Biology Reference
In-Depth Information
Interaction Between C-P1 Peptide and Hat/NA Protein by Yeast Two Hybrid
Assay
The yeast two-hybrid assay was employed to validate the HA-P1 interaction and also
to identify any interaction between NA-P1. To eliminate the false positive results (the
possibility of binding domain (BD)-P1, activation domain (AD)-HA
t
and AD-NA
fusion proteins themselves bringing about activation of the reporter genes), various
combinations of the recombinant plasmids along with the parental vector were co-
transformed into the yeast competent cells (Table 3). Three independent clones from
each co-transformation were analyzed for the activation of the β-galactosidase (β-gal)
reporter genes. As shown in Table 3, the co-transformed parental vectors did not show
any β-gal activities. When BD-P1 and AD-HA
t
or BD-P1 and AD-NA fusion con-
structs were co-transformed separately along with their respective parental vectors,
no β-gal activity was detected either. The co-transformed BD-P1 and AD-HA
t
as well
as BD-P1 and AD-NA showed comparatively high level of β-gal activity (25 and 3.5
Miller Units respectively). This observation showed that the P1 peptide bind with both
with HA glycoprotein as well as the NA glycoprotein. The P1 interaction with HA
glycoprotein support the previous experimental observation of hemagglutination inhi-
bition. As the yeast two-hybrid assay provided ambiguous result regarding the NA-P1
interaction, further experimental analysis (co-immunoprecipitation) was carried out.
Table 3.
P1: HAt/NA interactions in the yeast two-hybrid system.
DBD Vectors
a
AD Vectors
a
β-gal activity
b
Background
BD
AD
0.05
BD-PI
AD
0.07
BD
AD-HA
t
0.05
BD
AD-NA
0.03
PI: HA/NA interactions
BD-PI
AD-HA
t
25
BD-PI
AD-NA
3.5
a
pHyblex/Zeo and pYESTryp2 are vectors encoding the LexA DNA binding domain (BD) and B42 transcriptional
activation domain (AD), respectively.
b
Average ~-gal activity (Miller Units of 3 independent colonies for each co-transformation (The SD value is not shown
as there was very little variation between repeated experiment).
HAt-P1, NA-P1 Interaction Study by Co-immunoprecipitation
In order to verify the binding ability of the peptide P1 with HA
t
and NA proteins
through Co-IP method, these three proteins were initially synthesized by
in vitro
tran-
scription and translation methods. The P1 peptide was mixed either with the HA
t
pro-
tein or NA protein separately to allow the binding overnight or after incubation, the
