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Engineering BspQI n...
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Zhang, Penghua
(författare)
Engineering BspQI nicking enzymes and application of N.BspQI in DNA labeling and production of single-strand DNA
- Artikel/kapitelEngelska2010
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Nummerbeteckningar
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LIBRIS-ID:oai:gup.ub.gu.se/139229
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https://gup.ub.gu.se/publication/139229URI
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https://doi.org/10.1016/j.pep.2009.09.003DOI
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Ämneskategori:ref swepub-contenttype
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Ämneskategori:art swepub-publicationtype
Anmärkningar
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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).
Ämnesord och genrebeteckningar
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iis restriction-endonucleases
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escherichia-coli
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homing endonuclease
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modification system
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crystal-structure
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site
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amplification
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cloning
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binding
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expression
Biuppslag (personer, institutioner, konferenser, titlar ...)
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Too, Priscilla Hiu-Mei
(författare)
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Samuelson, James C.
(författare)
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Chan, Siu-Hong
(författare)
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Vincze, Tamas
(författare)
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Doucette, Stephanie
(författare)
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Bäckström, Stefan,1969Gothenburg University,Göteborgs universitet,Institutionen för biomedicin, avdelningen för medicinsk kemi och cellbiologi,Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology(Swepub:gu)xbacst
(författare)
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Potamousis, Konstantinos D.
(författare)
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Schramm, Timothy M.
(författare)
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Forrest, Dan
(författare)
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Schwartz, David C.
(författare)
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Xu, Shuang-yong
(författare)
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Göteborgs universitetInstitutionen för biomedicin, avdelningen för medicinsk kemi och cellbiologi
(creator_code:org_t)
Sammanhörande titlar
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Ingår i:Protein Expression and Purification: Elsevier BV69:2, s. 226-2341046-5928
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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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visa fler...
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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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Protein Expressi ...
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Göteborgs universitet