Year: 2025
Type: article
Abstract: Recombinant protein production in Escherichia coli is a fundamental aspect of biotechnology. Fusion tags are commonly used to enhance solubility and facilitate purification. However, these tags can lead to challenges such as low yields, complicated purification processes, and the necessity for tag removal, especially when dealing with peptides. This study introduces a novel fusion tag called SmallTalk, a truncated version of the small metal‐binding protein SmbP. Weighing in at 5 kDa, SmallTalk includes two of the four α‐helices found in SmbP. It retains the ability to bind Ni(II) ions, which enables purification through IMAC. In this work, we assessed the efficiency of SmallTalk in expressing and purifying both a model protein, the green fluorescent protein, and the antimicrobial peptide Bin1b. Both proteins were effectively expressed and purified using IMAC, demonstrating SmallTalk's value as an affinity tag, yielding 7.2 mg·L −1 of cell culture for the green fluorescent protein and up to 9.8 mg·L −1 for Bin1b. Antimicrobial assays conducted with SmallTalk‐tagged Bin1b showed activity against Staphylococcus aureus , Escherichia coli , Klebsiella pneumoniae , and Pseudomonas aeruginosa , with minimum inhibitory concentrations ranging from 7.5 to 22.5 μ m . Importantly, SmallTalk enabled the full retention of Bin1b's antimicrobial activity without the need for its removal, significantly simplifying the production process. These findings indicate that SmallTalk provides a promising strategy for the recombinant production of peptides. This tag has the potential to enhance the expression, purification, and functional analysis of antimicrobial peptides, which are increasingly being pursued as alternatives to antibiotics in the fight against antimicrobial resistance.
Source: FEBS Open Bio
Authors: Atika Tariq, Nestor G. Casillas‐Vega, Alma Gomez‐Loredo, Xristo Zarate
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,210
Award: G2810790111
OpenAlex ID: W4416128601