Pilot-scale extraction of an intracellular recombinant cutinase from E. coli cell homogenate using a thermoseparating aqueous two-phase system.
A thermoseparating aqueous two-phase system for extraction of a recombinant cutinase fusion protein from Escherichia coli homogenate has been scaled up to pilot scale. The target protein ZZ-cutinase-(WP)(4) was produced in a fed batch process at 500 l to a concentration of 12% of the total protein and at a cell concentration of 19.7 g l(-1). After harvest and high-pressure homogenisation a first extraction step was performed in an EO(50)PO(50) (50% (w/w) ethylene oxide and 50% (w/w) propylene oxide) thermopolymer/amylopectin rich Waxy barley starch system. The (WP)(4) tag was used for enhanced target protein partitioning to the EO(50)PO(50) phase while the cell debris was collected in the starch phase. A second extraction step followed where the recovered EO(50)PO(50) phase from the first step was supplemented with a non-ionic detergent (C(12-18)EO(5)) and heated to the cloud point (CP) temperature (45 degrees C). One polymer-rich liquid phase and one almost pure aqueous phase were formed. The target protein could be obtained in a water phase after the thermal phase separation at a total recovery over the extraction steps of 71% and a purification factor of 2.5. We were able to demonstrate that a disk-stack centrifugal separator could be adapted for rapid separation of both primary and thermoseparated phase systems.
Kepka
Cecilia
C
Department of Biochemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden.
Collet
Eric
E
Persson
Josefine
J
Ståhl
Ake
A
Lagerstedt
Torgny
T
Tjerneld
Folke
F
Veide
Andres
A
D001671:Biochemistry / Q000379:methods*; D019149:Bioreactors; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D010865:Pilot Projects; D011108:Polymers; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012492:Salts; D013696:Temperature
D011108:Polymers; D011993:Recombinant Fusion Proteins; D012492:Salts; D002265:Carboxylic Ester Hydrolases; C019952:cutinase