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High-level expression and characterization of Galactomyces geotrichum (BT107) lipase I in Pichia pastoris.

Authors
  • Fernández, Layla1
  • Pérez-Victoria, Ignacio
  • Zafra, Alberto
  • Benítez, Pedro L
  • Morales, Juan C
  • Velasco, Javier
  • Adrio, José L
  • 1 Department of Biotechnology, Puleva Biotech, Camino de Purchil, 66, 18004-Granada, Spain. , (Spain)
Type
Published Article
Journal
Protein expression and purification
Publication Date
Oct 01, 2006
Volume
49
Issue
2
Pages
256–264
Identifiers
PMID: 16884921
Source
Medline
License
Unknown

Abstract

The mature lipI gene, encoding the lipase I from Galactomyces geotrichum BT107, was obtained by PCR from genomic DNA, sequenced and cloned into a Pichia pastoris expression vector. Clones containing multiple copies of lipI integrated in their genome were analyzed to achieve high-level expression of the recombinant lipase I. One strain with four or more copies of the expression cassette was able to produce more than 200mg/L of extracellular heterologous protein. The lipase I was partially purified using anion exchange chromatography and its activity on monounsaturated (triolein) and polyunsaturated (triEPA) triglycerides was analyzed by a novel HPLC-MS assay.

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