title: Coupling of chemical extraction and expanded-bed adsorption for simplified inclusion-body processing: optimization using surface plasmon resonance.
abstract: Integration of the chemical extraction of recombinant inclusion-body protein from Escherichia coli, and its recovery by metal-affinity expanded-bed adsorption (IMAC-EBA) under denaturing conditions, was investigated. The viral coat protein L1 with a hexa-histidine tag was expressed in Escherichia coli HMS174(DE3) as a model protein. Interference of released host DNA with adsorbent fluidization in the EBA step was solved by selective precipitation using spermine and low-speed centrifugation. However, the capacity and selectivity of the adsorbent for L1 remained lower than anticipated. The binding of L1 to immobilized Ni(2+) was therefore studied in detail using surface plasmon resonance (SPR). The Tris buffer and ethylene-diamine tetraacetic acid (EDTA) used in the extraction mixture were found to interfere significantly with the L1-Ni(2+) interaction. The SPR studies suggest that L1 binding could be improved by replacing the Tris buffer with HEPES and by adding CaCl(2) to inactivate the EDTA. The modified chemical extraction conditions resulted in effective L1 extraction from cytoplasmic inclusion bodies, at high cell density (OD(600 )= 80) and without the use of reducing agent, into a medium optimized for subsequent IMAC recovery. The modified buffer conditions resulted in an improved binding capacity and a good L1 purification factor (12.7) and recovery yield (71%). This work demonstrates that it is possible to reduce the complexity and hence the cost associated with traditional processes used to prepare purified denatured protein, ready for refolding, from cytoplasmic inclusion bodies.
authors.affiliation: Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, UK.
authors.forename: Woo-Seok
authors.initials: WS
authors.lastname: Choe
authors.forename: Robert H
authors.initials: RH
authors.lastname: Clemmitt
authors.forename: Howard A
authors.initials: HA
authors.lastname: Chase
authors.forename: Anton P J
authors.initials: AP
authors.lastname: Middelberg
mesh_terms: D000327:Adsorption; D002213:Capsid / Q000378:metabolism; D036022:Capsid Proteins; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000821:virology; D006639:Histidine / Q000737:chemistry*; D002479:Inclusion Bodies / Q000737:chemistry*; D007700:Kinetics; D009532:Nickel / Q000737:chemistry; D009856:Oncogene Proteins, Viral; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D020349:Surface Plasmon Resonance / Q000379:methods*; D014764:Viral Proteins
chemical_list: D036022:Capsid Proteins; D009856:Oncogene Proteins, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D006639:Histidine; C091560:L1 protein, Human papillomavirus type 6; D009532:Nickel