Purification of his-tagged proteins using printed monolith adsorption columns

Area of Science:

• Biotechnology
• Biochemical Engineering
• Separation Science

Background:

• Traditional bio-separation methods like bead-based and expanded bed chromatography have limitations.
• Printed monolith adsorption (PMA) utilizes 3D-printed monolithic structures with ordered flow channels as a novel alternative.
• PMA allows direct purification from crude samples and can be functionalized for affinity chromatography.

Purpose of the Study:

• To combine PMA technology with immobilized metal affinity ligand (iminodiacetic acid) for selective binding of polyhistidine-tagged proteins.
• To evaluate the protein-binding capacity of different PMA structures.
• To demonstrate the efficacy of PMA for direct purification of tagged proteins from crude lysates.

Main Methods:

• Fabrication and testing of two distinct PMA structures.
• Assessment of both static and dynamic protein-binding capacities.
• Purification of a polyhistidine-tagged protein from crude lysate using functionalized PMA.

Main Results:

• Both PMA columns exhibited a dynamic binding capacity of approximately 3 mg/mL at comparative linear flow rates.
• Static binding capacity varied based on column voidage.
• Direct purification of polyhistidine-tagged proteins from crude lysate yielded results comparable to commercial immobilized metal affinity chromatography (IMAC) methods.
• Purification time was substantially reduced compared to conventional methods.

Conclusions:

• Functionalized PMA with immobilized metal affinity ligands is an effective method for purifying polyhistidine-tagged proteins.
• PMA offers a faster and efficient alternative for direct purification from crude biological samples.
• This technology holds promise for advancing bio-separation processes in biotechnology and biochemical engineering.