title: In vitro reconstitution of the bacteriophage T4 clamp loader complex (gp44/62).
abstract: The clamp loader complex (CLC) of bacteriophage T4 is essential for viability and has analogs in both prokaryotes and eukaryotes. The gp44 and gp62 subunits of the T4 CLC, in a 4:1 ratio, tightly associate such that the two proteins co-purify. Using transformed Escherichia coli, we were able to demonstrate for the first time purification of the unique protein gp62 in the absence of gp44. We experimentally determined the isoelectric point for the individual subunits. An in vitro physical interaction could be observed between the native subunits, which resulted in a reconstituted CLC that displayed the signature pattern of the ATPase functions of native CLC. Thus we demonstrate that the CLC forms via a self-assembly pathway rather than through a translational capture mechanism.
authors.affiliation: Chemistry Department, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201-0413, USA.
authors.forename: D M
authors.initials: DM
authors.lastname: Janzen
authors.forename: M Y
authors.initials: MY
authors.lastname: Torgov
authors.forename: M K
authors.initials: MK
authors.lastname: Reddy
mesh_terms: D017122:Bacteriophage T4 / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D014176:Protein Biosynthesis; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014764:Viral Proteins; C074855:gene 44 protein, Enterobacteria phage T4; C088659:gp62 protein, bacteriophage T4