A new pH-responsive peptide tag for protein purification.

Area of Science:

• Biochemistry
• Protein Engineering
• Biotechnology

Background:

• Protein purification often relies on affinity ligands, which can be costly and may damage sensitive proteins.
• Developing novel purification strategies that are efficient, cost-effective, and preserve protein integrity is crucial for biopharmaceutical development.

Purpose of the Study:

• To introduce a new pH-responsive peptide tag for reversible protein precipitation and redissolution.
• To evaluate the efficacy of this tag for protein purification, particularly for unstable pharmaceutical proteins.

Main Methods:

• Genetic fusion of proinsulin with N-terminal fragments (6, 17, 50, or 250 amino acids) of cell surface protein B (CspB).
• Characterization of pH-dependent precipitation and redissolution properties of the fusion proteins.
• Assessment of a representative tag (CspB50) with pharmaceutical proteins like Teriparatide and Bivalirudin.

Main Results:

• The CspB peptide tag demonstrated pH-dependent reversible precipitation and redissolution within a narrow, physiological pH range.
• A CspB50 tag showed efficient aggregation below pH 6.8 and complete redissolution above pH 7.3 for CspB50-Teriparatide fusion proteins.
• Similar pH-responsive behavior was observed for CspB50-Bivalirudin fusion proteins, indicating broad applicability.

Conclusions:

• The CspB peptide tag provides a novel, ligand-free method for protein purification based on pH-controlled liquid-solid separation.
• This pH-responsive tag is particularly useful for purifying unstable proteins that are sensitive to non-physiological pH conditions.
• The sharp pH transition around neutral pH makes this tag a valuable tool in bioprocessing and protein engineering.