosomal accumulation of heparan and dermatan sulfates. Currently, there are several cases of genetic diseases treated with enzyme replacement therapy, which have generated a great interest in the development of systems for recombinant protein expression. In this work we expressed the human recombinant IDS-Like enzyme (hrIDS-Like) in Escherichia coli DH5α. The enzyme concentration revealed by ELISA varied from 78.13 to 94.35 ng/ml and the specific activity varied from 34.20 to 25.97 nmol/h/mg. Western blotting done after affinity chromatography purification showed a single band of approximately 40 kDa, which was recognized by an IgY polyclonal antibody that was developed against the specific peptide of the native protein. Our 100 ml-shake-flask assays allowed us to improve the enzyme activity seven fold, compared to the E. coli JM109/pUC13-hrIDS-Like system. Additionally, the results obtained in the present study were equal to those obtained with the Pichia pastoris GS1115/ pPIC-9-hrIDS-Like system (3 L bioreactor scale). The system used in this work (E. coli DH5α/pGEX-3X-hrIDS-Like) emerges as a strategy for improving protein expression and purification, aimed at recombinant protein chemical characterization, future laboratory assays for enzyme replacement therapy, and as new evidence of active putative sulfatase production in E. coli.

Keywords: iduronate 2-sulfate sulfatase;E. coli DH5α;recombinant protein expression;affinity chromatography

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Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12

J. Microbiol. 2013;51(2):213-221. Published online April 27, 2013

DOI: https://doi.org/10.1007/s12275-013-2416-2

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