Functional polymer affinity matrix for purifying hexahistidine-tagged recombinant protein

Area of Science:

• Biochemistry
• Chromatography
• Protein Purification

Background:

• Metal chelate affinity chromatography is a powerful technique for protein purification.
• Nickel ions (Ni2+) are commonly used for chelating proteins with histidine tags.
• Development of novel adsorbents can improve the efficiency and specificity of this technique.

Purpose of the Study:

• To prepare a functional polyacrylic acid (PAA) adsorbent for metal chelate affinity chromatography.
• To evaluate the PAA adsorbent's ability to chelate nickel ions and bind proteins with neighboring histidine residues.
• To demonstrate the purification of a hybrid protein using this Ni2+-PAA affinity chromatography system.

Main Methods:

• Synthesis of a functional polyacrylic acid (PAA) adsorbent.
• Evaluation of nickel ion (Ni2+) chelation by PAA.
• Construction and expression of a hybrid protein (His6-OmpTS) by fusing DNA elements coding for adjacent histidines to the Aeromonas hydrophila ompTS gene.
• Purification of His6-OmpTS using Ni2+-PAA affinity chromatography.

Main Results:

• The PAA adsorbent demonstrated strong chelation of nickel ions (Ni2+).
• The Ni2+-PAA adsorbent effectively bound proteins containing neighboring histidine residues.
• A hybrid protein, His6-OmpTS, was successfully purified using Ni2+-PAA affinity chromatography.
• High specificity was observed in the purification process.

Conclusions:

• A functional polyacrylic acid (PAA) adsorbent has been developed for metal chelate affinity chromatography.
• The Ni2+-PAA adsorbent exhibits strong nickel ion binding and specific protein purification capabilities.
• This adsorbent represents an attractive and effective tool for purifying histidine-tagged proteins.