Co-immunopurification of multiprotein complexes containing RNA-degrading enzymes.
Co-immunopurification is a classical technique in which antiserum raised against a specific protein is used to purify a multiprotein complex. We describe work from our laboratory in which co-immunopurification was used to characterize the RNA degradosome of Escherichia coli, a multiprotein complex involved in RNA processing and mRNA degradation. Polyclonal rabbit antibodies raised against either RNase E or PNPase, two RNA degrading enzymes in the RNA degradosome, were used in co-immunopurification experiments aimed at studying the assembly of the RNA degradosome and mapping protein-protein interactions within the complex. In E. coli, this method has been largely supplanted by approaches in which proteins are engineered to contain tags that interact with commercially available antibodies. Nevertheless, we believe that the method described here is valid for the study of bacteria in which the genetic engineering needed to introduce tagged proteins is difficult or nonexistent. As an example, we briefly discuss ongoing work in our laboratory on the characterization of RNase E in the psychrotolerant bacterium Pseudoalteromonas haloplanktis.
Carpousis
Agamemnon J
AJ
Laboratoire de Microbiologie et Génétique Moléculaire, Centre National de la Recherche Scientifique and Université Paul Sabatier, Toulouse, France.
Khemici
Vanessa
V
Aït-Bara
Soraya
S
Poljak
Leonora
L
D000906:Antibodies / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005095:Exoribonucleases / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D024101:Mitochondrial Proteins
D000906:Antibodies; D004722:Endoribonucleases; D005095:Exoribonucleases; D012313:RNA; C476417:PNPT1 protein, human; D024101:Mitochondrial Proteins; C022537:ribonuclease E