title: Effect of imidazole on the solubility of a his-tagged antibody fragment.
abstract: A number of studies have demonstrated the limited solubility of single-chain Fv antibody fragments and its improvement by genetic engineering. This limits the stability of recombinant protein upon storage and the efficiency of chemical modification. The RAFT3 scFv used in the present work is specific for melanoma-associated proteoglycan and an attractive candidate for clinical radioimaging studies because of its unusual radiolabelling properties. However, when expressed with a c-terminal his(6) IMAC purification tag, the recombinant protein starts to precipitate after column elution and dialysis against PBS and reaches a concentration of soluble protein of approximately 150 microg/mL within a few days upon storage at 4 degrees C. We tested several commonly used buffer modifications (addition of detergents, high salt, amino acids) to improve the solubility and stability of the protein but without any major improvement. However, we found that, when the final dialysis step was omitted and the protein left in IMAC column elution buffer (PBS containing imidazole), it remained soluble. Furthermore, several months old and precipitated protein could be redissolved in this buffer without loss of antigen binding. This observation and the largely pH-independent nature of protein solubility suggest that neither salt bridges formed by the his(6) tail nor cross-linking of his(6) tails mediated by metal ions leached from the column during elution are responsible for the limited solubility of the protein in the absence of imidazole. The presence of imidazole did not interfere with radiolabelling and in vivo tumor targeting in a mouse model. The solubilizing and stabilizing effect of imidazole could be of use for his(6) tagged and poorly soluble recombinant proteins other than scFvs.
authors.affiliation: RAFT Institute of Plastic Surgery and Cancer Research Trust, Gray Cancer Institute, Mount Vernon Hospital, Northwood, Middlesex HA6 2RN, UK.
authors.forename: Stephen
authors.initials: S
authors.lastname: Hamilton
authors.forename: Joy
authors.initials: J
authors.lastname: Odili
authors.forename: Marc D
authors.initials: MD
authors.lastname: Pacifico
authors.forename: George D
authors.initials: GD
authors.lastname: Wilson
authors.forename: Jörg-M
authors.initials: JM
authors.lastname: Kupsch
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D000912:Antibodies, Neoplasm / Q000737:chemistry; D000941:Antigens / Q000737:chemistry; D000951:Antigens, Neoplasm / Q000737:chemistry; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology*; D007128:Immunoglobulin Fragments / Q000737:chemistry; D007135:Immunoglobulin Variable Region; D051379:Mice; D008807:Mice, Inbred BALB C; D009368:Neoplasm Transplantation; D011485:Protein Binding; D011509:Proteoglycans / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012492:Salts / Q000737:chemistry; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature
chemical_list: D000906:Antibodies; D000912:Antibodies, Neoplasm; D000941:Antigens; D000951:Antigens, Neoplasm; D004791:Enzyme Inhibitors; D007093:Imidazoles; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011509:Proteoglycans; D011994:Recombinant Proteins; D012492:Salts; C069150:melanoma-associated proteoglycan antigen, human; D012965:Sodium Chloride; C029899:imidazole