Transform pET-28a(+) constructs into BL21(DE3) E. coli strain for optimal expression of T7-driven constructs

Select transformants on kanamycin plates and validate by colony PCR or restriction digest

Express proteins in selected colonies (IPTG induction, 18°C overnight recommended for complex proteins)

Purify constructs via Ni-NTA affinity chromatography targeting 6xHis-tags Complex Assembly & Functional Testing

Combine SpyTag/SpyCatcher components in optimized ratios (ferritin variant + nanobody/Zif268 variant) and incubate to allow covalent coupling

Assess complex formation via SDS-PAGE under non-reducing conditions to visualize SpyTag-SpyCatcher linkage

Functional testing: ◦ For GFP capture: Mix with GFP solution, spin down complexes, measure supernatant fluorescence reduction ◦ For DNA capture: Mix with fluorescently-labeled Zif268 target sequence, perform gel-shift assay or filter-binding assay Optional Optimization

Test different ratios of taggedferritin subunits to optimize complex assembly and target binding

1. Transform pET-28a(+) constructs into BL21(DE3) E. coli strain for optimal expression of T7-driven constructs

2. Select transformants on kanamycin plates and validate by colony PCR or restriction digest

3. Express proteins in selected colonies (IPTG induction, 18°C overnight recommended for complex proteins)

4. Purify constructs via Ni-NTA affinity chromatography targeting 6xHis-tags

1. Combine SpyTag/SpyCatcher components in optimized ratios (ferritin variant + nanobody/Zif268 variant) and incubate to allow covalent coupling

2. Assess complex formation via SDS-PAGE under non-reducing conditions to visualize SpyTag-SpyCatcher linkage

3. Functional testing:

• ◦ For GFP capture: Mix with GFP solution, spin down complexes, measure supernatant fluorescence reduction
• ◦ For DNA capture: Mix with fluorescently-labeled Zif268 target sequence, perform gel-shift assay or filter-binding assay

1. Test different ratios of taggedferritin subunits to optimize complex assembly and target binding