Gravity-Flow Ni-NTA Affinity Purification of AsCas12a v1

<p>This protocol describes the expression of recombinant His-tagged AsCas12a in Escherichia coli BL21(DE3) by IPTG induction, followed by purification using gravity-flow Ni-NTA affinity chromatography. The workflow includes cell lysis, high-salt washing to reduce nucleic acid contamination, protein elution, and assessment of protein purity and recovery by SDS-PAGE. Successful completion of this protocol typically yields approximately 10 mg of ~90% pure AsCas12a per litre of culture, suitable for most biochemical and functional assays. Higher purity can be achieved through an additional heparin affinity chromatography polishing step, which is not described in this protocol.</p>