Characterization of an extremely thermostable restriction enzyme, PspGI, from a Pyrococcus strain and cloning of the PspGI restriction-modification system in Escherichia coli.
An extremely thermostable restriction endonuclease, PspGI, was purified from Pyrococcus sp. strain GI-H. PspGI is an isoschizomer of EcoRII and cleaves DNA before the first C in the sequence 5' CCWGG 3' (W is A or T). PspGI digestion can be carried out at 65 to 85 degrees C. To express PspGI at high levels, the PspGI restriction-modification genes (pspGIR and pspGIM) were cloned in Escherichia coli. M.PspGI contains the conserved sequence motifs of alpha-aminomethyltransferases; therefore, it must be an N4-cytosine methylase. M.PspGI shows 53% similarity to (44% identity with) its isoschizomer, M.MvaI from Micrococcus variabilis. In a segment of 87 amino acid residues, PspGI shows significant sequence similarity to EcoRII and to regions of SsoII and StyD4I which have a closely related recognition sequence (5' CCNGG 3'). PspGI was expressed in E. coli via a T7 expression system. Recombinant PspGI was purified to near homogeneity and had a half-life of 2 h at 95 degrees C. PspGI remained active following 30 cycles of thermocycling; thus, it can be used in DNA-based diagnostic applications.
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New England Biolabs, Inc., Beverly, Massachusetts 01915, USA.
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D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D019711:Pyrococcus / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D011994:Recombinant Proteins; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific