Cell-free expression and characterization of multivalent rhamnose-binding lectins using bio-layer interferometry.

Area of Science:

• Biochemistry
• Synthetic Glycobiology
• Protein Expression

Background:

• Lectins are crucial for glycan binding, but their recombinant expression is challenging, hindering discovery.
• Existing methods limit the rapid characterization of novel lectins and their functions.

Purpose of the Study:

• To develop an efficient workflow for expressing and characterizing challenging lectin classes.
• To enable high-throughput screening of new and engineered multivalent lectins.

Main Methods:

• Bacterial cell-free expression for small-scale production of disulfide bond-rich lectins.
• Direct coupling of cell-free expressed lectins with bio-layer interferometry (BLI) for interaction analysis.
• Label-free analysis of lectin-carbohydrate binding without protein purification.

Main Results:

• Efficient expression of multivalent, rhamnose-binding lectins was achieved using cell-free systems.
• Cell-free expressed lectins were directly analyzed by BLI to determine substrate specificity.
• Binding affinity estimation was performed without the need for lectin purification.

Conclusions:

• Bacterial cell-free expression coupled with BLI provides a rapid and efficient method for lectin discovery and characterization.
• This workflow facilitates the screening and engineering of lectins for synthetic glycobiology applications.
• The presented method overcomes expression challenges, accelerating the pace of lectin research.