Expression and Purification of XI Restriction Endonuclease and Engineering New Specificity From XI Specificity Subunit.

It is stated that XI is a Type IIB restriction endonuclease (REase) that cleaves both sides of its recognition sequence 5'↓N9 AC N5 CTCC N10↓ 3' (complement strand 5' ↓N7 GGAG N5 GT N12↓ 3'), creating 3-base 3' overhangs. Here we report the cloning and expression of and genes in . The XI activity was successfully reconstituted by mixing the XI RM fusion subunit with the XI S subunit and the enzyme complex further purified by chromatography over 6 columns. As expected, the S subunit consisted of two subdomains encoding TRD1-CR1 [target recognition domain (TRD), conserved region (CR)] for 5' AC 3', and TRD2-CR2 presumably specifying 5' CTCC 3'. TRD1-CR1 (TRD2-CR2 deletion) or duplication of TRD1 (TRD1-CR1-TRD1-CR2) both generated a new specificity 5' AC N5 GT 3' when the S variants were complexed with the RM subunits. The circular permutation of TRD1 and TRD2, i.e., the relocation of TRD2-CR2 to the N-terminus and TRD1-CR1 to the C-terminus generated the same specificity with the RM subunits, although some wobble cleavage was detected. The TRD2 domain in the XI S subunit can be substituted by a close homolog (∼59% sequence identity) and generated the same specificity. However, TRD2-CR2 domain alone failed to express in , but CR1-TRD2-CR2 protein could be expressed and purified which showed partial nicking activity with the RM subunits. This work demonstrated that like Type I restriction systems, the S subunit of a Type IIB system could also be manipulated to create new specificities. The genome mining of XI TRD2 homologs in GenBank found more than 36 orphan TRD2 homologs, implying that quite a few orphan TRD2s are present in microbial genomes that may be potentially paired with other TRDs to create new restriction specificities.