Comparison of three commonly used fusion tags for the expression of nanobodies in the cytoplasm of Escherichia coli

Nanobodies (Nbs) are variable domains of the heavy chain of the heavy-chain antibodies (VHHs) found only in camelids and sharks. They have the same binding capability as full-length immunoglobulin G (IgG) but exhibit improved thermal stability and permeability, pointing to potential scientific, medical, and industrial applications. The aim of the present study was to identify an alternative method for more efficient production of Nbs in the Escherichia coli-based expression system. Several fusion tags, including maltose-binding protein (MBP), small ubiquitinrelated modifier (SUMO) and hexahistidine (6 £ His), were examined to determine the optimal tag for the expression and purification of VHHs. In general, the SUMO fusion tag could improve the solubility, yield and antigen-binding activity of the VHHs, while the other two tags either decreased the antigen-binding activity (MBP) or decreased the yield (6 £ His) of the VHHs. What is more, the SUMO tag was easily removed by SUMO protease 1 to release the native VHHs. These advantages indicated that SUMO fusion could be considered an alternative strategy to express VHHs in the cytoplasm of E. coli.