Expression, purification and characterisation of recombinant chromatin assembly factor 1

Chromatin Assembly Factor 1 (CAF-1) is the only known replication dependant histone chaperone, responsible for the deposition of the histone H3/H4 tetramer onto DNA. Found in all eukaryotes, CAF-1 consists of three subunits, p150, p60 and p48. Since its identification work on CAF-1 has mainly focused on in vivo studies due to the lack of a reliable method to produce large quantities of recombinant protein for biochemical studies. Herein the cloning, production and purification of the three subunits of recombinant CAF-1 is described. The proteins were expressed as complexes and individually in insect cells and Escherichia coli, optimised protocols are described for maximum protein recovery and purity. Constructs of p150 and p60 were also produced and used to analyse the binding regions and modes of both the p48 and p60 proteins to p150. It is shown that the two smaller subunits of CAF-1 do not interact in the absence of p150 and that the p150 subunit of CAF-1 acts as a scaffold for assembly of the complex, binding directly to both p48 and p60. The stoichiometry of the CAF-1 complex was also investigated and a basis for further work, including structural studies, discussed.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2013

Author dc:creator

• Royle, Nikki

• Chromatin
• Biochemistry
• Chromatin assembly
• CAF1

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/65d913bc-4439-4b68-9143-074115ada025/download
• http://creativecommons.org/licenses/by-nc-sa/2.0/uk/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.16526

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/244243