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A P P L I C A T I O N N O T E
NTCP and LHBsAg
Gaining more insights into the NTCP-LHBsAg interaction is critical for developing new therapeutic
strategies, as it could lead to targeted treatments that prevent HBV entry into hepatocytes and re-
duce the viral load in infected individuals. Understanding this interaction in detail could also provide
valuable information for combating HBV-associated conditions such as cirrhosis and liver cancer
(22). This is the first study in which both proteins were purified as unaltered full-length versions to
keep the approach as native as possible.
As illustrated in Figure 8A, the copolymer nanodisc-stabilized NTCP and LHBsAg had a hydrodynam-
ic radius (rH) of 6.57 ± 0.22 nm and 5.80 ± 0.29 nm respectively. The polydispersity index (PDI) was
demonstrated to be 0.20 ± 0.04 and 0.16 ± 0.04 for NTCP and LHBsAg. The sizing information con-
firmed the anticipated formation of the nanodiscs, and the PDI indicated that the purified samples
were monodisperse for further biophysical analyses. Both samples remained thermal stable up to
approximately 53 °C (NTCP) and 79°C (LHBsAg).
The Cy5 analogue-labeled NTCP nanodisc was maintained at a constant concentration of 20 nM,
while the concentration of non-labeled L-HBsAg nanodisc was varied between 0.2 nM and 9.3 µM
(Figure 3B cyan). A decrease of nanodiscs with L-HBsAg inside was complemented with empty nano-
discs to preserve its constant molarity while only L-HBsAg had changing concentrations. The Kd was
determined by analyzing the TRIC signal using 670 nm at 2.5 s yielding a Kd of 967 nM. The black data
shows the negative control using the same assay settings only with a dilution series of a non-specific
membrane protein.
Figure 7: Western blot and SDS PAGE of NTCP sol-
ubilized with Native MP
TM
. NTCP is a protein with a
theoretical size of 39,3 kDa and was detected at an
apparent height of ~ 48 kDa (marked with arrows).
Detection of proteins in SDS Page via specific pri-
mar y Rho1D4 antibody and secondar y HRP anti-
body.
