Recombinant Expression of a Ready-to-Use EGF Variant Equipped With a Single Conjugation Site for Click-Chemistry

Area of Science:

• Biochemistry
• Molecular Biology
• Cancer Therapy

Background:

• Epidermal growth factor (EGF) receptor is a common target in cancer therapy due to its overexpression in malignant cells.
• A significant challenge in targeted therapy is achieving homogeneous coupling of targeting moieties with drug molecules.

Purpose of the Study:

• To engineer a recombinant EGF variant with a single conjugation site for versatile payload attachment.
• To develop a homogeneous and efficient platform for targeted drug delivery.

Main Methods:

• Recombinant expression of K-EGF^RR in E. coli.
• Sulfo-cyanine dye labeling for in vitro tracking.
• Enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) for interaction kinetics and affinity.
• Flow cytometry and high-content imaging for cellular uptake studies.
• Attachment of heterobifunctional polyethylene glycol linkers for orthogonal click-conjugation.

Main Results:

• K-EGF^RR was expressed with a yield of 4-6 mg/L.
• Dye loading achieved 0.65 dye per ligand, confirming functional group accessibility.
• K-EGF^RR exhibited slightly higher affinity (K_D: 5.9 nM) compared to wild-type EGF (7.3 nM).
• Cellular uptake and ligand-receptor interactions were successfully evaluated.

Conclusions:

• Engineered K-EGF^RR provides a versatile platform for targeted drug delivery by enabling homogeneous conjugation of various payloads.
• The single conjugation site and orthogonal click-chemistry approach offer a significant advancement in developing targeted cancer therapies.