title: Purification of recombinant chimeric B72.3 Fab' and F(ab')2 using streptococcal protein G.
abstract: Streptococcal protein G has been used extensively for the purification of antibodies using the interaction of the Fc region with protein G. Many antibodies also interact with protein G through a low-affinity binding site for the Fab region. The exploitation of this low-affinity interaction for the purification of Fab' fragments is described here. Chimeric mouse-human B72.3 Fab' and F(ab')2 fragments were expressed by CHO cells and purified from CHO cell supernatant using protein G-Sepharose. Since chimeric B72.3 Fab' bound weakly to the protein G-Sepharose it could be separated from F(ab')2 and eluted with a pH 7 wash whereas B72.3 F(ab')2 required elution at pH 2. Both Fab' and F(ab')2 were recovered with full immunoreactivity and could be further purified using gel-filtration chromatography to greater than 99% purity. This method allows the simple purification of directly expressed Fab' or F(ab')2 fragments from CHO cell supernatant.
authors.affiliation: Celltech Ltd., Slough, United Kingdom.
authors.forename: K A
authors.initials: KA
authors.lastname: Proudfoot
authors.forename: C
authors.initials: C
authors.lastname: Torrance
authors.forename: A D
authors.initials: AD
authors.lastname: Lawson
authors.forename: D J
authors.initials: DJ
authors.lastname: King
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D016466:CHO Cells; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D007147:Immunoglobulin Light Chains / Q000235:genetics; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D013291:Streptococcus / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007140:Immunoglobulin Fab Fragments; D007147:Immunoglobulin Light Chains; D011993:Recombinant Fusion Proteins