Expression and purification of recombinant human histones

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Recombinant nucleosomes are crucial for studying histone variants, mutants, and post-translational modifications.
• Bacterial expression systems like E. coli are widely used for protein production.

Purpose of the Study:

• To develop an improved method for expressing and purifying recombinant human histones (H2A, H2B, H3, H4) and CENP-A in E. coli.
• To optimize the expression of the poorly expressed human histone H4 gene in E. coli.

Main Methods:

• Codon optimization of human histone H4 and CENP-A genes for E. coli.
• Expression of hexahistidine-tagged recombinant histones in E. coli.
• One-step purification using nickel-nitrilotriacetic acid agarose chromatography in 6 M urea.
• Refolding of histone dimers and tetramers by dialysis and tag removal via thrombin protease digestion.
• Nucleosome reconstitution using the salt dialysis method.

Main Results:

• Successfully expressed and purified recombinant human histones H2A, H2B, H3, and H4, with optimized H4 showing improved expression.
• Developed a refolding and purification protocol yielding functional H2A/H2B dimers and H3/H4 tetramers capable of nucleosome formation.
• Constructed a codon-optimized CENP-A gene, and the purified recombinant CENP-A formed nucleosome-like structures with other histones.

Conclusions:

• The developed method enables efficient expression and purification of recombinant human histones and CENP-A in E. coli.
• Optimized histone H4 and CENP-A genes facilitate the production of key nucleosome components for structural and functional studies.
• This technique provides a valuable tool for investigating nucleosome dynamics and epigenetic regulation.