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. 2011 Jul 20;6(7):e22554. doi: 10.1371/journal.pone.0022554

Figure 2. Engineering scheme for the isolation of polyQ cleaving variants.

Figure 2

Screening of the S2 pocket variants for P2-Q cleavage yielded 54 positive candidates (12 from HKKL, 23 from VMHK, and 19 from MHKK libraries). Screening of the S1' pocket variants for P1'-Q cleavage resulted in 23 clones from the LPL library. These variants were combined through shuffling to generate a pool of variants in which each protease possesses S2 as well as S1' pocket mutations. These variants were then screened for Q8 substrate cleavage yielding 11 candidates with low activity. These mutants were further subjected to two rounds of error prone PCR and shuffling coupled with selection through GASP to generate 44 variants with moderate activity.