Separation of copurifying GroEL from glutathione-S-transferase fusion proteins.
The purification of overexpressed fusion proteins using bacterial expression systems is a useful tool for the study of many proteins. One problem that can occur is the formation of stable interactions between the expressed fusion protein and certain endogenous bacterial proteins, such as the molecular chaperone GroEL. Such interactions may result in the copurification of contaminating bacterial proteins. Here we describe an efficient and inexpensive method for the removal of contaminating GroEL from a bacterially expressed GST fusion protein. In this method, denatured bacterial proteins are added to the bacterial lysates prior to the addition of glutathione Sepharose resin. The denatured proteins compete for GroEL binding, thereby releasing the GroEL contaminants from the expressed fusion protein.
Rohman
M
M
Institute of Cancer Research, Chester Beatty Laboratories, CRC Centre for Cell and Molecular Biology, 237 Fulham Road, London, SW3 6JB, United Kingdom. mattias@icr.ac.uk
Harrison-Lavoie
K J
KJ
D000255:Adenosine Triphosphate / Q000737:chemistry; D018834:Chaperonin 60 / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000737:chemistry / Q000302:isolation & purification*; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry*
D018834:Chaperonin 60; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase