<p>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 <i>Mycobacterium tuberculosis</i>, 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&#xa0;hours and retains long-term functionality after storage at -80&#xa0;°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.</p> Graphical Abstract <p></p>