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

## TL;DR

Scientists engineered a modified EGF protein with a single site for attaching drugs, enabling precise and efficient targeted cancer therapy.

## Contribution

A novel EGF variant with a single conjugation site for orthogonal click-chemistry was engineered and validated for drug delivery.

## Key findings

- The recombinant EGF variant K-EGFRR was produced in E. coli with a yield of 4–6 mg/L.
- K-EGFRR showed higher affinity (KD: 5.9 nM) compared to wild-type EGF (7.3 nM).
- The variant enabled orthogonal click-conjugation for targeted drug delivery.

## Abstract

The epidermal growth factor (EGF) receptor is commonly targeted in cancer therapy because it is overexpressed in many malignant cells. However, a general problem is to couple the targeting moieties and the drug molecules in a way that results in a homogeneous product. Here, we overcome this issue by engineering a variant of EGF with a single conjugation site for coupling virtually any payload. The recombinant EGF variant K‐EGFRR was expressed in E. coli Rosetta with a 4–6 mg/L yield. To confirm the accessibility of the introduced functional group, the ligand was equipped with a sulfo‐cyanine dye with a loading of 0.65 dye per ligand, which enables tracking in vitro. The kinetics and affinity of ligand–receptor interaction were evaluated by enzyme‐linked immunosorbent assay and surface plasmon resonance. The affinity of K‐EGFRR was slightly higher when compared to the wild‐type EGF (K
D: 5.9 vs. 7.3 nM). Moreover, the ligand–receptor interaction and uptake in a cellular context were evaluated by flow cytometry and quantitative high‐content imaging. Importantly, by attaching heterobifunctional polyethylene glycol linkers, we allowed orthogonal click‐conjugation of the ligand to any payload of choice, making K‐EGFRR an ideal candidate for targeted drug delivery.