The Dac-tag, an affinity tag based on penicillin-binding protein 5

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Penicillin-binding protein 5 (PBP5) from Escherichia coli exhibits beta-lactamase activity.
• PBP5 hydrolyzes beta-lactam antibiotics through ester bond formation and ring opening.
• This enzymatic activity occurs over several minutes.

Purpose of the Study:

• To develop a novel reversible affinity tag utilizing PBP5's enzymatic properties.
• To create a tool for efficient and specific purification of fusion proteins.
• To demonstrate the utility of the tag across diverse expression systems.

Main Methods:

• A fragment of PBP5 was engineered to function as an affinity tag (Dac-tag).
• The Dac-tag was immobilized on ampicillin Sepharose for protein binding.
• Fusion proteins were purified by exploiting the tag's specific binding to ampicillin.
• Protein release was achieved using ampicillin elution or cold incubation with glycerol.

Main Results:

• The PBP5 fragment (Dac-tag) binds rapidly and specifically to ampicillin Sepharose at ambient temperatures.
• The tag facilitates reversible and monomeric affinity purification.
• Remarkably pure fusion proteins were isolated from various expression systems, including eukaryotic ones.

Conclusions:

• The Dac-tag offers a novel and effective method for reversible protein purification.
• Its application is demonstrated across diverse expression systems, highlighting its versatility.
• This technology provides a valuable tool for protein biochemistry and molecular biology research.