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

Abstract

It is stated that BsaXI 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 bsaXIS and bsaXIRM genes in Escherichia coli. The 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 [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 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. The 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.

Keywords: BsaXI specificity; RM fusion and S subunits; TRD1-CR1; Type IIB restriction endonuclease; circular permutation of BsaXI S subunit.

FIGURE 1

Purification of BsaXI RM and S subunits and BsaXI enzyme complex. (A) A BsaXI enzyme complex (RM + S) purified by 6-steps chromatography (see Materials and Methods section). The BsaXI was analyzed on SDS-PAGE by 3-fold serial dilutions. The predicted molecular mass of the RM and S subunits are 107 and 55 kDa, respectively. M, protein molecular mass ladder (NEB). (B) An SDS-PAGE analysis of partially purified BsaXI S and RM subunits. The BsaXI RM and S subunits were purified separately by chromatography through chitin and heparin columns.

FIGURE 2

Recombinant BsaXI activity assays. (A) A BsaXI restriction activity by co-purified BsaXI enzyme (RM + S). The specific activity of BsaXI was estimated at 22,000 U/mg protein (see unit definition in Materials and Methods section). A complete digestion pattern was observed at 1/64 enzyme dilutions. At high enzyme concentration, the DNA was strongly bound and shifted upward, suggesting enzyme aggregation with the DNA. (B) A BsaXI restriction activity reconstituted by mixing purified RM (chitin/DEAE/Heparin columns) and S (6xHis) × (Ni agarose column) subunits in restriction digestion of λ-DNA. Fixed amount of RM subunit (1 μg, at ∼93.5 nM, lanes 1–3) was mixed with varying amount of S (6xHis) (0.25, 1, and 2 μg, at 90.9, 363.6, and 727.2 nM). The RM to S ratio approximately at 1/1, 1/4, and 1/8 in lanes 1–3; lane 4, BsaXI RM subunit only; lane 5, BsaXI positive control (4 U); lane 6, uncut DNA; Lane 7, 2-log, DNA size ladder (0.1 to 10 kb, NEB).

FIGURE 3

Reconstitution of restriction activity by mixing WT RM subunits with S variants. (A) DNA cleavage patterns of pBR322 generated by reconstitution of BsaXI RM subunit with S variants TRD1-CR1 or TRD1-CR1 deletion variants (8-aa, 15-aa, 32-aa deletions in CR1). Fixed amount of RM subunit (1 μg, 94 nM) was mixed with two concentrations of the S variants (185 and 370 nM of 8-aa, 15-aa, 32-aa deletions in CR1, respectively). (B) The DNA digestion patterns of pBR322 by reconstitution of BsaXI RM (1 μg, 94 nM) subunit with S variants TRD1-CR1 (370, 185, and 93 nM, lanes 1–3), TRD1-CR2 (CR2 replacing CR1) (1450, 725, 363, 181, and 91 nM, lanes 4–8). High concentration of TRD1-CR2 inhibited activity (lane 4). Digestions were carried out in 1 × CutSmart buffer at 37°C for 1 h. No BsaXI sites are present in pBR322, so the observed cleavage suggested a new restriction specificity. Lane 9, RM subunit only. Lane 10, uncut DNA. (C) Computer generated virtual cleavage of pBR322 in the sites 5′ AC N5 GT 3′ by NEBcutter.

FIGURE 4

The DNA run-off sequencing of 2xTRD1 (TRD1-CR1-TRD1-CR2)/RM digested pBR322 DNA to show the cut sites outside of 5′ AC N5 GT 3′. When the template strand is cut, the sequencing Taq DNA polymerase adds an extra peak “A” (adenine) to the sequencing read after the broken backbone, creating doublets such as A/T, A/C, or A/G. If the original base call is “A”, then the extra overlapping “A” will create a high “A” peak. The sequencing peaks after the broken template will significantly drop off (DNA run-off). In a partially digested template, some DNA molecules are cut, while others remain intact and the base calls sometimes continue after the sudden peak drop off. Three examples of cut sites from partial digestion are shown near AC N5 GT sites (cleavage taking place either upstream or downstream or on both sides N9-12). Panels 1 and 3 show cleavage upstream at N12 and panel 2 shows cleavage downstream at N9 as indicated by the high “A” peak or a sudden drop in peak height.

FIGURE 5

DNA run-off sequencing of digested pBR322 by circular permutated TRD2-CR2-TRD1-CR1 + RM subunits. Examples of two cut sites are shown near 5′ GGAG N5 GT 3′. The recognition sequence is identical to the WT enzyme. But the cleavage distance is slightly variable (N12-13 and N10-13). Sequence shaded in yellow: poor sequence read as indicated by the sequence editing software (DNAStar/lasergene). Sequence in the black box indicates the recognition sequence 5′ GGAG N5 GT 3′.