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Applied and Environmental Microbiology, October 1998, p. 3669-3673, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Characterization of an Extremely Thermostable Restriction Enzyme, PspGI, from a Pyrococcus Strain and Cloning of the PspGI Restriction-Modification System in Escherichia coli

Richard Morgan, Jian-ping Xiao, and Shuang-yong Xu*

New England Biolabs, Inc., Beverly, Massachusetts 01915

Received 8 June 1998/Accepted 31 July 1998

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°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°C. PspGI remained active following 30 cycles of thermocycling; thus, it can be used in DNA-based diagnostic applications.

* Corresponding author. Mailing address: New England Biolabs, Inc., 32 Tozer Rd., Beverly, MA 01915. Phone: (978) 927-5054. Fax: (978) 921-1350. E-mail: xus{at}neb.com.

Applied and Environmental Microbiology, October 1998, p. 3669-3673, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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