Expression, purification, and structural characterization of human histone H4

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Expression

Background:

• Histone H4 is a core component of nucleosomes, crucial for DNA packaging and regulation.
• Understanding histone structure and function is vital for studying gene regulation and chromatin dynamics.

Purpose of the Study:

• To produce recombinant human histone H4 (hH4) in Escherichia coli.
• To characterize the secondary structure of purified recombinant hH4.
• To investigate methods for restoring the native conformation of isolated hH4.

Main Methods:

• Recombinant hH4 expression in E. coli using the pQE30 vector.
• Purification via nickel-chelating chromatography and gel filtration.
• Secondary structure analysis using circular dichroism spectroscopy.
• Conformation restoration attempts using varying ionic strengths, trifluoroethanol, and DNA addition.
• Thermal stability assessment via spectropolarimetry.

Main Results:

• Milligram quantities of pure recombinant hH4 were obtained with high yield (1 mg/L).
• SDS-PAGE confirmed the correct molecular weight of recombinant hH4.
• Isolated recombinant hH4 exhibited an altered secondary conformation compared to its state within the nucleosome.
• Increasing ionic strength, adding trifluoroethanol, or including DNA partially restored the native-like conformation.

Conclusions:

• Recombinant hH4 can be efficiently produced and purified from E. coli.
• The secondary structure of histone H4 is sensitive to its environment and can be altered when isolated.
• Environmental modifications can partially induce a more physiological conformation in recombinant hH4, aiding structural studies.