Arginine-tail method, an affinity tag procedure utilizing anhydrotrypsin agarose.

Area of Science:

• Biochemistry
• Protein purification
• Molecular biology

Background:

• Recombinant protein purification is crucial for research and therapeutics.
• Existing methods may struggle with protein activity loss or low yields.
• Novel affinity tag strategies are needed for efficient protein enrichment.

Purpose of the Study:

• To introduce and evaluate the arginine-tail method for recombinant protein enrichment.
• To assess the impact of the arginine tag on protein structure and function.
• To demonstrate the efficacy of this method in purifying proteins from complex lysates.

Main Methods:

• Engineered three model proteins with C-terminal arginine-based tags (Arg, Gly-Arg, Gly-Gly-Arg).
• Utilized immobilized anhydrotrypsin-agarose chromatography for specific protein adsorption.
• Employed denaturants (urea, HCl) to expose C-terminal tags for binding.
• Eluted bound proteins using benzoylglycylarginine (Bz-Gly-Arg) or HCl.

Main Results:

• Engineered proteins retained sugar-binding activity and antigenicity.
• Arginine-tailed proteins were successfully adsorbed and eluted from the anhydrotrypsin column.
• Achieved over ten-fold enrichment of a target lectin from urea-denatured Escherichia coli lysate.
• Demonstrated that even a minimal arginine tag can facilitate purification.

Conclusions:

• The arginine-tail method is an effective affinity tag strategy for recombinant protein purification.
• This method offers significant enrichment while minimizing impact on protein structure and function.
• The technique is applicable to various recombinant proteins, including those with altered activity due to mutagenesis.