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 his6 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 μg/mL within a few days upon storage at 4°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 his6 tail nor cross-linking of his6 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 his6 tagged and poorly soluble recombinant proteins other than scFvs.
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