Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Summary

Here we present a method to express acetylated histone proteins using genetic code expansion and assemble reconstituted nucleosomes in vitro.

Abstract

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N^ε-acetyl-lysine (AcK)-specific Methanosarcina mazi pyrrolysine tRNA-synthetase (MmAcKRS1) and its cognate tRNA (tRNA^Pyl) to assemble reconstituted mononucleosomes with site specific acetylated histones. MmAcKRS1 and tRNA^Pyl deliver AcK at an amber mutation site in the mRNA of choice during translation in Escherichia coli. This technique has been used extensively to incorporate AcK at H3 lysine sites. Pyrrolysyl-tRNA synthetase (PylRS) can also be easily evolved to incorporate other noncanonical amino acids (ncAAs) for site specific protein modification or functionalization. Here we detail a method to incorporate AcK using the MmAcKRS1 system into histone H3 and integrate acetylated H3 proteins into reconstituted mononucleosomes. Acetylated reconstituted mononucleosomes can be used in biochemical and binding assays, structure determination, and more. Obtaining modified mononucleosomes is crucial for designing experiments related to discovering new interactions and understanding epigenetics.

Introduction

We have utilized PylRS and tRNA^Pyl to synthesize and assemble reconstituted mononucleosomes with site specific acetylated histones. PylRS has proven invaluable as a genetic code expansion tool to produce proteins with post translational modifications (PTMs) and has been genetically evolved to incorporate about 200 different ncAAs. PylRS incorporates at an amber stop codon, removing competition from other amino acids during translation. PylRS was first discovered in methanogenic archaea, and has since been utilized in chemical biology to incorporate novel reactive chemical groups into proteins^1,2.

Protocol

1. Plasmid construction

1. Begin by deciding which histone protein will be acetylated and at which lysine site. Mutate the site to the amber stop codon (TAG) using site directed mutagenesis.
   ​NOTE: There are four previously designed plasmids utilized for expression of histone proteins. All four histone proteins were cloned into the pETDuet-1 vector with an N-terminal histidine tag. Histone H4 also includes a SUMO tag, the origin of replication colE1 with a copy number of approximately 40, ampicillin resistant, and a T7 promoter.
   1. Design forward and reverse primers that contain a TAG mutation at the desired site (replacing....

Results

Dimers, tetramers, and octamers can be assessed by running a 12% SDS PAGE gel (Figure 1 and Figure 2). Here you can see that some of the acetylated tetramers have a lower yield than others (Figure 1). In fact, the closer to the core region, the lower the yield observed. This is most likely due to the acetylation interfering with the assembling of the tetramer the closer you get to the core regions. A.......

Discussion

It is essential to follow this protocol in every detail during an experiment. Nucleosomes are not very stable and much trial and error has gone into determining this protocol. It is key to remove precipitates at every step (or whenever observed) because particulates can easily interfere with the assembling processes. Always keep histone samples on ice. If nucleosomes are stored at 4 °C for too long, they can spontaneously disassemble. Be sure to check any samples by Native PAGE if stored at 4 °C for more than 2.......

Materials

Name	Company	Catalog Number	Comments
0.5 M TE Buffer	NA	NA	0.5 M NaCl, 20 mM Tris, 1 mM EDTA, pH 7.8
1 M TE Buffer	NA	NA	1 M NaCl, 20 mM Tris, 1 mM EDTA, pH 7.8
100x TE Buffer	NA	NA
2 M TE Buffer	NA	NA	2 M NaCl, 20 mM Tris, 1 mM EDTA, pH 7.8
20 mM TE Buffer	NA	NA	20 mM NaCl, 20 mM Tris, 1 mM EDTA, pH 7.8
6 M GuHCl			6M guanidinium chloride, 20 mM Tris, 500 mM NaCl, pH 8.0
Acetyllysine
Column Wash Buffer	NA	NA	6 M urea, 500 mM NaCl, 20 mM Tris, 20 mM imidazole pH 7.8
Elution Buffer	NA	NA
Fisherbrand Variable-Flow Chemical Transfer Pump	Fischer Scientific	15-077-67
His-TEV protease
Histone Lysis Buffer	NA	NA	60 mM Tris, 100 mM NaCl, 0.5% Triton-X100 (v/v), 1 mM PMSF pH 8.0
Ni-NTA Resin			6 M urea, 500 mM NaCl, 20 mM Tris, 250 mM imidazole, pH 7.8
PCR Clean-Up Kit	Epoch Life Sicences	2360050
Pellet Wash Buffer	NA	NA	60 mM Tris, 100 mM NaCl, pH 8.0
petDUET-His-SUMO-TEV-H4
petDUET-His-TEV-H2A
petDUET-His-TEV-H2B
petDUET-His-TEV-H3
pEVOL-AckRS	Addgene	137976
pGEM-3z/601	Addgene	26656
Storage Buffer	NA	NA	20 mM NaCl, 20 mM Tris, 20 mM NaCl, 1 mM EDTA, 20% glycerol, pH 7.8
Thermocycler