EGF-mCherry Fusion Protein Expressed in E. coli Shows Product Heterogeneity but a High Biological Activity.

Abstract:

:The epidermal growth factor receptor (EGFR) is a transmembrane protein involved in cell signaling processes, and dysregulation of its activity often drives tumor growth. EGFR is a clinically validated tumor marker and target for antibodies and tyrosine kinase inhibitors. We demonstrate that a fusion protein of the natural ligand epidermal growth factor (EGF) with the fluorescent reporter mCherry can be expressed in the cytosol of E. coli in high yields and with a high biological activity. Biophysical characterization by mass spectrometry analysis confirmed three disulfide bonds that are crucial for protein structure. Biolayer interferometry data of the protein-protein interaction of EGF-mCherry with the soluble EGFR are comparable to that of unmodified EGF. Cell culture experiments demonstrated that this fusion replicates all important features of the natural ligand. Finally, fluorescent assays based on EGF-mCherry provided a simple and convenient method to compare EGFR levels on cells and to determine competition of EGFR-binding molecules. These assays will help to rank competitive properties of EGFR inhibitors.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Feiner RC,Pennè I,Müller B,Müller KM

doi

10.1021/acs.biochem.9b00021

subject

Has Abstract

pub_date

2019-02-26 00:00:00

pages

1043-1047

issue

8

eissn

0006-2960

issn

1520-4995

journal_volume

58

pub_type

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