Expression and Purification of BsaXI Restriction Endonuclease and Engineering New Specificity from BsaXI Specificity (S) Subunit

BsaXI is a Type IIB restriction endonuclease (REase) that cleaves both sides of its recognition sequence 5 {downarrow}N9 AC N5 CTCC N10{downarrow} 3 (complement strand 5 {downarrow}N7 GGAG N5 GT N12{downarrow} 3), creating 3-base 3 overhangs. Here we report the cloning and expression of bsaXIS and bsaXIRM genes in E. coli. BsaXI activity was successfully reconstituted by mixing the BsaXI RM fusion subunit with the BsaXI 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 (TRD, target recognition domain, CR, conserved region) 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. Circular permutation of TRD1 and TRD2, i.e. 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 BsaXI 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 E. coli, 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. Genome mining of BsaXI 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.