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Engineering BspQI n...
Engineering BspQI nicking enzymes and application of N.BspQI in DNA labeling and production of single-strand DNA
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Zhang, Penghua (author)
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Too, Priscilla Hiu-Mei (author)
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Samuelson, James C. (author)
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Chan, Siu-Hong (author)
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Vincze, Tamas (author)
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Doucette, Stephanie (author)
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- Bäckström, Stefan, 1969 (author)
- Gothenburg University,Göteborgs universitet,Institutionen för biomedicin, avdelningen för medicinsk kemi och cellbiologi,Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
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Potamousis, Konstantinos D. (author)
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Schramm, Timothy M. (author)
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Forrest, Dan (author)
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Schwartz, David C. (author)
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Xu, Shuang-yong (author)
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(creator_code:org_t)
- Elsevier BV, 2010
- 2010
- English.
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In: Protein Expression and Purification. - : Elsevier BV. - 1046-5928. ; 69:2, s. 226-234
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Abstract
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- BspQI is a thermostable Type IIS restriction endonuclease (REase) with the recognition sequence 5′GCTCTTC N1/N4 3′. Here we report the cloning and expression of the bspQIR gene for the BspQI restriction enzyme in Escherichia coli. Alanine scanning of the BspQI charged residues identified a number of DNA nicking variants. After sampling combinations of different amino acid substitutions, an Nt.BspQI triple mutant (E172A/E248A/E255K) was constructed with predominantly top-strand DNA nicking activity. Furthermore, a triple mutant of BspQI (Nb.BspQI, N235A/K331A/R428A) was engineered to create a bottom-strand nicking enzyme. In addition, we demonstrated the application of Nt.BspQI in optical mapping of single DNA molecules. Nt or Nb.BspQI-nicked dsDNA can be further digested by E. coli exonuclease III to create ssDNA for downstream applications. BspQI contains two potential catalytic sites: a top-strand catalytic site (Ct) with a D-H-N-K motif found in the HNH endonuclease family and a bottom-strand catalytic site (Cb) with three scattered Glu residues. BlastP analysis of proteins in GenBank indicated a putative restriction enzyme with significant amino acid sequence identity to BspQI from the sequenced bacterial genome Croceibacter atlanticus HTCC2559. This restriction gene was amplified by PCR and cloned into a T7 expression vector. Restriction mapping and run-off DNA sequencing of digested products from the partially purified enzyme indicated that it is an EarI isoschizomer with 6-bp recognition, which we named CatHI (CTCTTC N1/N4).
Keyword
- iis restriction-endonucleases
- escherichia-coli
- homing endonuclease
- modification system
- crystal-structure
- site
- amplification
- cloning
- binding
- expression
Publication and Content Type
- ref (subject category)
- art (subject category)
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- By the author/editor
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Zhang, Penghua
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Too, Priscilla H ...
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Samuelson, James ...
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Chan, Siu-Hong
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Vincze, Tamas
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Doucette, Stepha ...
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show more...
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Bäckström, Stefa ...
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Potamousis, Kons ...
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Schramm, Timothy ...
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Forrest, Dan
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Schwartz, David ...
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Xu, Shuang-yong
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show less...
- Articles in the publication
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Protein Expressi ...
- By the university
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University of Gothenburg