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A systematic analysis of affinity tags in the haloarchaeal expression system, Haloferax volcanii for protein purification

Karan, Ram; Renn, Dominik; Allers, Thorsten; Rueping, Magnus

A systematic analysis of affinity tags in the haloarchaeal expression system, Haloferax volcanii for protein purification Thumbnail


Authors

Ram Karan

Dominik Renn

Magnus Rueping



Abstract

Extremophilic proteins are valuable in various fields, but their expression can be challenging in traditional hosts like Escherichia coli due to misfolding and aggregation. Haloferax volcanii (H. volcanii), a halophilic expression system, offers a solution. This study examined cleavable and non-cleavable purification tags at both the N- and C-termini when fused with the superfolder green fluorescent protein (sfGFP) in H. volcanii. Our findings reveal that an N-terminal 8xHis-tag or Strep-tag®II significantly enhances protein production, purity, and yield in H. volcanii. Further experiments with mCherry and halophilic alcohol dehydrogenase (ADH) showed improved expression and purification yields when the 8xHis-tag or Strep-tag®II was positioned at the C-terminus for mCherry and at the N-terminus for ADH. Co-positioning 8xHis-tag and Twin-Strep-tag® at the N-terminus of sfGFP, mCherry, and ADH yielded significantly enhanced results. These findings highlight the importance of thoughtful purification tag design and selection in H. volcanii, providing valuable insights for improving protein production and purification with the potential to advance biotechnological applications.

Citation

Karan, R., Renn, D., Allers, T., & Rueping, M. (2024). A systematic analysis of affinity tags in the haloarchaeal expression system, Haloferax volcanii for protein purification. Frontiers in Microbiology, 15, Article 1403623. https://doi.org/10.3389/fmicb.2024.1403623

Journal Article Type Article
Acceptance Date May 15, 2024
Online Publication Date May 30, 2024
Publication Date May 30, 2024
Deposit Date Aug 1, 2024
Publicly Available Date Aug 19, 2024
Journal Frontiers in Microbiology
Electronic ISSN 1664-302X
Publisher Frontiers Media
Peer Reviewed Peer Reviewed
Volume 15
Article Number 1403623
DOI https://doi.org/10.3389/fmicb.2024.1403623
Keywords halophiles, His-tag, purification tags, Haloferax volcanii, extremophiles, bioengineering, Strep-tag®II, archaea
Public URL https://nottingham-repository.worktribe.com/output/36281409
Publisher URL https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2024.1403623/full

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Publisher Licence URL
https://creativecommons.org/licenses/by/4.0/

Copyright Statement
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.





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