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Figures
CC
Ang2
C C C
S SCD
1 18
CCOD
70
L
258 281
CCC C
FLD
496
Figure 1. Schematic diagram of the protein structure of Ang2. Amino acids 1–18 are the
secretory signal sequences (S), amino acids 19–70 are the superclustering domain (SCD),
amino acids 71–258 are the coiled-coil oligomerization domain (CCOD), amino acids 259–
281 are the linker region (L), and amino acids 282–496 are the fibrinogen-like domain (FLD)
of Ang2. Ang2 contains cysteines (C) at positions 41, 54, 265, 286, 315, 435, 437, 439, and
452. Muted Cys residues are shown in red.
B
Cell count
A
cMyc expression (PE)
C
Cell count
Figure
2:
Yeast
displayed
Ang2mut expression and binding
to Tie2-Fc. The Ang2mut protein is
expressed on the surface of yeast
(B) in comparison to the negative
control (A). The expressed Ang2
also binds to the Tie2-Fc receptor at
receptor concentrations of 10nM
(red) and 100 nM (light blue) (D)
relative to the negative control (C).
The expression was detected using
the Ms anti-cMyc 9E10 antibody
and the anti-Ms-PE antibody.
Binding to Tie2 was detected using
Tie2-Fc and an anti-Fc-FITC
antibody.
Tie2 binding (FITC)
D
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Normalized binding
Figure 3: Determination of the affinity of yeast
displayed Ang2mut to Tie2. Binding titration curve
for the reaction of yeast displayed Ang2mut and
different concentrations of Tie2. The data was fitted
and a KD was calculated and found to be 68 nM
which is in good agreement with the value obtained
from binding experiments with pure proteins taken
from the literature.
Tie2 concentration (nM)
Figure 4: Random mutagenesis
Ang2mut library screening. FACS
dot plots showing sorting of
Ang2mut error-prone mutagenesis
library (D). X-axis indicates cMyc
expression and y-axis indicates Tie2
receptor binding (20 nM). Polygons
indicate sort gate used to collect
yeast cells. Negative control (A),
library expression (B) and library
binding to Tie2 (C) are also shown.
Tie2 binding (FITC)
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A
B
C
D
Figure 5: Random mutagenesis
Ang2wt library screening. FACS
dot plots showing sorting of Ang2wt
error-prone mutagenesis library (D).
X-axis indicates cMyc expression
and y-axis indicates Tie2 receptor
binding (20 nM). Polygons indicate
sort gate used to collect yeast cells.
Negative control (A), library
expression (B) and library binding
to Tie2 (C) are also shown.
Tie2 binding (FITC)
cMyc expression (PE)
A
B
C
D
cMyc expression (PE)