posted on 2021-08-20, 20:13authored byJia Fu, Qianqian Jia, Peida Liang, Saisai Wang, Huaxin Zhou, Liyang Zhang, Chunlei Gao, Hong Wang, Yanni Lv, Shengli Han
Membrane protein immobilization is particularly significant in
in vitro drug screening and determining drug–receptor interactions.
However, there are still some problems in the immobilization of membrane
proteins with controllable direction and high conformational stability,
activity, and specificity. Cell membrane chromatography (CMC) retains
the complete biological structure of membrane proteins. However, conventional
CMC has the limitation of poor stability, which results in its limited
life span and low reproducibility. To overcome this limitation, we
propose a method for the specific covalent immobilization of membrane
proteins in cell membranes. We used the SNAP-tag as an immobilization
tag fused to the epidermal growth factor receptor (EGFR), and Cys145
located at the active site of the SNAP-tag reacted with the benzyl
group of O6-benzylguanine (BG). The SNAP-tagged
EGFR was expressed in HEK293 cells. We captured the SNAP-tagged EGFR
from the cell membrane suspension onto a BG-derivative-modified silica
gel. Our immobilization strategy improved the life span and specificity
of CMC and minimized loss of activity and nonspecific attachment of
proteins. Next, a SNAP-tagged EGFR/CMC online HPLC-IT-TOF-MS system
was established to screen EGFR antagonists from Epimedii
folium. Icariin, magnoflorine, epimedin B, and epimedin
C were retained in this model, and pharmacological assays revealed
that magnoflorine could inhibit cancer cell growth by targeting the
EGFR. This EGFR immobilization method may open up possibilities for
the immobilization of other membrane proteins and has the potential
to serve as a useful platform for screening receptor-binding leads
from natural medicinal herbs.