Hydroxyapatite-based immobilized metal affinity adsorbents for protein purification

Area of Science:

• Biochemistry
• Protein Purification
• Biotechnology

Background:

• Recombinant protein purification is crucial for biotechnology.
• Poly(His)-tagged proteins are widely used but require efficient purification methods.
• Immobilized metal affinity chromatography (IMAC) is a common technique.

Purpose of the Study:

• To investigate the use of metal ion-charged hydroxyapatite for one-step purification of poly(His)-tagged proteins.
• To evaluate the selectivity and stability of different metal ions.
• To compare the performance of hydroxyapatite-based IMAC with commercial adsorbents.

Main Methods:

• Metal ion (Fe(III)) adsorption onto hydroxyapatite.
• One-step purification of poly(His)-tagged D-hydantoinase from crude lysate.
• Optimization of buffer conditions (pH, NaCl, NaF concentrations).
• Protein recovery using phosphate buffer.
• Purification of poly(His)-tagged N-acetyl-D-glucosamine 2-epimerase under denaturing conditions.

Main Results:

• Fe(III)-loaded hydroxyapatite demonstrated the highest selectivity and operational stability.
• Optimal purification conditions were identified (20 mM pH 8.0 buffer with 150 mM NaCl and 50 mM NaF).
• Quantitative protein recovery was achieved using 150 mM pH 8.0 phosphate buffer.
• The capacity of Fe(III)-hydroxyapatite was 4.9 mg/g, comparable to commercial Ni-NTA adsorbents.
• Achieved >95% purity for D-hydantoinase in a single step.
• Demonstrated successful purification under denaturing conditions.

Conclusions:

• Hydroxyapatite charged with Fe(III) is a promising and cost-effective adsorbent for IMAC.
• This method enables efficient, one-step purification of poly(His)-tagged proteins.
• The technique is applicable to both native and denatured proteins.