Expression and purification of BmrI restriction endonuclease and its N-terminal cleavage domain variants.
BmrI (ACTGGG N5/N4) is one of the few metal-independent restriction endonucleases (REases) found in bacteria. The BmrI restriction-modification system was cloned by the methylase selection method, inverse PCR, and PCR. BmrI REase shows significant amino acid sequence identity to BfiI and a putative endonuclease MspBNCORF3798 from the sequenced Mesorhizobium sp. BNC1 genome. The EDTA-resistant BmrI REase was successfully over-expressed in a pre-modified E. coli strain from pET21a or pBAC-expIQ vectors. The recombinant BmrI REase shows strong promiscuous activity (star activity) in NEB buffers 1, 4, and an EDTA buffer. Star activity was diminished in buffers with 100-150 mM NaCl and 10 mM MgCl(2). His-tagged BmrI192, the N-terminal cleavage domain of BmrI, was expressed in E. coli and purified from inclusion bodies. The refolded BmrI192 protein possesses non-specific endonuclease activity. BmrI192 variants with a single Ser to Cys substitution (S76C or S90C) and BmrI200 (T200C) with a single Cys at the C-terminal end were also constructed and purified. BmrI200 digests both single-strand (ss) and double-strand (ds) DNA and the nuclease activity on ss DNA is at least 5-fold higher than that on ds DNA. The Cys-containing BmrI192 and BmrI200 nuclease variants may be useful for coupling to other DNA binding elements such as synthetic zinc fingers, thio-containing locked nucleic acids (LNA) or peptide nucleic acids (PNA).
Bao
Yongming
Y
New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
Higgins
Lauren
L
Zhang
Penghua
P
Chan
Siu-Hong
SH
Laget
Sophie
S
Sweeney
Suzanne
S
Lunnen
Keith
K
Xu
Shuang-Yong
SY
D000595:Amino Acid Sequence; D001410:Bacillus megaterium / Q000201:enzymology; D003001:Cloning, Molecular; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D010957:Plasmids; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013014:SOS Response, Genetics; D013696:Temperature
D011993:Recombinant Fusion Proteins; D015252:Deoxyribonucleases, Type II Site-Specific