Recombinant Ubiquitin Mutants and Histone Constructs

To prepare ubiquitin-aminoethanethiol (Ub-SH), we used a previously described pTXB1-Ub-AvaDnaE-AAFN-His₆ plasmid.^{24 (link)} Ubiquitin double mutants (Ub1 – E16A, E18A; Ub2 – I44A, F45A) were generated using a Quick Change Mutagenesis kit with the Pfu Ultra II polymerase (Agilent) and the following primers: forward Ub1 5-TTCTCAATGGTGTCACTCGGAGCCACAGCGAGAGTGATGGTCTTACCAG-3; reverse Ub1 5-CTGGTAAGACCATCACTCTCGCTGTGGCTCCGAGTGACACCATTGAGAA-3; forward Ub2 5-CATCTTCCAGCTGTTTCCCAGCAGCAGCCAACCTCTGCTGGTCAGGAGGG-3; reverse Ub2 5-CCCTCCTGACCAGCAGAGGTTGGCTGCTGCTGGGAAACAGCTGGAAGATG-3; forward Ub^Bpa 5-CCAGCTGTTTCCCAGCCTAGATCAACCTCTGCTGG-3; reverse Ub^Bpa 5-CCAGCAGAGGTTGATCTAGGCTGGGAAACAGCTGG-3; forward K119C 5-GTCCGTGCTGCTGCCCAAGTGTACCGAGAGTTCCAAGTCGG-3; reverse K119C 5-CCGACTTGGAACTCTCGGTACACTTGGGCAGCAGCACGGAC-3.
The correct sequence was confirmed by gene sequencing. The plasmids for wild-type Xenopus histones, H2B K120C, H2A N110C, and all DNA constructs have been described before.^{15 (link),25 (link),45 ,46} pEVOL-pBpF (used for Ub^Bpa expression) was a gift from Peter Schultz (Addgene plasmid # 31190).^{30 (link)}