selection of xid target site ani-wt: t g a g g a g g t t t c t c t g t a a m-xid: a g t g c c t g t...
TRANSCRIPT
Selection of XID target site
Ani-wt: T G A G G A G G T T T C T C T G T A Am-XID: A G T G C C T G T T T C T C T T G A C
-10 -9 -8 –7 –6 –5 -4 –3 –2 –1 +1 +2 +3 +4 +5 +6 +7 +8 +9
Ani wt oligo-APC
XID
N o
lig
o-P
E
3216854 bp
pCTCON2 REOAni-Myc
STS1 STS2
Oligo PCR and yeast transformation Error prone PCR (2 rounds) Yeast tranformation to generate library
3216854 bp
pCTCON2 REOAni-Myc
STS1 STS2
Computational design (169)
Yeast surface
Aga1pss
Aga2p
I-AniI
MycHA
Biotin
ss
Surface expression selection Counter Selection
Ani Myc-FITC
Generation of XID sequence specific I-Anil variants through yeast surface display libraries
Error prone PCR mutation rate
0
1
2
3
4
5
6
7
8
9
1 2
Mu
tati
on
s/kb
EP-PCR Control59 mutation/7.44 kb 1 mutation/6 kb
0
10
20
30
40
50
60
1 2
An
i Su
rfa
ce
ex
pre
ss
ion
%Input library Output library
I-Anil yeast surface expression after error prone PCR
Ani wt oligo-PE
XID
N o
lig
o-A
PC
Round 1 counter selection Y2 Ani XID N2 Lib4
Generation of Y2 Ani XID N2 Libraries
Ani wt oligo-PE
XID
N o
lig
o-A
PC
Round 3 counter selection Y2 Ani XID N2 Lib10
Ani wt oligo-PE
XID
N o
lig
o-A
PC
Round 5 counter selection Y2 Ani XID N2 Lib14
Ani wt oligo-PE
XID
N o
lig
o-A
PC
Round 7 counter selection Y2 Ani XID N2 Lib18
Ani wt oligo-PE
XID
N o
lig
o-A
PC
Round 8 counter selection Y2 Ani XID N2 Lib21
Ani wt oligo-PE
XID
N o
lig
o-A
PC
Round 9 counter selection Y2 Ani XID N2 Lib24
Titration of WT Ani, XID Lib7, 11, 18 and 23 towards XID -5 oligo and WT oligo.
6000
5000
4000
3000
2000
1000
0
MF
I
1 10 100 1000
WT Oligo[nm]
WT Lib7 Lib11 Lib18
5000
4000
3000
2000
1000
0
MF
I
1 10 100 1000-5 XID Oligo[nM]
WT Lib7 Lib11 Lib18
18
16
14
12
10
8
6
4
2
0
MF
I
1 10 100 1000Substrate (nM)
Ani WT oligo XID -5 oligo
XID Lib23
Sequence analysis from Ani XID N2 Lib24 (32 colonies)
Desgin site WT STS2 region WT
STS1 region 53 I A 1 Q 7 V 24
16 Y Y 3 Q 1 S 28 55 S R 25 Y 7
18 S S 3 H 1 K 1 Q 26 R 1 57 R R 7 V 1 Y 24
20 T T 32 59 R R 32
22 K N 32 61 E K 32
23 G G 32 62 I E 28 K 4
24 K K 32 64 M R 32
25 Y Y 32 66 A Q 32
27 T H 1 Q 31 68 R A 1 V 31
29 E S 31 T 1 70 R R 32
31 G R 32 Random mutation
33 E E 32 73 N N 3 D 29
120 D D 17 V 15
XID oligo binding affinity
Selected for continuous culture and mutation
DT40 B lymphocytes
I-Anil
Biotin
XID oligo
I-Anil DT 40 surface display and somatic hypermutation
Generation of XID sequence specific I-Anil variants through B cell somatic hypermutation
XID N Oligo-PE
WT
oli
go
-AP
C
Generation of XID sequence specific I-Anil variants through B cell somatic hypermutation
-3 oligo 2nd round sorting
Oligo-PE Oligo-PE
-3 oligo 5th round sorting
Oligo-PE
-5 oligo 4th round Sorting
XID N Oligo-PE
WT
oli
go
-AP
C
-5 oligo 3rd round counter selection.
Generation of XID sequence specific I-Anil variants through B cell somatic hypermutation
XID N Oligo-PE
WT
oli
go
-AP
C
-5 oligo 4th round counter selection.
XID N Oligo-PE
WT
oli
go
-AP
C
-5 oligo 5th round counter selection.
XID Oligo-PE
WT
oli
go
-AP
C
XID oligo 1st round counter selection.
XID Oligo-PE WT Oligo-PE
I-Anil coding sequence in DT40 cells
WT
WT
XID Oligo-PE
9th rounds binding sorting
XID Oligo-PE
WT
oli
go
-AP
C
XID oligo 6th round counter selection.
Sid
e s
ca
tte
r