A rapid and universal tandem-purification strategy for recombinant proteins

Area of Science:

• Biochemistry
• Protein purification
• Recombinant protein expression

Background:

• Efficient recovery of pure, functional therapeutic and recombinant proteins is crucial.
• Current purification methods can be complex and time-consuming.

Purpose of the Study:

• To design and validate a novel tandem purification tag, His-tag and calmodulin (HiCaM), for simplified protein purification.
• To combine immobilized metal affinity chromatography (IMAC) and hydrophobic interaction chromatography (HIC) into a single, efficient procedure.

Main Methods:

• A novel tandem (His)(6)-calmodulin (HiCaM) fusion tag was designed.
• The HiCaM tag was fused to enhanced green fluorescent protein (eGFP) and human p53.
• Fusion constructs were expressed in E. coli and purified using tandem IMAC/HIC.

Main Results:

• The HiCaM tag facilitated rapid purification of eGFP and p53 to near homogeneity under native conditions.
• Cleavage at a thrombin site yielded untagged, functional proteins with >97% purity.
• The method proved effective for both large-scale and small-scale protein purification.

Conclusions:

• The HiCaM purification strategy is rapid, cost-effective, and utilizes widely available matrices.
• This approach offers an excellent method for large-scale recombinant protein purification.
• The HiCaM tag is suitable for protein array designs and producing pure, functional proteins.