Multiple Site-Specific Installations of N~ε-Monomethyl-l-Lysine into Histone Proteins by Cell-Based and Cell-Free Protein Synthesis

摘要

Lysine methylation is one of the important post-translational modifications of histones, and produces an N~ε-mono-, di-, or trimethyllysine residues. Multiple and site-specific lysine methylations of histones are essential to define epigenetic statuses and control heterochromatin formation, DNA repair, and transcription regulation. A method was previously developed to build an analogue of N~ε-monomethyllysine, with cysteine substituting for lysine. Here, we have developed a new method of preparing histones bearing multiple Ne-monomethyllysine residues at specified positions. Release factor 1-knockout (RFzero) Escherichia coli cells or a cell-free system based on the RFzero cell lysate was used for protein synthesis, as in RFzero cells UAG is redefined as a sense codon for noncanonical amino acids. During protein synthesis, a tert-butyloxycarbonyl-protected N~ε-monomethyllysine analogue is ligated to Methanosarcina mazei pyrrolysine tRNA (tRNA~(Pyl)) by M. mazei pyrrolysyl-tRNA synthetase mutants, and is translationally incorporated into one or more positions specified by the UAG codon. Protecting groups on the protein are then removed with trifluoroacetic acid to generate N~ε-monomethyllysine residues. We installed Ne-monomethyllysine residues at positions 4, 9, 27, 36, and/or 79 of human histone H3. Each of the N~ε-monomethyllysine residues within the produced histone H3 was recognized by its specific antibody. Furthermore, the antibody recognized the authentic N~ε-monomethyllysine residue at position 27 better than the N~ε-monomethyllysine analogue built with cysteine. Mass spectrometry analyses also confirmed the lysine modifications on the produced histone H3. Thus, our method enables the installation of authentic N~ε-monomethyllysines at multiple positions within a protein for largescale production.