BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism

Abstract

The GGCC-specific restriction endonuclease BspRI is one of the few Type IIP restriction endonucleases, which were suggested to be a monomer. Amino acid sequence information obtained by Edman sequencing and mass spectrometry analysis was used to clone the gene encoding BspRI. The bspRIR gene is located adjacently to the gene of the cognate modification methyltransferase and encodes a 304 aa protein. Expression of the bspRIR gene in Escherichia coli was dependent on the replacement of the native TTG initiation codon with an ATG codon, explaining previous failures in cloning the gene using functional selection. A plasmid containing a single BspRI recognition site was used to analyze kinetically nicking and second-strand cleavage under steady-state conditions. Cleavage of the supercoiled plasmid went through a relaxed intermediate indicating sequential hydrolysis of the two strands. Results of the kinetic analysis of the first- and second-strand cleavage are consistent with cutting the double-stranded substrate site in two independent binding events. A database search identified eight putative restriction-modification systems in which the predicted endonucleases as well as the methyltransferases share high sequence similarity with the corresponding protein of the BspRI system. BspRI and the related putative restriction endonucleases belong to the PD-(D/E)XK nuclease superfamily.

Identification and characterization of CbeI, a novel thermostable restriction enzyme from Caldicellulosiruptor bescii DSM 6725 and a member of a new subfamily of HaeIII-like enzymes

“…The cloning and expression of CbeI in E. coli presented some challenges, as does the expression of other toxic genes including other restriction endonucleases [12, 24]. In addition to using BL21-CodonPlus(DE3)-RIPL cells to compensate for differences in codon usage between E. coli and C. bescii , we took advantage of the fact that CbeI is from an extreme thermophile and would be expected to have minimal activity at temperatures significantly below the growth temperature of C. bescii ( T opt ≈ 80°C).…”

Type II Restriction-Modification System from Gardnerella vaginalis ATCC 14018

“…A BLASTp search with the R.Gva14018I sequence found 39% identity and 57% similarity with the restriction endonuclease BspRI (GenBank CBE66553.1), a single protein with a known function among multiple matches. BspRI is an experimentally determined Type IIP REase that cleaves DNA at the sequence GGCC to produce blunt-end fragments [ 38 ]. Sequence analysis of R.Gva14018I using the PDEXK server [ 39 ] showed a probability of 0.95 that the nuclease belongs to the PD-(D/E)XK superfamily.…”

“…Moreover, similarity searches for M.Gva14018I showed many C-5 cytosine-specific DNA MTases, which generate GGm5CC methylation on both DNA strands. G. vaginalis ATCC 14018 possesses a HaeIII-like R-M system, which is widespread among bacterial species [ 38 , 40 , 43 ]. A HaeIII-like restriction endonuclease activity is an effective tool against foreign DNA because the short cleavage site frequently occurs [ 43 ].…”

“…Recombinant M.Gva14018I was active in vivo. Cloning and expression of R.Gva14018I in E. coli cells was challenging due to toxicity, which is common for REases [ 38 , 44 ]. R.Gva14018I mutants were successfully expressed in E. coli .…”

Thermostable proteins bioprocesses: The activity of restriction endonuclease-methyltransferase from Thermus thermophilus (RM.TthHB27I) cloned in Escherichia coli is critically affected by the codon composition of the synthetic gene

“…It has been shown that replacing the TTG initiation codon with an ATG codon resulted in high-level expression of a previously silent heterologous gene in E . coli [ 19 ]. To address the potential problems mentioned above selection of properly engineered recombinant host, silencing endogenous proteolytic enzymes or co-expression of cloned chaperons may highly stimulate recombinant protein production [ 20 ].…”