Engineering of Methylation State Specific 3xMBT Domain Using ELISA Screening.
The ε-amino group of lysine residues may be mono-, di- or tri-methylated by protein lysine methyltransferases. In the past few years it has been highly considered that methylation of both histone and non-histone proteins has fundamental role in development and progression of various human diseases. Thus, the establishment of tools to study lysine methylation that will distinguish between the different states of methylation is required to elucidate their cellular functions. The 3X malignant brain tumor domain (3XMBT) repeats of the Lethal(3)malignant brain tumor-like protein 1 (L3MBTL1) have been utilized in the past as an affinity reagent for the identification of mono- and di-methylated lysine residues on individual proteins and on a proteomic scale. Here, we have utilized the 3XMBT domain to develop an enzyme-linked immunosorbent assay (ELISA) that allows the high-throughput detection of 3XMBT binding to methylated lysines. We demonstrated that this system allows the detection of methylated peptides, methylated proteins and PKMT activity on both peptides and proteins. We also optimized the assay to detect 3XMBT binding in crude E. coli lysates which facilitated the high throughput screening of 3XMBT mutant libraries. We have utilized protein engineering tools and generated a double site saturation 3XMBT library of residues 361 and 411 that were shown before to be important for binding mono and di-methylated substrates and identified variants that can exclusively recognize only di-methylated peptides. Together, our results demonstrate a powerful new approach that will contribute to deeper understanding of lysine methylation biology and that can be utilized for the engineering of domains for specific binders of other post-translational modifications.
The Shraga Segal Department of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, Be'er Sheva, Israel.|The National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev, Be'er Sheva, Israel.
Dan Od
DO
Cohen
Department of Life Sciences, Ben-Gurion University of the Negev, Be'er Sheva, Israel.|The National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev, Be'er Sheva, Israel.
Shai
S
Duchin
The Shraga Segal Department of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, Be'er Sheva, Israel.|The National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev, Be'er Sheva, Israel.
Michal
M
Feldman
Department of Life Sciences, Ben-Gurion University of the Negev, Be'er Sheva, Israel.|The National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev, Be'er Sheva, Israel.
Raz
R
Zarivach
Department of Life Sciences, Ben-Gurion University of the Negev, Be'er Sheva, Israel.|The National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev, Be'er Sheva, Israel.
Amir
A
Aharoni
The Shraga Segal Department of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, Be'er Sheva, Israel.|The National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev, Be'er Sheva, Israel.
Dan
D
Levy
D000595:Amino Acid Sequence; D001681:Biological Assay; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D000072417:Protein Domains; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D054852:Small Molecule Libraries / Q000378:metabolism; D025521:Tumor Suppressor Proteins
D002868:Chromosomal Proteins, Non-Histone; C120887:L3MBTL1 protein, human; D058619:Maltose-Binding Proteins; D010455:Peptides; D011994:Recombinant Proteins; D012097:Repressor Proteins; D054852:Small Molecule Libraries; D025521:Tumor Suppressor Proteins; D008239:Lysine