Recovery of intracellular recombinant proteins from the yeast Pichia pastoris by cell permeabilization.
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.
Shepard
Scot R
SR
Diosynth RTP, Inc, 3000 Weston Parkway, Cary, NC 27513, USA. scot.shepard@diosynth-rtp.com
Stone
Charmaine
C
Cook
Susan
S
Bouvier
Anne
A
Boyd
Gregory
G
Weatherly
Gresham
G
Lydiard
Debra
D
Schrimsher
Jeffrey
J
D001905:Botulinum Toxins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D019274:Botulinum Toxins, Type A / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D002463:Cell Membrane Permeability / Q000187:drug effects*; D004795:Enzyme Stability; D010843:Pichia / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D011312:Pressure; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D011994:Recombinant Proteins; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A