High yield production of a mutant Nippostrongylus brasiliensis acetylcholinesterase in Pichia pastoris and its purification

Sven Richter, Jens Nieveler, Holger Schulze, Till T Bachmann, Rolf D Schmid

Research output: Contribution to journalArticlepeer-review

Abstract

The mutant M301A of the acetylcholinesterase B from Nippostrongylus brasiliensis (NbAChE) was produced in a high-cell-density fermentation of a recombinant methylotrophic yeast Pichia pastoris. Dissolved oxygen (DO) spikes were used as an indicator for feeding the carbon source. Wet cell weight (WCW) reached after 8 days a maximum value of 316 g/L and the OD600 at this time was 280. The acetylcholinesterase activity increased up to 6,600 U/mL corresponding to an expression rate of 2 g of NbAChE per liter supernatant. The specific activity of the mutant NbAChE was determined after purification as 3,300 U/mg. Active site titration with chlorpyrifos, a strong AChE inhibitor, yielded in a specific activity of 3,400 U/mg. The enzyme was secreted by Pichia pastoris. Therefore, it could be concentrated from culture broth by cross-flow-filtration (50 kDa cut-off membrane). It was further purified in one-step anion-exchange chromatography, using a XK 50/20 column filled with 125 mL Q Sepharose HP. Mutant NbAChE was purified 1.9-fold up to a purity of 97% and a yield of 87%. The isolated enzyme was nearly homogenous, as seen on the silver stained SDS-PAGE as well as by a single peak after gel filtration. This extraordinary high expression rate and the ease of purification is an important prerequisite for their practical application, for example in biosensors for the detection of neurotoxic insecticides.
Original languageEnglish
Pages (from-to)1017-22
Number of pages6
JournalBiotechnology and Bioengineering
Volume93
Issue number5
DOIs
Publication statusPublished - 2006

Keywords

  • Acetylcholinesterase
  • Animals
  • Biomass
  • Catalytic Domain
  • Chlorpyrifos
  • Cholinesterase Inhibitors
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Electrophoresis, Polyacrylamide Gel
  • Fermentation
  • Methanol
  • Mutation
  • Nippostrongylus
  • Oxygen
  • Pichia
  • Recombinant Proteins
  • Time Factors
  • Transfection

Fingerprint Dive into the research topics of 'High yield production of a mutant Nippostrongylus brasiliensis acetylcholinesterase in Pichia pastoris and its purification'. Together they form a unique fingerprint.

Cite this