Purification of the coat and scaffolding proteins from procapsids of bacteriophage P22

Abstract

The procapsid intermediate in phage P22 morphogenesis is a double shell structure with the gene 5 coat protein on the outside and the gene 8 scaffolding protein on the inside. Scaffolding subunits were released from within purified procapsids by treatment with 0.5 M guanidine hydrochloride, and separated from the remaining empty coat protein shells by gel filtration. The scaffolding subunits probably exit through the coat protein lattice. Scaffolding protein which had not interacted with coat protein was also purified without guanidine hydrochloride from extracts of 5 −-infected cells lacking the coat protein. This isolated protein was in the form of soluble subunits, rather than organized shells, and behaved similarly to the subunits obtained by dissociation of procapsids. Electron micrographs of uranyl acetate stained scaffolding protein displayed pentameric aggregates. Empty procapsid shells were dissociated into coat subunits with 3 M guanidine hydrochloride, and the subunits further purified by gel filtration. Both coat and scaffolding subunits isolated from procapsids remained soluble under conditions in which the procapsids themselves were stable. Preliminary amino acid composition analysis revealed no distinctly basic or acidic composition of either protein. These coat and scaffolding subunits may represent the precursor conformations of these proteins.