expanding the pool
DESCRIPTION
Expanding the Pool. Characterizing LAGLIDADG Homing Endonuclease Orthologs. Picking the ORF Regions to Use. OK active sites 50-60+ % homology Regions homologous to Ani Optimization Yeast expression, restriction Surface expression?. Intron. ORF. Expression & Relatedness. - PowerPoint PPT PresentationTRANSCRIPT
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Expanding the Pool
Characterizing LAGLIDADG Homing Endonuclease Orthologs
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Picking the ORF Regions to Use
Intron
ORF• OK active sites• 50-60+ % homology• Regions homologous to Ani
• Optimization– Yeast expression, restriction– Surface expression?
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Expression & Relatedness
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Expression problems
• Mutagenesis & directed evolution to regain & improve expression
• Glycosylation changes?– Tas Tin and Vin are predicted to be lacking
glycosylation in regions glycosylated in Pno, Ach, Hje, and Ani
– Tas and Tin are predicted to be glycosylated at DNA binding regions
• Poor surface expressers but otherwise OK
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Target Determination• Target for Ani at intron/exon junctions
• By Homology…
• Central 4 are hard to predict
Ach cytb gene2423 bp
Exon 1 Exon 2Sequence Used
Ani apocytB (cobA) Gene2694 bp
Exon 1 Ani Exon2
TGAGGAGGTT T CT CTGTAAA
TGGGGAGG TTT TTCAGTATC
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Binding Vs Ani Target &Predicted Targets
0.1 1 10 100 10000
100
200
300
400
500
600
700
800
900
1000Against Ani target
Ach
Hje
Pno
Tas
Tin
Vin
Ani (WT)
Ani (E148D)
concentration (nM)
MFI
(APC
)
0.1 1 10 100 10000
500
1000
1500
2000
2500Against own target
Ach
Hje
Pno
Tas
Tin
Vin
Ani (WT, against SCID)
Ani (E148D)
concentration (nM)
MFI
(APC
)
Conclusion: Tas, Tin, and Vin are not producing a viable surfac-expressed HE
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CleavageAc
h
Hje
Pno
Ani
E148
D
Ach
Hje
Pno
Ach
Hje
Pno
Ani
37°C, against Predicted Targ 37°C, against Ani Targ
30°C, againstPredicted Targ
Target Only
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What Changes Are Responsible?
• Variance at DNA-interacting domains– Insertions relative to Ani
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Differences at DNA-Binding Regions
• 5Å interactions as spheres• Unchanged residues in green• Conserved Residues in Purple
+5, +9 & +10 positions (white) +2 & +5 positions (white)
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Insertions• Directly change DNA-interacting regions (left)• May realign directly interacting regions (right)
– Harder to obtain by directed evolution alone
+5, +9 & +10 positions (white) -8 position (white)
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Rosetta Predictions
• -8 A -> G• K24N & T29K
• D73N Conserved residue changed
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Future Directions• Mutagenesis & directed evolution to improve surface
expression and/or activity• Comparing Ani ability to cleave predicted targets to the
corresponding enzyme’s cleavage to evaluate actual changes• Determine specificity with 1-off panels• DNA shuffling to generate hybrid HE’s• Begin to look at which changes are tolerated and which aren’t
Take-home Message• We can use homology searches to find functional homing
endonucleases with different targets• This can help us determine what AA changes affect what target
specificities