Multiple Site‐Specific Installations of Nε‐Monomethyl‐L‐Lysine into Histone Proteins by Cell‐Based and Cell‐Free Protein Synthesis. Issue 12 (25th July 2014)

• Record Type: Journal Article
• Authors: Yanagisawa, Tatsuo; Takahashi, Mihoko; Mukai, Takahito; Sato, Shin; Wakamori, Masatoshi; Shirouzu, Mikako; Sakamoto, Kensaku; Umehara, Takashi; Yokoyama, Shigeyuki
• Is Part Of: Chembiochem. Volume 15:Issue 12(2014)
• Page Start: 1830
• Page End: 1838
• Publication Date: 2014-07-25
• Subjects: Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201402291
• Languages: English
• ISSNs: 1439-4227

Abstract

<abstract abstract-type="main" xml:lang="en"> <title>Abstract</title> <p>Lysine methylation is one of the important post‐translational modifications of histones, and produces an <italic>N</italic><sup><italic>ε</italic></sup>‐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 <italic>N</italic><sup><italic>ε</italic></sup>‐monomethyllysine, with cysteine substituting for lysine. Here, we have developed a new method of preparing histones bearing multiple <italic>N</italic><sup><italic>ε</italic></sup>‐monomethyllysine residues at specified positions. Release factor 1‐knockout (RFzero) <italic>Escherichia coli</italic> 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 non‐canonical amino acids. During protein synthesis, a <italic>tert</italic>‐butyloxycarbonyl‐protected <italic>N</italic><sup><italic>ε</italic></sup>‐monomethyllysine analogue is ligated to <italic>Methanosarcina mazei</italic> pyrrolysine tRNA (tRNA<sup>Pyl</sup>) by <italic>M. mazei</italic> 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 <italic>N</italic><sup><italic>ε</italic></sup>‐monomethyllysine residues. We installed <italic>N</italic><sup><italic>ε</italic></sup>‐monomethyllysine residues at positions 4, 9, 27, 36, and/or 79 of human histone H3. Each of the <italic>N</italic><sup><italic>ε</italic></sup>‐monomethyllysine residues within the produced histone H3 was recognized by its specific antibody. Furthermore, the antibody recognized the authentic <italic>N</italic><sup><italic>ε</italic></sup>‐monomethyllysine residue at position 27 better than the <italic>N</italic><sup><italic>ε</italic></sup>‐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 <italic>N</italic><sup><italic>ε</italic></sup>‐monomethyllysines at multiple positions within a protein for large‐scale production.</p> </abstract>