AUTHOR=Karan Ram , Renn Dominik , Allers Thorsten , Rueping Magnus 

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

JOURNAL=Frontiers in Microbiology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2024.1403623

DOI=10.3389/fmicb.2024.1403623

ISSN=1664-302X

ABSTRACT=<p>Extremophilic proteins are valuable in various fields, but their expression can be challenging in traditional hosts like <italic>Escherichia coli</italic> due to misfolding and aggregation. <italic>Haloferax volcanii</italic> (<italic>H. volcanii</italic>), 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 <italic>H. volcanii</italic>. Our findings reveal that an N-terminal 8xHis-tag or Strep-tag<sup>®</sup>II significantly enhances protein production, purity, and yield in <italic>H. volcanii</italic>. Further experiments with mCherry and halophilic alcohol dehydrogenase (ADH) showed improved expression and purification yields when the 8xHis-tag or Strep-tag<sup>®</sup>II was positioned at the C-terminus for mCherry and at the N-terminus for ADH. Co-positioning 8xHis-tag and Twin-Strep-tag<sup>®</sup> 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 <italic>H. volcanii</italic>, providing valuable insights for improving protein production and purification with the potential to advance biotechnological applications.</p>