title: Antibody-mediated purification of co-expressed antigen-antibody complexes.
abstract: Immunoaffinity is an established chromatographic method for isolating macromolecules independently on the presence of specific tags while the tight interaction between antigen and antibody has been exploited to stabilize proteins during crystallization trials. Therefore, it seems reasonable to try to combine the two protocols, namely to co-express the target proteins together with their specific antibodies to obtain stable complexes suitable for direct purification and further analyses. Using the variable region of single domain llama antibodies, we showed that the co-expression of antigen-antibody pairs is feasible in both the periplasm and the cytoplasm of bacteria. Moreover, the complexes that were formed in vivo could be purified using a tag fused to the recombinant antibody and remained stable during gel-filtration. The co-expression and co-purification strategy significantly increased the final protein yields promoting the accumulation of functional intrabodies. The described method may offer a suitable alternative for the purification of proteins intended for crystallization trials and it may also be used as a general purification protocol for both antigens and recombinant antibodies.
authors.affiliation: Protein Chemistry Unit, Cogentech - IFOM-IEO Campus for Oncogenomics, via Adamello 16, 20139 Milano, Italy.
authors.forename: Sergio
authors.initials: S
authors.lastname: Bossi
authors.forename: Benedetta
authors.initials: B
authors.lastname: Ferranti
authors.forename: Chiara
authors.initials: C
authors.lastname: Martinelli
authors.forename: Paola
authors.initials: P
authors.lastname: Capasso
authors.forename: Ario
authors.initials: A
authors.lastname: de Marco
mesh_terms: D000906:Antibodies / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D001419:Bacteria / Q000166:cytology / Q000235:genetics; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000906:Antibodies; D000936:Antigen-Antibody Complex; D011994:Recombinant Proteins