Expression and purification of CFM2 and filamin A repeat 10 domain : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatu, New Zealand
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Date
2012
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Massey University
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Abstract
Filamins are a group of proteins that interact with over 60 other proteins. Mutations to
the Filamin A gene results in a spectrum of disorders including Otoplatodigital
spectrum disorder type 1, Otoplatodigital spectrum disorder type 2,
Frontometaphyseal dysplasia, Melnick-Needles syndrome and Periventricular Nodular
Heterotopia. All cases of Melnick-Needles syndrome can be accounted for by
mutations in repeat 10. Using a yeast-2 hybrid assay Professor S.P Robertson identified
the protein FAM101A (the protein is alternatively named CFM2) that associated with
Filamin A repeat 10. CFM2 was found to interact with itself in a yeast-2-hybrid screen,
suggesting homo-dimerisation properties in addition to Filamin A repeats 10 and 21
binding affinity. If CFM2 dimerises and binds to repeat 10 and 21 it is possible that
Filamin A’s function will alter, thus altering the properties of the cytoskeleton. To
investigate the interaction between Filamin A repeat 10 and CFM2, each was
subcloned into an E.coli plasmid vector fused to a purification tag. Purification of CFM2
failed due to misfolding, this upholds later work that claims CFM2 cannot fold correctly
without the presence of vertebrate Filamin. Filamin A repeat 10 purification went well
but the fusion was unable to be concentrated without precipitating out of solution.
Also the GST purification tag could not be cleaved without secondary cleavage
products forming. Pull-down of C2C12 mouse fibroblast cell lysate using the GSTFilamin
A repeat 10 fusion as the probe did not identify any other proteins that bind
Filamin A repeat 10.
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Keywords
Filamins, Protein purification, Genetic mutation, Otoplatodigital spectrum disorder, Otopalatodigital spectrum disorder, Frontometaphyseal dysplasia, Melnick-Needles syndrome, Filamin A