Recombinant Expression of Mif2 Proteins

Expression of core histones was described previously (Mizuguchi et al. 2007 (link); Xiao et al. 2011 (link)). Plasmids expressing full-length Mif2 and truncated forms, residues 256–530 of the Mif2c dimer, residues 345–549, and residues 365–530, were a generous gift from S.C. Harrison (Cohen et al. 2008 (link)). Plasmids expressing Mif2 256–365 and 256–356 and the Mif2c^ath dimer with the AT-hook motif (residues 357–364) GRPRGRPK changed to AAADADAA were synthesized and cloned into the expression vector pET28a (Celtek Genes). Additional DHBD and human CENP-C motif constructs were also synthesized and cloned into pET28a (GenScript USA, Inc.). For more efficient removal of the His tag, the thrombin cleavage site of the original Mif2(256–530) plasmid was replaced with a precision proteinase cleavage site. Details are available on request.

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Xiao H., Wang F., Wisniewski J., Shaytan A.K., Ghirlando R., FitzGerald P.C., Huang Y., Wei D., Li S., Landsman D., Panchenko A.R, & Wu C. (2017). Molecular basis of CENP-C association with the CENP-A nucleosome at yeast centromeres. Genes & Development, 31(19), 1958-1972.

Publication 2017

pET28a

Cenp c Cleavage Genes Histones Human Motif at hook Plasmid Proteinase a Thrombin Vector

Corresponding Organization :

Other organizations : National Institutes of Health, National Cancer Institute, Janelia Research Campus, Howard Hughes Medical Institute, National Center for Biotechnology Information, National Institute of Diabetes and Digestive and Kidney Diseases, Center for Cancer Research, Johns Hopkins Medicine, Johns Hopkins University

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Variable analysis

independent variables

Expression of full-length Mif2 and truncated forms, including residues 256–530 of the Mif2c dimer, residues 345–549, and residues 365–530
Expression of Mif2 256–365 and 256–356
Expression of the Mif2c^ath dimer with the AT-hook motif (residues 357–364) GRPRGRPK changed to AAADADAA
Expression of additional DHBD and human CENP-C motif constructs

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

controls

None specified

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