Author/Creator ORCID






Biomedical Science

Citation of Original Publication


Public Domain Mark 1.0


Solubility is a recurrent challenge when overexpressing non-native proteins in Escherichia coli (E. coli) for purification. N-utilizing substance A (NusA) is often used as a fusion tag to enhance the solubility of its partner protein. However, NusA is not always successful in rescuing solubility. We sought a bacterial homologue with stronger solubility enhancing properties and discovered that the NusA from a closely related species, Yersinia enterocolitica (Y. ent), was completely insoluble as a fusion partner when expressed in E. coli. To investigate the cause of the marked difference in solubility, we made chimeras between the sequences of the two homologues and tested the effects both with and without the burden of a fusion partner. We narrowed the region of interest down to the portion of the N-terminal domain between amino acids 62 and 121. We then used site directed mutagenesis to investigate the seven amino acid substitutions that were present in that region of the Y. ent NusA. We identified amino acids 63 and 64 as the pair that were responsible for the difference in solubility. Replacing alanine in position 64 of the Y. ent NusA with valine results in a protein that is much more soluble than the wild type Y. ent, but is not as soluble as the V63L/A34V double mutant previously mentioned. We then made random mutants at those locations to see if we could improve the solubility. We were unable to significantly improve the solubility enhancement capabilities of NusA beyond that of the native E. coli protein, but we discovered that an isoleucine at position 64 in the E. coli sequence was equally as soluble as the wildtype E. coli protein. We will discuss how the characteristics of the amino acids in both sequences and their location within those sequences may affect solubility.