C9orf72 hexanucleotide repeat expansion transcript is the foundation on which toxic
membraneless G4C2 foci are built. These are common among patients with familial
amyotrophic lateral sclerosis and frontotemporal dementia. Mechanisms underlying the
formation of toxic G4C2 foci are unknown and could represent the basis for new
treatments. To research G4C2 foci proteins that localize and form these structures are
needed. For that reason, this work focuses on the production of plasmids for in vitro
transcription/translation and expression of SFPQ, NONO, PABPC1, TDP-43 and FUS
proteins in E. coli BL21(DE3) bacteria. A fluorescent NeonGreen protein was fused to
the wild type proteins along with maltose binding protein for increased construct
solubility. The plasmids also contained a hexahistidine tag and a TEV cleavage site to
assist with protein purification. SFPQ, PABPC1, TDP-43 and FUS were expressed at 37
oC with 1 mM IPTG for three hours and at 30 oC, with 0,5 mM IPTG for five hours.
Lower temperature expression yielded a better result since most of the protein was in the
soluble fraction of the cell lysate. Both SFPQ and SFPQ-NeonGreen were expressed in
too low a quantity for further protein isolation. Further work needs to check the expression
of NONO, NONO-NeonGreen, NeonGreen-PABPC1 and purify all of the constructs to
achieve our goal.
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