Automated two step purification of 6xHis-tagged GFP

Importance of the topic

A reliable and efficient purification strategy is essential for producing high-quality recombinant proteins. His-tag affinity chromatography offers strong specificity and binding capacity, but often requires a polishing step to achieve the purity and buffer conditions needed for downstream applications. Automation of multi-step purification workflows can significantly reduce hands-on time, improve reproducibility, and accelerate protein production.

Objectives and overview of the study

This application note demonstrates an automated two-step purification of 6xHis-tagged green fluorescent protein (GFP) using the AZURA® Bio purification system. The goal was to capture GFP with immobilized metal ion affinity chromatography (IMAC) and seamlessly perform a subsequent buffer exchange via size-exclusion chromatography, all without manual intervention.

Methodology

The workflow comprises five phases: column equilibration, sample injection and capture of 6xHis-GFP on a Ni-NTA column, washing to remove nonspecific binders, elution of GFP into a sample loop, and re-injection onto a desalting column for buffer exchange. Key buffer conditions include a low-imidazole loading buffer (10 mM) to minimize contaminants and a high-imidazole elution buffer (500 mM) for efficient protein release. The desalting step replaces the imidazole-rich eluate with phosphate-buffered saline free of imidazole.

Used instrumentation

• AZURA P 6.1L HPG pump
• AZURA ASM 2.1L autosampler with dual 6-port/3-channel valves
• AZURA MWD 2.1L multi-wavelength detector (280 nm and 395 nm)
• AZURA CM 2.1S conductivity monitor
• Column switching valve and Foxy R1 fraction collector
• Sepapure FF Ni-NTA 1 mL affinity column
• Sepapure Desalting 5 mL gel filtration column

Main results and discussion

Chromatograms recorded at 280 nm and 395 nm revealed clear separation of flow-through impurities (peak A) and the eluted 6xHis-GFP (peaks B1 and B2). The parking of the eluate in a sample loop enabled precise transfer to the desalting column without volume loss. Conductivity traces confirmed effective buffer exchange. SDS-PAGE analysis showed a strong 27 kDa band in the elution fraction with minimal contaminating bands, verifying high purity.

Benefits and practical applications

• Fully automated two-step protocol eliminates manual sample handling between affinity capture and buffer exchange.
• Multi-wavelength detection provides both total protein (280 nm) and GFP-specific (395 nm) monitoring.
• Adaptable to various tagged proteins and purification scales.
• Reduces risk of sample loss and cross-contamination while improving reproducibility.

Future trends and opportunities

Integration of additional chromatographic modes (e.g., ion exchange, hydrophobic interaction) and on-line analytical tools (mass spectrometry, light scattering) could further streamline workflows. Advances in software-driven method optimization, continuous-flow purification, and miniaturized single-use systems are poised to enhance throughput and flexibility in protein production.

Conclusion

The AZURA Bio system successfully performed an uninterrupted two-step purification of 6xHis-tagged GFP, combining IMAC capture with size-exclusion buffer exchange. The automated process yielded high-purity protein with minimal user involvement, demonstrating a versatile approach for protein purification in research and industrial settings.