Expression of recombinant galactose oxidase by Pichia pastoris

Mei M. Whittaker, James Whittaker

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

Galactose oxidase catalyzes the oxidation of a variety of primary alcohols, producing hydrogen peroxide as a product. Among hexose sugars, the enzyme exhibits a high degree of specificity for the C6-hydroxyl of galactose and its derivatives, underlying a number of important bioanalytical applications. Galactose oxidase cDNA has been cloned for expression in Pichia pastoris both as the full-length native sequence and as a fusion with the glucoamylase signal peptide. Expression of the full-length native sequence results in a mixture of partly processed and mature galactose oxidase. In contrast, the fusion construct directs efficient secretion of correctly processed galactose oxidase in high-density, methanol-induced fermentation. Culture conditions (including induction temperature and pH) have been optimized to improve the quality and yield (500 mg/L) of recombinant enzyme. Lowering the temperature from 30 to 25°C during the methanol induction phase results in a fourfold increase in yield. A simple two-step purification and one-step activation produce highly active galactose oxidase suitable for a wide range of biomedical and bioanalytical applications. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)105-111
Number of pages7
JournalProtein Expression and Purification
Volume20
Issue number1
DOIs
StatePublished - 2000

Fingerprint

Galactose Oxidase
Pichia
Methanol
Fusion reactions
Glucan 1,4-alpha-Glucosidase
Temperature
Hexoses
Enzymes
Protein Sorting Signals
Galactose
Sugars
Hydroxyl Radical
Fermentation
Hydrogen Peroxide
Purification
Complementary DNA
Chemical activation
Alcohols
Derivatives
Oxidation

Keywords

  • Galactose oxidase
  • Pichia pastoris

ASJC Scopus subject areas

  • Biochemistry

Cite this

Expression of recombinant galactose oxidase by Pichia pastoris. / Whittaker, Mei M.; Whittaker, James.

In: Protein Expression and Purification, Vol. 20, No. 1, 2000, p. 105-111.

Research output: Contribution to journalArticle

Whittaker, Mei M. ; Whittaker, James. / Expression of recombinant galactose oxidase by Pichia pastoris. In: Protein Expression and Purification. 2000 ; Vol. 20, No. 1. pp. 105-111.
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