Penghua Zhang
New England Biolabs
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Featured researches published by Penghua Zhang.
Nucleic Acids Research | 2007
Shuang-yong Xu; Zhenyu Zhu; Penghua Zhang; Siu-Hong Chan; James C. Samuelson; Jian-ping Xiao; Debra Ingalls; Geoffrey G. Wilson
BsrDI and BtsI restriction endonucleases recognize and cleave double-strand DNA at the sequences GCAATG (2/0) and GCAGTG (2/0), respectively. We have purified and partially characterized these two enzymes, and analyzed the genes that encode them. BsrDI and BtsI are unusual in two respects: each cleaves DNA as a heterodimer of one large subunit (B subunit) and one small subunit (A subunit); and, in the absence of their small subunits, the large subunits behave as sequence-specific DNA nicking enzymes and only nick the bottom strand of the sequences at these respective positions: GCAATG (−/0) and GCAGTG (−/0). We refer to the single subunit, the bottom-strand nicking forms as ‘hemidimers’. Amino acid sequence comparisons reveal that BsrDI and BtsI belong to a family of restriction enzymes that possess two catalytic sites: a canonical PD-Xn-EXK and a second non-canonical PD-Xn-E-X12-QR. Interestingly, the other family members, which include BsrI (ACTGG 1/−1) and BsmI/Mva1269I (GAATGC 1/−1) are single polypeptide chains, i.e. monomers, rather than heterodimers. In BsrDI and BtsI, the two catalytic sites are found in two separate subunits. Site-directed mutagenesis confirmed that the canonical catalytic site located at the N-terminus of the large subunit is responsible for the bottom-strand cleavage, whereas the non-canonical catalytic site located in the small subunit is responsible for hydrolysis of the top strand. Top-strand specific nicking variants, Nt.BsrDI and Nt.BtsI, were successfully engineered by combining the catalytic-deficient B subunit with wild-type A subunit.
Protein Expression and Purification | 2008
Yongming Bao; Lauren Higgins; Penghua Zhang; Siu-Hong Chan; Sophie Laget; Suzanne Sweeney; Keith D. Lunnen; Shuang-yong Xu
BmrI (ACTGGG N5/N4) is one of the few metal-independent restriction endonucleases (REases) found in bacteria. The BmrI restriction-modification system was cloned by the methylase selection method, inverse PCR, and PCR. BmrI REase shows significant amino acid sequence identity to BfiI and a putative endonuclease MspBNCORF3798 from the sequenced Mesorhizobium sp. BNC1 genome. The EDTA-resistant BmrI REase was successfully over-expressed in a pre-modified E. coli strain from pET21a or pBAC-expIQ vectors. The recombinant BmrI REase shows strong promiscuous activity (star activity) in NEB buffers 1, 4, and an EDTA buffer. Star activity was diminished in buffers with 100-150 mM NaCl and 10 mM MgCl(2). His-tagged BmrI192, the N-terminal cleavage domain of BmrI, was expressed in E. coli and purified from inclusion bodies. The refolded BmrI192 protein possesses non-specific endonuclease activity. BmrI192 variants with a single Ser to Cys substitution (S76C or S90C) and BmrI200 (T200C) with a single Cys at the C-terminal end were also constructed and purified. BmrI200 digests both single-strand (ss) and double-strand (ds) DNA and the nuclease activity on ss DNA is at least 5-fold higher than that on ds DNA. The Cys-containing BmrI192 and BmrI200 nuclease variants may be useful for coupling to other DNA binding elements such as synthetic zinc fingers, thio-containing locked nucleic acids (LNA) or peptide nucleic acids (PNA).
Protein Expression and Purification | 2010
Penghua Zhang; Priscilla Hiu-Mei Too; James C. Samuelson; Siu-Hong Chan; Tamas Vincze; Stephanie Doucette; Stefan Bäckström; Konstantinos Potamousis; Timothy M. Schramm; Dan Forrest; David C. Schwartz; Shuang-yong Xu
Archive | 2011
Zhenyu Zhu; Yu Zheng; Shengxi Guan; Hua Wang; Aine Quimby; Penghua Zhang; Lynne Apone
Archive | 2008
Zhenyu Zhu; Aine Blanchard; Shuang Yong Xu; Shengxi Guan; Hua Wei; Penghua Zhang; Dapeng Sun; Siu-Hong Chan
Archive | 2011
Zhenyu Zhu; Yu Zheng; Hua Wang; Aine Quimby; Lynne Apone; Penghua Zhang; Shengxi Guan
Archive | 2011
Zhu Zhenyu; Zheng Yu; Hua Wang; Aine Quimby; Lynne Apone; Penghua Zhang; Shengxi Guan
Archive | 2011
Zhu Zhenyu; Zheng Yu; Hua Wang; Aine Quimby; Lynne Apone; Penghua Zhang; Shengxi Guan
Archive | 2008
Zhenyu Zhu; Aine Blanchard; Shuang-yong Xu; Shengxi Guan; Hua Wei; Penghua Zhang; Dapeng Sun; Siu-Hong Chan
Archive | 2008
Zhenyu Zhu; Aine Blanchard; Shuang-yong Xu; Shengxi Guan; Hua Wei; Penghua Zhang; Dapeng Sun; Siu-Hong Chan