PURIFICATION AND INTERACTION STUDIES OF HISTONE, HMGB, AND PPI PROTEINS, FACILITATED BY THE DIPOLAR NATURE OF HMGBS

High mobility group box (HMGB) proteins are the most abundant non-histone proteins in the nuclei of eukaryotic cells and are highly conserved in animals. They bind dynamically to chromosomal DNA, interact with other proteins including transcription regulators, and have key roles in gene transcription and DNA repair. These fundamental activities require HMGB proteins to interact with the histone proteins found in chromatin, but the precise mechanisms remain unclear - the work here provides steps towards their clarification. Acid extraction of histones from chicken erythrocytes was explored, as this provides high yields without the use of ultracentrifugation. Histone protein markers (MW range 11.4 – 22.5kDa) suitable for use with SDS-PAGE were prepared using sulphuric acid extraction. For future research, almost pure histone octamers were also prepared, using phosphoric acid extraction combined with potassium chloride to ensure the octamers remained intact and in a nuclear-like environment. The remaining work was based on proteins extracted by mild methods from chicken erythrocytes to retain post-translational modifications. A nuclear protein set was isolated containing almost entirely histones and HMGB proteins, and was subject to cation-exchange chromatography, with phosphate buffers chosen to partially simulate nuclear conditions. HMGB molecules have a C-terminal acidic tail which in free solution is folded back onto the remaining basic part of the protein. Analysis of the chromatogram peaks suggested the HMGB proteins had unfolded into a dipolar configuration, with their basic parts binding to the cation-exchange column and their acidic tails binding to the histones. Gel filtration chromatography applied to fractions eluted from the cation-exchange column suggested the presence of one or more unidentified complexes. Native HMGB proteins where isolated using a novel method based on their dipolar nature. At high concentration, HMGB1 proteins were used to pull out a potential HMGB1/FKBP3 complex from a pool of nuclear proteins. Technology to significantly enhance the concentration of HMGB1 in chicken plasma was also developed, as the basis for prompt, low cost measurement of HMGB1 in biofluids (this has medical applications, such as in cancer diagnosis and prognosis). Further exploiting the dipolar nature of HMGB proteins, a method was developed for isolating the peptidyl-prolyl isomerases FKBP3 and Cyp B.

Name dc:type.qualificationname

mphil

Level dc:type.qualificationlevel

masters

Grantor dc:publisher.institution

Liverpool John Moores University

Year dc:date.issued

2018

Author dc:creator

• Smallman, H

Contributors dc:contributor

• Wood, C
• Evans, K
• Baldwin, J

• QD Chemistry
• QH301 Biology

DOI dc:identifier.doi

https://doi.org/10.24377/LJMU.t.00008566

OAI identifier oai:identifier

oai:researchonline.ljmu.ac.uk:8566