Protein derivitization-expressed protein ligation.

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Chemistry

Background:

• Expressed protein ligation (EPL) is a powerful technique for protein modification.
• Native chemical ligation (NCL) joins peptides via C-terminal thioesters and N-terminal cysteines.
• Intein chemistry provides essential reactive groups for NCL.

Purpose of the Study:

• To describe the methodology and advantages of expressed protein ligation.
• To highlight the use of inteins and NCL for synthesizing modified proteins.
• To explain the application of the IMPACT(TM)-System for protein purification and ligation.

Main Methods:

• Utilizing the IMPACT(TM)-System with intein-chitin-binding domain (CBD) fusions for protein purification.
• Generating a C-terminal thioester on a recombinant protein via intein cleavage with a thiol reagent (MESNA).
• Ligating the C-terminal thioester of the recombinant protein with an N-terminal cysteine-containing synthetic peptide.

Main Results:

• Intein-mediated purification yields highly purified recombinant proteins.
• The process generates a reactive C-terminal thioester intermediate.
• Successful ligation of synthetic peptides to recombinant proteins via a native peptide bond.

Conclusions:

• Expressed protein ligation is an effective strategy for creating large, modified proteins.
• The IMPACT(TM)-System facilitates efficient purification and subsequent ligation.
• This technique allows for the site-specific incorporation of synthetic moieties into proteins.