当前位置： SCI文献检索 > APPLIED MICROBIOLOGY AND BIOTECHNOLOGY期刊下所有文献 > Expression, purification, and characterization of the native intracellular domain of human epidermal growth factor receptors 1 and 2 in Escherichia coli.

Expression, purification, and characterization of the native intracellular domain of human epidermal growth factor receptors 1 and 2 in Escherichia coli.

Abstract：

: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.

journal_name

Appl Microbiol Biotechnol

journal_title

Applied microbiology and biotechnology

authors

Seetaha S,Ratanabanyong S,Choowongkomon K

doi

10.1007/s00253-019-10116-6

subject

Has Abstract

pub_date

2019-10-01 00:00:00

pages

8427-8438

issue

20

eissn

0175-7598

issn

1432-0614

pii

10.1007/s00253-019-10116-6

journal_volume

103

pub_type

杂志文章

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