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Cloning and
shake flask expression of hrIDS-Like in Pichia
pastoris
Patricia Landázuri1,2, Raúl A. Poutou-Piñales1,3,
Jovanna Acero-Godoy1, Henry A. Córdoba-Ruiz
1, Olga Y. Echeverri-Peña 1, Homero Sáenz1,
Julio M. Delgado1,5 and Luis A.
Barrera-Avellaneda1*
1Instituto
de Errores Innatos del Metabolismo, Pontificia Universidad
Javeriana. Bogotá D. C., Colombia.
2Laboratorio
de Investigaciones Biomédicas. Facultad de las Ciencias de
la Salud Universidad del Quindío, Armenia, Colombia.
3Laboratorio
de Biotecnología Aplicada, Grupo de Biotecnología Ambiental
e Industrial, Depto. Microbiología, Facultad de Ciencias,
Pontificia Universidad Javeriana, Bogotá, D. C., Colombia.
4Unidad
de Biologia Celular y Microscopía, Decanato de Ciencias de
la Salud, Universidad Centroccidental Lisandro Alvarado,
Barquisimeto, Venezuela.
5Biotechnova,
Bogotá, D. C., Colombia.
*Corresponding author. E-mail:
abarrera@javeriana.edu.co.
Fax:
(571) 338-4548.
Accepted 8 May, 2009 |
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The human Iduronate-2-sulfate sulfatase (hIDS-Like)
was cloned into the methylotrophic yeast Pichia pastoris
under the control of alcohol oxidase promoter (AOX1)
and the
α-mating factor signal peptide (a-factor).
Six clones were identified by PCR. Using clone
IDS28, the enzyme was secreted into the culture
medium, yielding a protein with an activity of 4.213
nmol.h-1.mg of total protein-1 at 72
h, in 0.5% v/v methanol. Several bands were revealed by
western-blot, indicating that a P. pastoris
processing was slightly different than in mammalian cells.
Key
words:
Iduronate-2-sulfate sulfatase, MPS II,
Pichia
pastoris,
human
recombinant protein, Hunter syndrome. |
