Purification of plasmid-expressed proteins which lack functional assay systems.
A general method for the purification of proteins whose genes are cloned into plasmid vectors, but whose biochemical and functional characteristics are unknown, is described. A cell-free transcription-translation system from Escherichia coli K-12 is used to synthesize in vitro radiolabeled protein expressed from recombinant plasmid vectors. The radiolabeled proteins are then fractionated and used as markers for the purification of nonradiolabeled proteins without recourse to functional assays. Biochemical analysis of the purified proteins can reveal information about their cellular localization, binding parameters, and physical, enzymatic, or regulatory properties. This information complements in vivo genetic analysis with the goal of identifying the gene and the function of its protein product. An example using this technique in which the product of the usg gene in the hisT operon of E. coli has been purified and biochemically characterized is described.
Marvel
C C
CC
University of Southern California School of Medicine, Albert Soiland Cancer Research Laboratory, Los Angeles 90015.
Kammen
H O
HO
D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D007553:Isotope Labeling; D008970:Molecular Weight; D010957:Plasmids; D011506:Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011506:Proteins