Lysis cassette genes from phages determine the final lytic event of the host cells. The lysis cassette genes are conserved in phages and prophages. The membrane associated holin from DLP12 prophage, available as a GFP fusion construct, was shown to be overexpressed, using confocal microscopy analysis, in bacterial cells. The protein expression caused cell death in E. coli AG1 strain suggesting the protein was functional. The His-tag HolinGFP protein was purified using cobalt affinity column and was eluted in the presence of different non-ionic detergents DDM (n-dodecyl-ß-d-maltoside), LDAO (Lauryldimethylamine-oxide), OG (n-octyl β-d-glucopyranoside) and C12E9 (dodecyl nonaoxyethylene ether). HolinGFP existed predominantly as a dimer in LDAO in Superdex S200 gel filtration chromatography. Circular dichroism and fluorescence spectroscopy of the fluorescent HolinGFP in all four detergents (C12E9, DDM, LDAO, and OG) confirmed the folded state. Both dithiobis succinimidyl propionate and gluteraldehyde crosslinking revealed the existence of higher order oligomers and dimers. HolinGFP has been functionally and biophysically characterised and is being explored for crystallographic structure determination.
Keywords: Crosslinking; DLP12; HolinGFP; Oligomerisation; Prophage.