Expression, purification, and characterization of the native intracellular domain of human epidermal growth factor receptors 1 and 2 in Escherichia coli.
Supaphorn SeetahaSiriluk RatanabanyongKiattawee ChoowongkomonPublished in: Applied microbiology and biotechnology (2019)
Human epidermal growth factor receptors (EGFR) are an important target in drug discovery in terms of both protein-small-molecule interactions and protein-protein interactions. In this work, the isolation of a stable soluble protein of the tyrosine kinase domain of EGFR in Escherichia coli expression has been accomplished. This successful study presents the expression and purification conditions to obtain a stable soluble protein of the active tyrosine kinase domain of EGFR (EGFR-TK) and ErbB2 (ErbB2-TK) in a bacterial system, albeit in relatively low yields. The recombinant gene was inserted into a pColdI vector and recombinant protein was expressed at low temperature. Purification of EGFR-TK and ErbB2-TK took place under the same conditions by purified supernatant using a diethylaminoethyl sepharose column followed by anion exchange and size-exclusion chromatography columns. The final yields of purified EGFR-TK and ErbB2-TK were 8.4 and 9.5 mg per liter of culture, respectively. Determination of EGFR-TK and ErbB2-TK was performed via enzyme activity with commercial drugs. The IC50 values of erlotinib and afatinib against EGFR-TK were 13.09 nM and 2.36 nM respectively, while the IC50 values of lapatinib and afatinib against ErbB2-TK were 24.69 nM and 1.36 nM, respectively. These results confirmed that soluble proteins of the active intracellular domain of the HERs family were successfully expressed and purified in a bacterial system. The new protein expression and purification protocol will greatly facilitate the enzymatic inhibition and structural studies of this protein for drug discovery.
Keyphrases
- tyrosine kinase
- epidermal growth factor receptor
- growth factor
- drug discovery
- advanced non small cell lung cancer
- escherichia coli
- small molecule
- protein protein
- binding protein
- poor prognosis
- endothelial cells
- photodynamic therapy
- small cell lung cancer
- mass spectrometry
- nitric oxide
- liquid chromatography
- cell free
- reactive oxygen species
- dna methylation
- recombinant human
- induced pluripotent stem cells
- klebsiella pneumoniae
- high speed
- high performance liquid chromatography
- copy number
- pluripotent stem cells
- transcription factor
- genome wide analysis