Journal of Biotechnology 2002-10-23

Recovery of intracellular recombinant proteins from the yeast Pichia pastoris by cell permeabilization.

Scot R Shepard, Charmaine Stone, Susan Cook, Anne Bouvier, Gregory Boyd, Gresham Weatherly, Debra Lydiard, Jeffrey Schrimsher

Index: J. Biotechnol. 99(2) , 149-60, (2002)

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Abstract

A cell permeabilization method for the release of intracellular proteins from microbial cells was developed. The method was applied to the recovery of recombinant botulinum neurotoxin fragments, expressed intracellularly in the yeast Pichia pastoris, by suspending the cells in an aqueous solution containing N,N-dimethyltetradecylamine. For the botulinum neurotoxin serotype B C-terminal heavy chain fragment, 1.8 mg g(-1) biomass were recovered. For the botulinum neurotoxin serotype A C-terminal heavy chain fragment, 3.7 mg g(-1) biomass were recovered. The concentration of recombinant protein in the cell extracts remained stable for up to 48 and 24 h for the serotype B and serotype A fragments, respectively. The permeabilization method was compared with high-pressure homogenization; the permeabilization method proved to be both more selective and more efficient.Copyright 2002 Elsevier Science B.V.