An Affinity Chromatography Technique for the Purification of a Recombinant Bacterial Protein

JoVE Encyclopedia of Experiments

Microbiologie

0 weergaven • 2:54 min • November 28th, 2025

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Take a suspension of bacterial cells expressing a polyhistidine-tagged target protein.

Sonicate on ice to minimize protein degradation. High-frequency sound waves lyse the cells, releasing intracellular contents, including the target protein.

Centrifuge to separate debris and transfer the supernatant containing proteins into a syringe.

Filter the supernatant to remove aggregates and prevent clogging of the chromatography column.

Take a nickel-based affinity chromatography column and equilibrate it for optimal protein purification.

Load the filtrate onto the column. The polyhistidine tag binds to nickel ions on the column resin, immobilizing the target proteins.

Wash the column with a buffer to remove unbound proteins, and monitor UV absorbance of the eluate to confirm the their removal of non-target proteins.

Add an elution buffer containing imidazole, which competes with the polyhistidine tag for nickel binding, releasing the proteins.

A second spike in UV absorbance confirms the elution of the target protein, which is ready for downstream use.

Sonicate the cells on ice using five cycles of a 15 second pulse and a 30 second pause.

After centrifuging the cell lysate at 21,000 g and four degrees Celsius for 15 minutes, filter the supernatant using a sterile syringe filter system. Use an automated purification system, equipped with a five milliliter nickel-based affinity column to purify the HIS-tagged RTA fraction from the sterile filtered E.coli supernatant. In general, use an elution speed of one milliliter per minute and cool the whole purification system to prevent non-efficient toxin binding and loss of toxin activity.

Briefly equilibrate the affinity column with 20 milliliters of binding buffer to remove the storage buffer. Then, apply the sterile, filtered supernatant onto the affinity column using a syringe. Wash the column with 25 to 35 milliliters of binding buffer to remove the unbound proteins from the column.

Perform the washing step until the UV absorbance at 280 nanometers is close to the initial UV value. Then, elute the bound RTA fraction with 20 to 35 milliliters of elution buffer and keep the sample on ice. It's important to start the RTA fraction sampling shortly after the UV 280 value increases and stop the RTA fraction sampling when it reaches the original baseline again.

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Laatst bijgewerkt: 1 augustus 2026