Phizicky Lab
Protocols
Growth of GST-ORF strains and purification of proteins
DIRECTIONS FOR GST-ORF STRAINS
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Note that the genotype of the host strain is: strain EJ 758 [MATa his3-delta200, leu2-3,112, ura3-52, pep4::HIS3], a derivative of JHRY-20-2Ca and NOT pep4::URA3, as stated in the article (our apologies): |
1. For Strains arriving in microtiter dishes: To pull out the cells from an entire plate, the microtiter plates are thawed. Generally, we find that the cells have settled to the bottom of the wells so we first mix the material in the individual wells using a multichannel pipettor. Then the cells are transferred to a large (150 mm) SD - Ura plate using either a 96 well pinner or a multichannel pipettor (2 microliter/well).
2. For strains arriving on either microtiter plates or agar (-Ura) plates: Always use a mixture of cells from any well (or plate). When we made the library, the entire set of transformants from each transformation was pooled and saved; so it is likely that individual cells in each well contain plasmids expressing GST alone, the GST-ORF, and likely also some GST-ORF* (mutation).
When we get a hit, we purify the plasmid from the yeast mix, obtain several transformants in E. coli, purify the plasmids from E. coli, confirm identity by restriction and sequence analysis, followed by re-transforming a single plasmid back into the parent strain, purification and assay.
GROWTH PROTOCOL:
The 96 strains on a microtiter plate are grown in patches on a 150 mm SD -Ura plate (2 microliter from each well), and patches are mixed together in SD-Ura medium before growth. Cells are scraped into 5 ml YPD, (aliqoted at this point for saving, if desired), and diluted into 5 ml SD-Ura liquid medium at A600=0.2. After growth overnight, they are inoculated into 50 ml of SD -Ura -Leu medium at A600= 0.2 and grown overnight, re-inoculated into 500 ml SD -Ura -Leu medium at A600=0.2, grown to A600=0.8 (to further amplify the plasmid, which has an inefficiently expressed leu2-d marker), and supplemented with 0.5 mM copper sulfate for two hours to induce expression of the GST-ORF before harvest (I. G. Macreadie, O. Horaitis, A. J. Verkuylen and K. W. Savin Gene 104, 107 (1991). The cells are harvested, washed with cold water, and frozen in two 2059 (Falcon) tubes at -70oC. One tube (from 250 ml culture) was used for the preps.Alternative: Frequently, we dilute the 50 ml -leu -ura culture into the large culture the night before. We calculate the inoculum size so that the large culture will be at OD600 = 0.8 the next morning and all we have to do is induce with copper, harvest, wash and freeze. In general we calculate using a 2.5 hr generation time; this is very close to the generation time at the settings on our shaking incubator.
GST-ORF purification.
Extraction Buffer: 50 mM Tris-HCl (pH 7.5)1 mM EDTA
4 mM MgCl2
5 mM DTT
10% glycerol
1 M NaCl
+ 2 ug/ml leupeptin (Add these just prior to use)
1 ug/ml pepstatin
Wash Buffer: (Also need No Salt Wash buffer, no NaCl)
50 mM Tris-HCl (pH 7.5)
4 mM MgCl2
1 mM DTT
10% glycerol
0.5 M NaCl
Elution buffer: (Make up right before use)
| Wash buffer | 29.7 ml |
| 20 mM NaOH | 0.3 ml of 2 M stock |
| 25 mM glutathione (Sigma #G4251) | 0.23 g |
Dialysis buffer:
20 mM Tris-HCl (pH 7.5)
2 mM EDTA
4 mM MgCl2
1 mM DTT
55 mM NaCl
50% (v/v) glycerol
Purification Procedure:
(Directions are for prep from 250 ml of cells grown as described above.)
Before you start: Use 100ul of GSH sepharose resin per extract. Pre wash GSH resin (Glutathione sepharose-4B from Pharmacia: code 17-0756-01) with wash buffer (5 washes) to remove free glutathione. We generally use very quick (20-40 sec) low speed microfuge spins to bring down the resin.
1. Resuspend frozen cell pellets in extraction buffer with leupeptin and pepstatin (1 ml extraction buffer /250 ml cell grown as described) Transfer to bead beater vial. Add glass beads (0.45-0.5 mm) to the top of the tube. [(S. M. McCraith and E. M. Phizicky, Mol.Cell. Biol. 10, 1049, (1990)]
2. To lyse cells, beat at 4oC in a Biospec mini Bead beater: 10 repeats of 20 sec beating followed by 1 min on ice. (Generally use an ice-water mix to facilitate cooling).
3. The extract is removed from the beads. We puncture the bottom of the tube with a hot (flamed) 25 gauge needle and gently force out the liquid with low pressure air. The beads are then washed with 0.5 ml Extraction buffer which is also pushed through the tube with air.
4. Add 2 microliter of 1 M PMSF to the extract.
5. Spin out junk for 10 min at 4oC in a microfuge. The crude extracts are generally about 20 mg/ml protein. Keep a sample of the crude extract to assess lysis.
6. Mix the crude extract with an equal volume of NO SALT wash buffer to bring the salt to 0.5 M NaCl. Add 100 microliter of pre-equilibrated GSH resin per 2 ml of final extract (after no salt dilution) GST-ORF fusion proteins were purified essentially as described [J. R. Nelson, C. W. Lawrence and D. C. Hinkle, Science 272, 1646 (1996)].
7. NUTATE (gentle mixing) in the cold room for 3 hrs.
8. Spin 20-30 sec at low speed (Near lowest setting 1-2).
9. Remove the unbound material.
10. Wash the GSH resin with 1 ml wash buffer; nutate 10 min, spin for 20 sec at low speed and remove the supernatant.
11. Repeat the wash except nutate for 20 min. because we make the glutathione buffer up during this step. Spin at low speed for 30 sec, remove the supernatant.
12. Elute by adding 2 ml Elution Buffer and nutating at 4oC for 40 min (can go longer).
13. Spin 20-30 sec.
14. Take off supernatant and dialyze it overnight against dialysis buffer.
15. Store at -20oC.
Yield: 0.5 ml of protein at about 0.25 mg/ml