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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 T T C T C T T G A C -10 -9 -8 –7 –6 –5 -4 –3 –2 –1.

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Presentation on theme: "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 T T C T C T T G A C -10 -9 -8 –7 –6 –5 -4 –3 –2 –1."— Presentation transcript:

1 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 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

2 Ani wt oligo-APC XID N oligo-PE pCTCON2 REOAni-Myc STS1 STS2 Oligo PCR and yeast transformation Error prone PCR (2 rounds) Yeast tranformation to generate library pCTCON2 REOAni- Myc STS1 STS2 Computational design (169) Yeast surface Aga1p s s Aga2p I-AniI Myc HA Biotin s s Surface expression selection Counter Selection Ani Myc-FITC Generation of XID sequence specific I-Anil variants through yeast surface display libraries

3 Error prone PCR mutation rate EP-PCR Control 59 mutation/7.44 kb 1 mutation/6 kb Input library Output library I-Anil yeast surface expression after error prone PCR

4 Ani wt oligo-PE XID N oligo-APC Round 1 counter selection Y2 Ani XID N2 Lib4 Generation of Y2 Ani XID N2 Libraries Ani wt oligo-PE XID N oligo-APC Round 3 counter selection Y2 Ani XID N2 Lib10 Ani wt oligo-PE XID N oligo-APC Round 5 counter selection Y2 Ani XID N2 Lib14 Ani wt oligo-PE XID N oligo-APC Round 7 counter selection Y2 Ani XID N2 Lib18 Ani wt oligo-PE XID N oligo-APC Round 8 counter selection Y2 Ani XID N2 Lib21 Ani wt oligo-PE XID N oligo-APC Round 9 counter selection Y2 Ani XID N2 Lib24

5 Titration of WT Ani, XID Lib7, 11, 18 and 23 towards XID -5 oligo and WT oligo. XID Lib23

6 Sequence analysis from Ani XID N2 Lib24 (32 colonies) Desgin siteWT STS2 region WT STS1 region 53 I A 1Q 7V 24 16 YY 3Q 1S 28 55 S R 25Y 7 18 SS 3H 1K 1Q 26R 157 RR 7V 1Y 24 20 TT 32 59 RR 32 22 KN 32 61 E K 32 23 GG 32 62 I E 28K 4 24 KK 32 64 M R 32 25 YY 32 66 A Q 32 27 T H 1Q 31 68 R A 1V 31 29 E S 31T 1 70 RR 32 31 G R 32 Random mutation 33 EE 32 73 NN 3D 29 120 DD 17V 15

7 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 oligo-APC

8 Generation of XID sequence specific I-Anil variants through B cell somatic hypermutation -3 oligo 2nd round sorting Oligo-PE -3 oligo 5th round sorting Oligo-PE -5 oligo 4th round Sorting XID N Oligo-PE WT oligo-APC -5 oligo 3 rd round counter selection.

9 Generation of XID sequence specific I-Anil variants through B cell somatic hypermutation XID N Oligo-PE WT oligo-APC -5 oligo 4 th round counter selection. XID N Oligo-PE WT oligo-APC -5 oligo 5 th round counter selection. XID Oligo-PE WT oligo-APC XID oligo 1 st round counter selection. XID Oligo-PEWT Oligo-PE

10 I-Anil coding sequence in DT40 cells WT

11 XID Oligo-PE 9th rounds binding sorting XID Oligo-PE WT oligo-APC XID oligo 6th round counter selection. Side scatter


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