Design and assessment of an active anti-epidermal growth factor receptor (EGFR) single chain variable fragment (ScFv) with improved solubility.

ScFv is emerging as a therapeutic alternative to the full-length monoclonal antibodies due to its small size and low production cost, but its low solubility remains a limiting factor toward wider use. Here, we increased the solubility of an Anti-epidermal growth factor receptor ScFv (Anti-EGFR ScFv) by attaching, a short 12-residue solubility enhancing peptide (SEP) tag at its C terminus. We first estimated the solubility increase by running 500-ns Brownian dynamics (BD) simulations. We then experimentally evaluated the predictions by producing recombinant Anti-EGFR ScFv with and without a SEP tag (called C9R) in E. coli. At 20 °C, ∼85% of Anti-EGFR ScFv-C9R expressed in the soluble fraction, whereas all of the Anti-EGFR ScFv remained in the insoluble fraction. The total yield of Anti-EGFR ScFv-C9R was 17.15 mg which was ∼3 times higher than that of Anti-EGFR ScFv refolded from the insoluble fraction. Static and dynamic light scattering demonstrated the higher solubility of the purified Anti-EGFR ScFv-C9R, and Circular Dichroism (CD) indicated its high thermal stability, whereas the untagged protein aggregated at 37 °C and pH 6. Finally, the binding activity of Anti-EGFR ScFv-C9R to EGFR was confirmed by surface plasmon resonance (SPR). Altogether, these results illustrate the improved biophysical and biochemical characteristics of Anti-EGFR ScFv-C9R and emphasize the potentials of SEP-tags for enhancing the solubility of aggregation-prone antibody fragments.

A SEP tag enhances the expression, solubility and yield of recombinant TEV protease without altering its activity.

Tobacco Etch Virus (TEV) protease is used in the purification of recombinant proteins, but its usage is often hampered by solubility issues. Here, we report a short, 12-residue solubility enhancing peptide (SEP) tag attached at the C-terminus of TEV (TEV-C9R). We assessed the effects of the C9R tag on the biophysical and biochemical characteristics of TEV. The yield of HPLC purified TEV-C9R expressed in E. coli grown in 200 mL LB or TB media was between 10 and 13 mg, which was up to 6.5 times higher than the yield of the untagged TEV (untagged-TEV). TEV-C9R was active over a pH range of 5-8, which was wider than that of the commonly used thrombin, and it remained active upon incubation at 60 °C much longer than the untagged-TEV, which aggregated at this temperature. Static and dynamic light scattering demonstrated the higher solubility of purified TEV-C9R. Furthermore, the thermal unfolding of TEV-C9R, as assessed by circular dichroism at pH 4.7, was almost perfectly reversible, in contrast to that of untagged-TEV, which aggregated at high temperature. These results demonstrate the improved biophysical and biochemical characteristics of TEV-C9R originating from higher solubility and provide another example of how SEP tags can enhance enzyme solubility without altering its activity.