GST融合蛋白的准备 Preparation of Glutathione-S-Transferase (GST) Fusion Proteins

Margret B. Einarson and Elena N. Pugacheva Foxx Chase Cancer Center, Philadelphia, PA 19111 Jason R. Orlinck Brigham & Women"s Hospital, Boston, MA 02115 Excerpted from Protein: Protein Interactions, Second Edition Edited by Erica A. Golemis and Peter D. Adams

ABSTRACT Glutathione-S-Transferase (GST) fusion proteins have a wide range of applications. This protocol is designed for IPTG-inducible bacterial expression vectors.

MATERIALS Buffers, Solutions, and Reagents

Reduced glutathione, 20 mM prepared in 50 mM Tris-HCl (pH 8.0) Isopropyl-β-D-thio-galactoside (IPTG) LB broth, containing appropriate antibiotic selection Lysis buffer: PBS + 1% Triton X-100, supplemented just before use with protease inhibitors (e.g, final concentrations of 2 µg/ml aprotinin , 1 µg/ml leupeptin , and 25 µg/ml phenylmethylsulfonyl fluoride [PMSF]) Phosphate-buffered saline (PBS), ice cold 150 mM NaCl 20 mM sodium phosphate (pH 7.4) PBS supplemented just prior to use with protease inhibitors (e.g., final concentrations of 2 µg/ml aprotinin, 1 µg/ml leupeptin , and 25 µg/ml PMSF) Appropriate antibiotics for plasmid selection (see step 1)

Bacterial Strains

Bacterial strain transformed with GST and GST fusion expression plasmids (see note to step 1)

Special Equipment

Centrifuge, precooled to 4°C Shaking incubator, preset to 37°C Rotator for end-over-end mixing Disposable chromatography column Glutathione-Sepharose beads Sonicator

Additional Reagents

This protocol also requires equipment and reagents for SDS-PAGE, including Coomassie blue (see Sambrook and Russell 2001) SDS-PAGE sample buffer, 2x (Sambrook and Russell 2001) 100 mM Tris-HCl (pH 6.8) 4%(w/v) SDS (electrophoresis grade) 0.2% (w/v) bromophenol blue 20% (v/v) glycerol 200 mM dithiothreitol ( DTT ) or β-mercaptoethanol

METHOD

Inoculate one colony of each bacterial strain expressing each construct (GST alone, GST fusion proteins) into individual 5-ml aliquots of LB broth containing appropriate antibiotic selection. Grow overnight at 37°C with shaking. Although variations of GST fusion protein expression vectors are available, the most commonly used versions include the sequence encoding the GST moiety followed by a multiple cloning site; an IPTG-inducible promoter; the ampicillin-resistance gene; the lacI gene for expression control; and a bacterial origin of replication. Many bacterial strains can be used, including those commonly used for cloning. Alternatively, protease-deficient strains such as BL21 have been commonly used for expression of recombinant proteins. Inoculate 1 liter of LB containing antibiotic selection with the 5-ml overnight culture from step 1. Grow at 37°C with shaking to an OD 600 of 0.5-1.0 (should take 3-6 hr). Induce expression of the protein by adding IPTG to a final concentration of 0.1 mM. Different proteins are optimally produced using different concentrations of IPTG. If protein expression is problematic, first titrate the amount of IPTG added to determine the optimal conditions for protein induction. Incubate for an additional 3 hr at 37°C with shaking. Centrifuge the bacterial culture at 3500g for 20 min at 4°C. Discard the supernatant. At this point, pellets can be stored frozen at -20°C if necessary. Resuspend the pellet in 20 ml of lysis buffer supplemented with protease inhibitors. Sonicate the bacterial suspension on ice, in short 10-sec bursts alternated with 10 sec resting on ice. Three cycles of sonication are usually sufficient.