Enzymatic protein fusions with 100% product yield

Area of Science:

• Biochemistry
• Molecular Biology
• Bioengineering

Background:

• The protein ligase Connectase enables fusion of proteins with various molecules.
• Connectase offers high substrate specificity and catalytic efficiency but has a reversible reaction limiting yield to 50%.

Purpose of the Study:

• To develop a method for achieving 100% fusion product yield in Connectase-mediated 1:1 conjugation reactions.
• To enable the generation of defined, completely labeled antibody conjugates.

Main Methods:

• A simple, efficient method for N- or C-terminal protein-protein or protein-peptide fusions was developed.
• The method utilizes short incubation times, minimal enzyme amounts, and is effective at low substrate concentrations and temperatures.

Main Results:

• Reliable generation of 100% fusion product in 1:1 conjugation reactions.
• Enables production of antibody conjugates with one fusion partner per chain.
• Effective even under challenging conditions like low substrate concentrations and temperatures.

Conclusions:

• The developed method significantly improves Connectase-mediated protein conjugation.
• Presents a valuable new tool for bioengineering applications, particularly for antibody conjugation.
• Offers enhanced control and efficiency in creating precisely labeled biomolecular constructs.