Feb 14, 2023
Version 1
Expression, purification and characterization of the GpC methyltransferase M.CviPI V.1
- Karine Lapouge1,
- Rozemarijn Kleinendorst2,
- Kasit Chatsirisupachai2,
- Nikolay Dobrev1,
- Kim Remans1,
- Arnaud Krebs2
- 1European Molecular Biology Laboratory (EMBL), Protein Expression and Purification Core Facility, 69117, Heidelberg, Germany;
- 2European Molecular Biology Laboratory (EMBL), Genome Biology unit, Meyerhofstraße 1, 69117 Heidelberg. 2European Molecular Biology Laboratory (EMBL)
Protocol Citation: Karine Lapouge, Rozemarijn Kleinendorst, Kasit Chatsirisupachai, Nikolay Dobrev, Kim Remans, Arnaud Krebs 2023. Expression, purification and characterization of the GpC methyltransferase M.CviPI. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly776plx9/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 02, 2022
Last Modified: February 14, 2023
Protocol Integer ID: 72195
Keywords: characterization of the gpc methyltransferase, gpc methyltransferase, endogenous dna methylation11, concentration compatible with dna footprinting application, binding of general transcription factor, dna footprinting application, transcription factor, general transcription factor, enzymes with various nucleotide specificity, rna pol ii6, dna contacts at the resolution, various nucleotide specificity, dna contact, rna, individual dna molecules1, genome, dna, protein, enzyme, purified enzyme, methyl, purification
Abstract
Methylation footprinting can be used to map protein-DNA contacts at the resolution of individual DNA molecules1. Enzymes with various nucleotide specificities have been successfully used to footprint genomes including GpC, CpG and A methyl-transfereases2–7. Among these M.CviPI methylate DNA in GpC context, that is distinct from CpGs that are endogenously methylated in mammals. This feature has been leveraged to profile nucleosome occupancy8,9; the binding of General Transcription Factors and RNA Pol II6; the co-occupancy of Transcription Factors (TFs)10 and the relation between TF binding and endogenous DNA methylation11. Here, we present a protocol for the production and purification of M.CviPI in E. coli. Our protocol routinely yields milligrams of protein at a quality and a concentration compatible with DNA footprinting applications. We characterize the purity and the activity of the purified enzyme, providing a benchmark for future production.
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