A refolding tag for purifying bioactive recombinant proteins from E. coli inclusion bodies

Recovery of bioactive proteins from insoluble inclusion bodies (IB) is a critical challenge when purifying recombinant proteins expressed in E. coli. One practical solution is to enhance the renaturability or refolding capability of the target protein after the insoluble proteins are solubilized with denaturants such as urea. While some methods, such as using chemical additives and manipulating the physical environment, have been reported to assist in protein refolding, their effectiveness remains limited. This work evaluated the potential of a highly conserved domain found in five biotin-dependent carboxylases as a fusion tag for purifying IB proteins. We found that this tag, comprised of 67 amino acids (named P67) as eight anti-parallel β-sheets that collectively form a flattened barrel structure, significantly improved the refolding process of its IB protein partner. The yield of bioactive proteins fused with this tag recovered from the dialysis stage was markedly higher than untagged ones. We further proved that this effect relies on the structural integrity of the tag's spatial conformations. Under natural conditions, soluble proteins can be purified using the P67 tag via the biotin/streptavidin affinity system. In conclusion, this tag offers a promising methodology for purifying both insoluble and soluble recombinant proteins.