Simultaneous clarification of Escherichia coli culture and purification of extracellularly produced penicillin G acylase using tangential flow filtration and anion-exchange membrane chromatography (TFF-AEMC).
Downstream purification often represents the most cost-intensive step in the manufacturing of recombinant proteins since conventional purification processes are lengthy, technically complicated, and time-consuming. To address this issue, herein we demonstrated the simultaneous clarification and purification of the extracellularly produced recombinant protein by Escherichia coli using an integrated system of tangential flow filtration and anion exchange membrane chromatography (TFF-AEMC). After cultivation in a bench-top bioreactor with 1L working volume using the developed host/vector system for high-level expression and effective secretion of recombinant penicillin G acylase (PAC), the whole culture broth was applied directly to the established system. One-step purification of recombinant PAC was achieved based on the dual nature of membrane chromatography (i.e. microfiltration-sized pores and anion-exchange chemistry) and cross-flow operations. Most contaminant proteins in the extracellular medium were captured by the anion-exchange membrane and cells remained in the retentate, whereas extracellular PAC was purified and collected in the filtrate. The batch time for both cultivation and purification was less than 24h and recombinant PAC with high purity (19 U/mg), yield (72% recovery), and productivity (41 mg of purified PAC per liter of culture) was obtained. Due to the nature of the non-selective protein secretion system and the versatility of ion-exchange membrane chromatography, the developed system can be widely applied for effective production and purification of recombinant proteins.
Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada.
Valerie
V
Orr
Jeno
J
Scharer
Murray
M
Moo-Young
C Howie
CH
Honeyman
Drew
D
Fenner
Lisa
L
Crossley
Shing-Yi
SY
Suen
C Perry
CP
Chou
D000838:Anions / Q000737:chemistry; D019149:Bioreactors; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D010399:Penicillin Amidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014462:Ultrafiltration / Q000379:methods*
D000838:Anions; D011994:Recombinant Proteins; D010399:Penicillin Amidase