ArticleApplied biochemistry and biotechnology2026
An Aerobic in Vitro Cell Lysate-based Method for Fe-S Cluster Reconstitution in Recombinant Rv1460 Protein from Mycobacterium tuberculosis H37Rv.
Article in Applied biochemistry and biotechnology, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Iron-sulfur (Fe-S) clusters serve as essential cofactors in many proteins performing diverse roles, yet their incorporation remains a major bottleneck in recombinant proteins, requiring strict anaerobic conditions. Here, we report a modified Fe-S reconstitution method for recombinant proteins using an in vitro cell lysate-based approach under aerobic conditions. In this study, Rv1460, a transcriptional regulator from Mycobacterium tuberculosis, is used as a model protein that binds 4Fe-4S cluster and has been characterized earlier under strict anaerobic conditions. In this approach, the Fe-S cluster incorporation step is performed after sonication by adding the Fe and S sources to the cell lysate, which likely contains endogenous expression host cell chaperones and associated factors required for Fe-S cluster synthesis, followed by affinity purification. This method does not require anaerobic chambers and also minimizes downstream purification steps. The Fe-S cluster incorporation was confirmed by UV-Visible, MALDI-TOF, and Circular Dichroism spectroscopy, Differential Scanning Calorimetry, Fourier transform infrared spectroscopy, and Thermogravimetry. Electrophoretic mobility shift assays confirmed that the cell lysate-based reconstituted protein maintains its DNA-binding activity for 6 hours and retains long-term functionality after storage at -80 °C. This aerobic cell lysate-based strategy leverages a native environment to enable cost-effective Fe-S cluster incorporation, distinguishing it from other conventional methods that require maintaining anaerobic conditions or using multiple purified enzymes. This approach enables Fe-S incorporation in near-physiological conditions, yielding a functionally active protein, and may potentially be extended to purify and characterize other Fe-S containing recombinant proteins across diverse life forms.
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