A general approach for purifying proteins encoded by cloned genes without using a functional assay: isolation of the uvrA gene product from radiolabeled maxicells.
The uvrA protein (UVRA) of E. coli has been extensively purified from a strain in which UVRA is overproduced and specifically labeled with 35S-methionine. This approximately 100-fold overproduction relative to normal strains is a result of having the uvrA gene present on a multicopy plasmid in a spr recA cell that makes defective lexA protein, the normal repressor of the uvrA gene, while the specific labeling of UVRA is done with maxicells. This approach facilitates the preparation of the protein since enzyme assays do not have to be carried out during the intermediate stages of purification. The purified UVRA binds to DNA and has ATPase activity but does not have intrinsic endonuclease activity. When added to extracts of uvrA- cells, the purified UVRA does promote the specific cutting of UV-irradiated DNA. Since this approach for working out rapid purification procedures by specifically labeling the proteins encoded by cloned genes does not require the use of a functional assay, it is a general one that can be applied to a wide variety of other gene products in addition to UVRA.
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004706:Endodeoxyribonucleases; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D007700:Kinetics; D009097:Multienzyme Complexes / Q000235:genetics; D009154:Mutation; D009699:N-Glycosyl Hydrolases; D010957:Plasmids
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; C032219:UV endonuclease; D004706:Endodeoxyribonucleases; D009699:N-Glycosyl Hydrolases; D000251:Adenosine Triphosphatases