title: Production of enzymatically active ketosteroid isomerase following insoluble expression in Escherichia coli.
abstract: Enzymatically active Delta(5)-3-ketosteroid isomerase (KSI) protein with a C-terminus his(6)-tag was produced following insoluble expression using Escherichia coli. A simple, integrated process was used to extract and purify the target protein. Chemical extraction was shown to be as effective as homogenization at releasing the inclusion body proteins from the bacterial cells, with complete release taking less than 20 min. An expanded bed adsorption (EBA) column utilizing immobilized metal affinity chromatography (IMAC) was then used to purify the denatured KSI-(His(6)) protein directly from the chemical extract. This integrated process greatly simplifies the recovery and purification of inclusion body proteins by removing the need for mechanical cell disruption, repeated inclusion body centrifugation, and difficult clarification operations. The integrated chemical extraction and EBA process achieved a very high purity (99%) and recovery (89%) of the KSI-(His(6)), with efficient utilization of the adsorbent matrix (9.74 mg KSI-(His(6))/mL adsorbent). Following purification the protein was refolded by dilution to obtain the biologically active protein. Seventy-nine percent of the expressed KSI-(His(6)) protein was recovered as enzymatically active protein with the described extraction, purification, and refolding process. In addition to demonstrating the operation of this intensified inclusion body process, a plate-based concentration assay detecting KSI-(His(6)) is validated. The intensified process in this work requires minimal optimization for recovering novel his-tagged proteins, and further improves the economic advantage of E. coli as a host organism.
authors.affiliation: Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, United Kingdom.
authors.forename: Matthew H
authors.initials: MH
authors.lastname: Hutchinson
authors.forename: Giacomo
authors.initials: G
authors.lastname: Morreale
authors.forename: Anton P J
authors.initials: AP
authors.lastname: Middelberg
authors.forename: Howard A
authors.initials: HA
authors.lastname: Chase
mesh_terms: D000327:Adsorption; D005591:Chemical Fractionation / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D002479:Inclusion Bodies / Q000201:enzymology / Q000382:microbiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D013251:Steroid Isomerases / Q000096:biosynthesis / Q000302:isolation & purification*
chemical_list: D011994:Recombinant Proteins; D013251:Steroid Isomerases