US 11,739,128 B2
Peptide ligase-mediated engineering of recombinant nucleosomes
Martis William Cowles, Chapel Hill, NC (US); Michael-Christopher Keogh, Cambridge, MA (US); Jonathan M. Burg, Durham, NC (US); and Zu-Wen Sun, Brentwood, TN (US)
Assigned to EPICYPHER, INC., Durham, NC (US)
Appl. No. 16/977,218
Filed by EPICYPHER, INC., Durham, NC (US)
PCT Filed Mar. 7, 2019, PCT No. PCT/US2019/021106
§ 371(c)(1), (2) Date Sep. 1, 2020,
PCT Pub. No. WO2019/173565, PCT Pub. Date Sep. 12, 2019.
Claims priority of provisional application 62/639,802, filed on Mar. 7, 2018.
Prior Publication US 2021/0040164 A1, Feb. 11, 2021
Int. Cl. C07K 14/47 (2006.01); C12N 9/00 (2006.01)
CPC C07K 14/47 (2013.01) [C12N 9/93 (2013.01)] 16 Claims
 
1. A method for ligating a histone peptide onto a fully assembled recombinant nucleosome, the nucleosome comprising core histones and a polynucleotide wrapped around the core histones; the method comprising:
(a) contacting a histone peptide with a transpeptidase to produce a histone peptide-transpeptidase intermediate, wherein the histone peptide comprises a C-terminal transpeptidase recognition site; and
(b) contacting the histone peptide-transpeptidase intermediate with a fully assembled recombinant nucleosome comprising a modified core histone protein in which the N-terminal tail has been deleted and an amino acid sequence suitable for transpeptidase mediated ligation is added to the N-terminus under conditions sufficient for ligation of the histone peptide to the modified core histone protein;
thereby producing a fully assembled recombinant nucleosome with a histone peptide ligated thereto.