来源物种?Schistosoma japonicum ?日本吸血虫?
分子量? 26 000?单体? 58 500?二体?
Km (glutathione)? 0.43?0.07 mM
以上信息请参考?GE GST Handbook
Genetic engineers have used glutathione S-transferase to create the so-called 'GST gene fusion system'. Here, GST is used to purify and detect proteins of interest. In a GST gene fusion system, the GST sequence is incorporated into an expression vector alongside the gene sequence encoding the protein of interest. Induction of protein expression from the vector's promoter results in expression of a fusion protein - the protein of interest fused to the GST protein. This GST-fusion
protein can then be purified from cells via its high affinity for glutathione.
Fusion proteins offer an important biological assay for direct protein-to-protein interactions. For instance, to demonstrate that caveolin
(a membrane protein) binds to eNOS (a catalytic protein) a 'GST-caveolin' fusion protein would be generated. Assay beads, coated with thetripeptide glutathione, strongly bind the GST fusion protein (GST-ca
veolin, in this example). It is noted that, if cavelin binds eNOS, then GST-caveolin will also bind eNOS, and this eNOS will therefore be present on assay beads.
GST is commonly used to create fusion proteins. The tag has the size of 220 amino acids, which, compared to other tags like the myc- or the FLAG-tag, is quite big. It is fused to the N-terminus of a protein.
However, many commercially-available sources of GST-tagged plasmids include a thrombin domain for cleavage of the GST tag during protein purification.
A GST-tag is often used to separate and purify proteins that contain the GST-fusion. GST-fusion proteins can be produced in Escherichia coli, as recombinant proteins. The GST part binds its substrate, glutathione. Agarose beads can be coated with glutathione, and such glutathione-Agarose beads bind GST-proteins. These beads are then washed, to
remove contaminating bacterial proteins. Adding free glutathione to beads that bind purified GST-proteins will release the GST-protein in
当GST柱材料?Glutathione Sepharose beads?的结合能力降低时?很可能是
1. 用两个柱体积?column volume?的6 M盐酸胍来清洗柱材料?
1. 用70%的乙醇清洗柱子3~4个柱体积?或用1%的Triton X-100清洗2个柱体
Margret B. Einarson, Elena N. Pugacheva, and Jason R. Orlinick
This protocol was adapted from "Identification of Protein-Protein Interactions with
Glutathione-S-Transferase Fusion Proteins," Chapter 6, in Protein-Protein Interact
, 2nd edition (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Coions
ld Spring Harbor, NY, USA, 2005.
Glutathione-S-transferase (GST) fusion proteins have had a widerange of applications since their introduction as tools forsynthesis of r ecombinant proteins in bacteria. One of theseapplications is their u se as probes for the identification ofprotein-protein interactions. The pull-down method describedin this protocol is fundamentally simi lar to immunoprecipitation.Immunoprecipitation is based on the abili ty of an antibody tobind to its antigen in solution, and the subsequ ent purificationof the immunocomplex by collection on protein A- or G-coupledbeads. Similarly, the GST pull-down is an affinity capture ofone or more proteins (either defined or unknown) in solutionby its interaction with the GST fusion probe protein and subsequentisolati on of the complex by collection of the interacting proteinsthrough t he binding of GST to glutathione-coupled beads.
An introduction to the use of GST fusion proteins for studyingprotei
n-protein interactions can be found in Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins.
35 This protocol is designed to use a S-labeled cell lysate asthe sour35 ce for interacting proteins. For S-labeling procedures,see Orlinick
and Chao (1996) and Spector et al. (1998). Additionally,if the inte racting protein of interest is known to be confinedto a specific cel lular compartment (e.g., the nucleus), a fractionof the cell lysate corresponding to that compartment (e.g.,a nuclear extract [to prepar Dignam et al. 1982]) canbe used in place of a total cell lysae, see te.
This procedure may require equipment or reagents for Westernanalysis, Step 13). Coomassie blue staining, and/or silver staining (see
35Cell lysate (unlabeled orlabeled with S, depending on experimenta l goal)
This experiment compares GST versus GST fusion protein, so itis nece of lysate in ssary to prepare enough lysate to provide equal amounts an interaction ieach reaction. The amount of lysate needed to detect 67to 1 x 10 to 1 x 10 s highly variable. Start with lysate equivalent tissue culture cells.
GST fusion protein (see Preparation of GST Fusion Proteins)
GSTpull-down lysis buffer, ice cold
Reagents for SDS-Polyacrylamide Gel Electrophoresis of Proteins(see Step 12)
2X SDS gel-loading buffer
Tris-Cl (50 mM, pH 8.0) containing20 mM reduced glutathione (optio nal; for Step 11 only)
Equipment for SDS-Polyacrylamide Gel Electrophoresis of Proteins(see Step 12)
Gel dryer (optional; see Step 13)
Glutathione-Sepharose beads (store at 4?C; do not freeze)
Beads are often supplied by commercial vendors in solutionscontainin in GST pull-g alcohols. It is important to wash the beads thoroughly of beads in GST pulldown lysis buffer and to generate a 50/50 slurry -down lysis buffer prior to use.
Microcentrifuge, precooled to 4?C
Microcentrifuge tubes, 0.5 mL (optional; for Steps 11.vi-11.ixonly) and 1.5 mL
Needle, small bore, sterile (optional; for Steps 11.vi-11.ixonly)
Rotator for end-over-end mixing
Water bath, boiling (optional; see Step 10)
X-ray film (optional; see Step 13)
1. Incubate the cell lysate with 50 μL of glutathione-Sepharos ebeads (50/50 slurry in lysis buffer) and 25 μg of GST(NOT th e GST fusion probe protein) for 2 h at 4?C with end-over-endmixing. Allow enough volume in the tube to permit liberal mixing; 500 μl to 1 mL is a good starting point. This step isdesigned to preclear from the lysate proteins that interactnonspecifically with the GST moiety or with the beadsa If the interaction will be detected primarily with antibolone. directed to a candidate interacting protein, it is not absdies necessary to preclear the lysates with GST or glutathioolutely 35beads. However, when S-labeled cell lysates are une-Sepharose novel protein-protein interactions, these stepssed to identify reduce background. can help to When detecting the interacting proteinwith antibodies to thatp "GST + beads" and "beadsalonrotein, it is important to include e" controls.
2. Centrifuge at13,000 rpm for 10 sec at 4?C in a microcentri fuge.
3.Transfer the supernatant (precleared cell lysate) to a fresh tube.
4. Set up two tubes containing equal amounts of thepreclearedc ell lysate. i. Add 50 μl of glutathione-Sepharosebeads (50/50 slurryin ly sis buffer) to each tube.
ii. Thenadd GST protein toone tube and the GST fusion probeto theother (~5-10 μgeach). The amount of protein added shouldbe equimolar inthe two react (i.e., the final molar concentrationof GSTshould be the sions as that of the GST probe protein).ame 5. Incubate the tubes for 2 h at 4?C with end-over-endmixing.
6. Centrifuge the samples at 13,000 rpm for 10 secat 4?Cin a microcentrifuge.
7. Transfer the supernatantsto fresh microcentrifuge tubesand reserve them for SDS-PAGE(see Troubleshooting).
8. Wash the beads four times with 1mL of ice-cold lysis buffer. Discard the washes.
9. At thispoint, proteins bound to the probe protein must bedi ssociatedfor analysis. Use either the boiling method (themore popularchoice; see Step 10) or one of the elution methodsdescr ibedin Step 11. The disadvantage of elution (Step 11) is thatif multiple eluti are necessary, the final sample volumemay be large. In thesons cases, it may be impossible to load morethan a small percentae of the sample on an SDS-polyacrylamidegel for analysis. Thisge is the reason most researchers chooseinstead to boil complexes off the beads in SDS sample buffer(Step 10).
10. If the samples are to be boiled off the beads,do as follow s: i. Add an equal volume of 2X SDS gel-loadingbuffer to the bead s. It is important to include a "glutathione-Sepharosebeadsonly" control. Proteins bound nonspecifically to the beadscanappear as bound to the fusion protein, even in comparisontoGST alone.
ii. Boil for 5 min in a water bath. Samplesarenow ready for SDS-PAGE (Step 12). 11. If the samplesare to be eluted from the beads, performone of the followingoptions: Option I:
i. Add 50 μl of 20 mM reduced glutathionein 50 mM Tris-Cl(pH 8.0).
ii. Flick the tube to mix the contents,and incubatethe samples at room temperature for 5 min.
iii.Centrifugethe samples at 13,000 rpm for 10 sec at roomtem peratureina microcentrifuge.
iv. Transfer the supernatant containingthe eluted protein toa new microcentrifuge tube.
v. If elutionis incomplete, repeat Steps 11.i-11.iv. Option II: vi.Add 50 μl of 20 mM reduced glutathione in 50 mM Tris-Cl(pH 8.0).
vii. Flick the tube to mix the contents, and incubatethe sampl esat room temperature for 5 min.
viii. Transferthe mixture to a fresh 0.5-mL tube. Carefully,us ing a sterile,small-bore needle, poke a hole in the bottomof t he 0.5-mL tube.Place it inside a 1.5-mL microcentrifugetube.
ix. Centrifugethe tubes together at 1000g in a microcentrifuge for 2 min atroom temperature. The eluted proteins will be deposited inthe 1.5-mL tube. 12. Analyze as much of the sample as possibleby SDS-PAGE (seeS DS-Polyacrylamide Gel Electrophoresis of Proteins).
13. Detect the proteins. The method of detection will dependon the experimental goal: 35 i. If the goal is to detect theS-labeled proteins associatedw ith the fusion protein afterSDS-PAGE, dry the gel on a geldrye r and expose it to X-rayfilm.
ii. If the goal is to detectspecific partners afterSDS-PAGE,t ransfer the proteins to amembrane and subject themto Westerna nalysis.
iii. If thegoal is to determine thesize and abundance of prote insassociatedwith the fusion proteinfrom a nonradioactive lys ate,subsequentto SDS-PAGE, stainthe gel with Coomassie blue o rsilver stain.
Problem: Conditions for pull-down are not optimal
Solution: Analyze aliquots of equal percentage volumes (e.g.,1%) fro m each of the following fractions generated during theprotocol:
Total cell lysate (Step 1)
Beads prior to elution (Step 9)
Eluate (Steps 10 or 11)
Beads post-elution (Steps 10 or 11)
Supernatant saved at Step 7
"Beads + GST" eluate (if applicable; Steps 10 or 11)
"Beads alone" eluate (if applicable; Steps 10 or 11)
After these samples are collected at the indicated steps, addan appr opriate volume of SDS-PAGE sample buffer. Snap-freezethe samples in a dry-ice ethanol bath for analysis by SDS-PAGE(during Step 12), and perform autoradiography, Western analysis,or Coomassie staining as appropriate (Step 13).
Results from this gel will show:
; The prevalence of the novel interactor in the total cell lysate (aliquot from Step 1).
; How much of the interactor is boundto the GST fusion protein(a liquot from Step 9).
; How muchGST fusion protein + associated proteins were elutedfr om thebeads (aliquot of eluate from Steps 10 or 11).
; What fractionof the interactors remained bound (beads remainin gfrom Steps10 or 11).
; How much of the interacting protein was depletedfrom the total cell lysate (aliquot from supernatant at Step7).
Problem: Low signal
Solution: If an interaction yields a low signal even thoughthe inter actor is abundant in the total cell lysate, this mayindicate that th e binding conditions are not optimal. A changein salt and detergent concentrations, in addition to increasingthe time allowed for associ ation, may improve the binding. Apoor signal can also be caused by i nefficient elution; i.e.,the complex being retained on the glutathio ne-Sepharose beads.This can be determined by SDS-PAGE comparison of the eluateversus the "beads post-elution" fraction (Steps 10 or 11). Ifthis proves to be the problem, it may be remedied by poolingmulti ple elutions or increasing the time for elution. Releasingthe intera ctors by boiling in sample buffer (Step 10), if appropriate,would be applicable in this case.
Problem: Nonspecific background
Solution: Preclearing a lysate with GST, or beads alone, canhelp to minimize nonspecific interactions (see Step 1). Titratingthe amount of lysate added and increasing the stringency ofthe binding and wash conditions can also reduce background.
When analyzing an interaction by Western blot (e.g., analyzinga pred icted interaction for which there are available antibodies),it is im portant to reprobe the membrane with anti-GST antibodiesafter probin g for the candidate interactor. This will determinewhether all sampl es were incubated with the same amount of GSTfusion protein, and it will help to determine whether the fusionprotein is undergoing degra dation while incubated with the celllysate. If the interactors are r adio- or biotin-labeled, oneshould also confirm equal amounts of GST fusion and GST proteinsby Western blot or Coomassie blue staining.