Presentation on theme: "Yupeng. Yeast surface Aga1p s s s s Aga2p I-AniI Myc 3’-ACTCCTCCAAAGAGACATT 5’-TGAGGAGGTTTCTCTGTAA Biotin Ani-wt: T G A G G A G G T T T C T C T G T A."— Presentation transcript:
Yeast surface Aga1p s s s s Aga2p I-AniI Myc 3’-ACTCCTCCAAAGAGACATT 5’-TGAGGAGGTTTCTCTGTAA Biotin 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 m-wt: C -10 -9 -8 –7 –6 –5 -4 –3 –2 –1 +1 +2 +3 +4 +5 +6 +7 +8 +9 Engineering I-AniI HEs for XID site I-Anil Yeast surface display HA
Ani XID C terminal Design (Jim Havranek from Baker Lab) STS 3 STS 4 Ani XID C variants yeast surface expression. Myc-Ani 1 2 3456
Vector digestion Yeast transformtion to establish Ani XID-C library (9×10 6 ) Six combo error prone PCR First round sorting based on Myc-Ani expression.
Second round sorting based on binding affinity toward Ani +6T+7G+9C. Ani wt oligo Ani XID oligo (+6T +7G +9C) Ani wt Library w/o sorting Ani-Myc sorting Ani XID +6T+7G+9C sorting Binding affinity of Ani variants toward Ani wt and Ani +6T+7G+9C oligos.
STS 3 STS 4 D Future experiments: Characterizing binding affinity and cleavage activity of individual variant. Engineering Ani XID N variants. JH Design 1 2 3 4 5 6 Copy number Ani XID variants sequence analysis (45 variants) P JH Design 1 2 3 4 5 6 Fold enrichment
90nM 25nM 15nM 4nM Ani WTE148DL156R E148D C150RK155N E148D C198R C150R I164V Q171HD173N C150S C198R CTD mutants of I-Ani1: Selection by YSD for higher affinity to target 10nM
YSD K d titration for Reo’s “Y2” mutant 90nM50nM10nM
Current Projects Expression of I-SceI (wt coding sequence) in S. cerevisiae Design of REO-I-SceI (Restriction Enzyme- saturated ORF) for target of canine X-SCID site Future projects: REO-I-CreI monomer and REO- I-MsoI monomer
Design of REO-I-SceI Optimized codon usage for S. cerevisiae expression Eliminated splice donors and acceptors Inserted silent restriction sites around desired motifs. I-SceI