Transcript Hoku`s Slides

Several coupled DNA and protein libraries
are constructed, randomizing 3 base pairs
and 5 contacting amino acids for each
NNNGGAGGTTTCTCTGTAAA TGANNNGGTTTCTCTGTAAA
TGAGGAGGTTTCTCTGTNNN
Library-on-library cleavage profiling
Double-stranded target pool
is used to stain yeast
Cleavable targets on
cleaving enzymes are
cut, rest remain intact
Biotinylated linker is
ligated to cleaved
targets
HE-expressing yeast
Streptavidin purification
removes yeast and
non-cleaved targets
Recovering sequences
PCR amplify ligated,
purified oligos
TA clone
Colony sequencing will result in a full target profile!
Can then extract plasmids and create dual-conjugated target
DNA for any sequence recovered
Matching proteins to targets
Create yeast library,
oligo pool
+
+
Recover cleavable
sequences
Choose desired sequence,
PCR amplify (with
conjugates for FACS)
Now haveclone(s)
Recover
which cleave this
matched
sequence
protein/target
pair