Cloning and expression of AatII restriction-modification system in Escherichia coli.
The genes encoding the AatII restriction endonuclease and methylase from Acetobacter aceti have been cloned and expressed in Escherichia coli. The nucleotide sequences of aatIIM and aatIIR genes were determined. The aatIIM and aatIIR genes are 996 bp and 1038 bp, respectively, encoding the 331-aa methylase with a predicted molecular mass of 36.9 kDa, and the 345-aa AatII restriction endonuclease with a predicted molecular mass of 38.9 kDa. The two genes overlap by 4 base pairs and are transcribed in the same orientation. The aatIIRM genes are located next to a putative gene for plasmid mobilization. A stable overproducing strain was constructed, in which the aatIIM gene was expressed from a pSC101-derived plasmid. The aatIIR gene was inserted into a modified T7 expression vector that carries transcription terminators upstream from the T7 promoter. The recombinant AatII restriction endonuclease was purified to near homogeneity by chromatography through DEAE Sepharose, Heparin Sepharose, and phosphocellulose columns.
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New England Biolabs, Inc., Beverly, MA 01915, USA.
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D000091:Acetobacter / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D002845:Chromatography; D003001:Cloning, Molecular; D015254:DNA Modification Methylases / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D011994:Recombinant Proteins; D015254:DNA Modification Methylases; D015252:Deoxyribonucleases, Type II Site-Specific; C069035:GACGTC-specific type II deoxyribonucleases