Soluble expression, two-step purification and tagmentation-compatible activity assessment of hyperactive Tn5 transposase using a GB1 fusion tag strategy.

BACKGROUND

Hyperactive Tn5 transposase is an essential reagent for tagmentation-based next-generation sequencing (NGS) library construction that includes assays for transposase-accessible chromatin using sequencing and cleavage under targets and tagmentation. However, recombinant expression of Tn5 in Escherichia coli frequently results in inclusion body formation, limiting yield and functional activity.

METHODS

In this study, we developed a streamlined expression and purification protocol for hyperactive Tn5 transposase by fusing the B1 immunoglobulin-binding domain of Streptococcal protein G (GB1) and a polyhistidine tag to enhance solubility and enable efficient nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The fusion protein was expressed in E. coli BL21(DE3) under low-temperature induction (20°C, 15 h) and purified using a two-step strategy that combined Ni-NTA affinity chromatography and size-exclusion chromatography (SEC).

RESULTS

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirmed robust expression of the ∼63-kDa GB1-Tn5 fusion protein with a substantial portion partitioning into the soluble fraction. Purification of Ni-NTA from 38 g of cell paste yielded a total of 685.5 mg of protein across 15 elution fractions, with peak concentrations reaching 17.3 mg/mL. Subsequent SEC polishing separated higher-order aggregate/oligomeric species from a dimer-enriched GB1-Tn5 pool selected for transposome assembly. Adapter-loaded in vitro assays using λDNA demonstrated dose-dependent fragmentation, providing qualitative evidence of transposome-dependent cleavage activity.

CONCLUSIONS

The GB1-Tn5 fusion strategy provides a cost-effective and reproducible method for producing milligram quantities of active Tn5 transposase, offering a practical alternative to intein-based and small ubiquitin-like modifier purification systems for further NGS reagent optimisation.