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Soluble expression, two-step purification and tagmentation-compatible activity assessment of hyperactive Tn5
Yuzhen Xie1, Yan Shi1, Heyao Zhang1
1College of Biotechnology, Suzhou Industrial Park Institute of Services Outsourcing, Suzhou, Jiangsu, 215123, China.
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.
