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. 2018 Nov 5;47(1):450–467. doi: 10.1093/nar/gky1059

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© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 7. — Cleavage activities of tethered, single-chain variants of BbvCI. The wild-type R1 and R2 genes were joined in-frame with oligonucleotide linkers that introduced 7, 14, or 21 additional codons between the two genes (Panels A, B and C,respectively). Two independent isolates of each construct were sequence-verified, and the fused protein each encoded was partially purified and titrated in 2-fold dilution steps on linear phage lambda DNA. Reactions were extracted with phenol/dichloromethane and analyzed by 1% agarose gel electrophoresis. The construct with the longest linker (panel C) displayed highest activity (∼6 × 10³ units/ml of extract), corresponding to a specific activity of ∼4 × 10⁶ units per mg of protein, or approximately the same as the two-subunit parental enzyme. The construct with the intermediate linker (panel B) displayed ∼15% of this activity (∼1 × 10³ units/ml extract), and the construct with the shortest linker (panel a) was essentially inactive (<1 unit/ml).