title: Comparative evaluation of two purification methods of anti-CD19-c-myc-His6-Cys scFv.
abstract: Different chromatographic methods have been used to purify bacterially expressed single chain antibodies in soluble or insoluble form. Here, we compared two methods for purification of anti-CD19-c-myc-His6-Cys scFv expressed in Escherichia coli as soluble protein. The protein-L-agarose purification method is a one step purification method that yielded significant amounts of pure protein compared to the two-step Ni-NTA-agarose plus Resource 15S purification method. However, the protein-L purification method exhibited an additional lower molecular weight protein contaminant. Based on results from in vitro gel digestion, mass spectrometry and database search results, we confirmed that the lower molecular weight protein contaminant, which could not be purified by Ni-NTA-agarose and 15S column method, is a degraded product of the full length scFv construct.
authors.affiliation: Department of Pharmacology, University of Alberta, Edmonton, AB, Canada T6G 2H7.
authors.forename: Dipankar
authors.initials: D
authors.lastname: Das
authors.forename: Theresa M
authors.initials: TM
authors.lastname: Allen
authors.forename: Mavanur R
authors.initials: MR
authors.lastname: Suresh
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D018941:Antigens, CD19 / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D015153:Blotting, Western; D002614:Chelating Agents / Q000494:pharmacology; D002846:Chromatography, Affinity / Q000379:methods*; D002847:Chromatography, Agarose / Q000379:methods*; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D005434:Flow Cytometry; D006639:Histidine / Q000737:chemistry; D006825:Hybridomas; D006863:Hydrogen-Ion Concentration; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D007475:Ion Exchange Resins / Q000494:pharmacology; D013058:Mass Spectrometry; D008970:Molecular Weight; D009532:Nickel / Q000737:chemistry; D009571:Nitrilotriacetic Acid / Q000494:pharmacology; D010957:Plasmids; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry; D012995:Solubility; D013696:Temperature; D013862:Thiogalactosides / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D018941:Antigens, CD19; D002614:Chelating Agents; D007135:Immunoglobulin Variable Region; D007475:Ion Exchange Resins; D016271:Proto-Oncogene Proteins c-myc; D013862:Thiogalactosides; D006639:Histidine; D009532:Nickel; D003545:Cysteine; D009571:Nitrilotriacetic Acid