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bioRxiv · 10.1101/2024.10.14.618345

Further characterization and engineering of a 11-amino acid motif for enhancing recombinant protein expression

Bi, J.·Tiong, E.·Zhou, W.·Wong, F. T.
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Abstract

BackgroundRecombinant protein production in Escherichia coli (E. coli) is a widely used system in both academic and industrial research owing to its low cost and wide availability of genetic tools. Despite its advantages, this system still struggles with soluble expression of recombinant proteins. To address this, various solubility-enhancing and yield-improving methods such as the addition of fusion tags have been developed. However, traditional tags such as small ubiquitin-related modifier (SUMO) and Glutathione S-transferase (GST) can interfere with protein folding or require removal post-translation, which adds complexity and cost to production. To circumvent these issues, smaller solubility tags (<10 kDa) are preferred. This study specifically focuses on an 11-amino acid solubility-enhancing tag (NT11) derived from the N-terminal domain of a duplicated carbonic anhydrase from Dunaliella species. ResultsA comprehensive analysis was performed to improve the characteristics of the 11-amino acid tag. By investigating the alanine-scan library of NT11, we increased its activity and identified key residues for further development. Screening with the alanine mutant library consistently led to at least a two-fold improvement in protein yield for three different proteins. We also discovered that the NT11 tag is not limited to the N-terminal position and can function at either the N- or C-terminal of the protein, providing flexibility in designing protein expression constructs. With these new insights, we have successfully doubled the recombinant protein yields of valuable growth factors, such as fibroblast growth factor 2 (FGF2) and an originally low-yielding human epidermal growth factor (hEGF), in E. coli. ConclusionThe further characterisation and development of the NT11 tag have provided valuable insights into the optimization process for such small tags and expanded our understanding of its potential applications. The ability of NT11 tag to be positioned at different locations within the protein construct without compromising its effectiveness to enhance recombinant protein yields, makes it a valuable tool across a diverse range of proteins. Collectively, these findings have the potential to simplify and enhance the efficiency of recombinant protein production.

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Bi, J., Tiong, E., Zhou, W., Wong, F. T.. 2024-10-16. Further characterization and engineering of a 11-amino acid motif for enhancing recombinant protein expression. https://doi.org/10.1101/2024.10.14.618345

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