Protein purification with light via a genetically encoded azobenzene side chain

Area of Science:

• Biochemistry
• Chromatography
• Molecular Biology

Background:

• Affinity chromatography is a key technique for protein purification from complex biological mixtures.
• Current methods often require chemical agents or harsh conditions, potentially affecting protein function.

Purpose of the Study:

• To introduce a novel, light-controlled affinity chromatography method for protein purification.
• To demonstrate the efficacy of the Azo-tag system for selective protein elution.

Main Methods:

• Development of the Azo-tag, a peptide incorporating p-(phenylazo)-L-phenylalanine (Pap) sensitive to UV light.
• Utilizing an α-cyclodextrin (α-CD) chromatography matrix that binds trans-Pap but not cis-Pap.
• Employing 355 nm UV light to trigger the cis-configuration and induce protein elution.

Main Results:

• Successful single-step purification of various proteins, including enzymes and antibody fragments, using the Azo-tag system.
• Demonstrated light-induced elution (Excitography) without competing agents or harsh buffers.
• Showcased the reversible nature of the trans-Pap to cis-Pap transition upon light exposure.

Conclusions:

• Excitography offers a novel, non-chemical approach to protein purification via light-controlled affinity chromatography.
• The Azo-tag and α-CD system provide a versatile platform for selective protein separation.
• This physical control principle enhances affinity chromatography, expanding its applicability in biotechnology and research.