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The Laboratory of Jim Kadonaga at the University of California, San Diego
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| This protocol describes the use of the baculovirus vector expression system for heterologous protein production. Autographa californica nuclear polyhedrosis virus is the most commonly used baculovirus for protein expression in the insect cells Spodoptera frugiperda. There are several advantages of this system over prokaryotic expression systems: the insect cells are capable of proper transcriptional slicing of genes with introns (but protein expression is less efficient) and of many post-translational modifications found in mammalians cells, including myristylation, phosphorylation, amidation, prenylation, and proteolytic processing. N- and O-linked glycosylation, but probably not sialylation, occur. Glycosylation patterns are similar, but not identical, to mammalian cells. Potential N-linked glycosylation sites are often either fully glycosylated or not glycosylated at all; the various glycoforms seen in mammalian cells are not found in the insect cell-produced proteins. Other advantages include the fact that the cytoplasmic environment of the insect cells allows for proper protein folding and disulfide bond formation, in contrast to that found in the reducing environment of the E. coli cytoplasm. Oligomerization of proteins can also occur. In addition, heterologous proteins can be directed to particular subcellular locations, including the cytoplasm, ER, Golgi, plasma membrane, or nucleus, or be secreted. While the potential yields of this system are 30% to 50%, it is more common to see much lower yields. Such theoretical high yields are possible because expression of the heterologous protein is under the control of the strong polyhedrin promoter in the baculovirus vector. Low levels of protein expression can often be increased by optimizing the time of expression and the multiplicity of infection. Improperly folded proteins and proteins aggregates may be the result of expression late in the infection cycle - harvesting cells at earlier times after infection may improve matters. Vectors are available that allow for production of fusion proteins with tag sequences for immunoaffinity chromatography or metal chelation chromatography. This protocol uses immunoaffinity chromatography, with a FLAG tag, to purify the protein (see Hint #4). |
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1. Dilute a log phase suspension culture of Spodoptera frugiperda (Sf9) insect cells with TMN-FH Medium to a final volume of 500 ml and a density of 1 x 106 cells/ml.
2. Infect the culture with freshly amplified recombinant FLAG baculovirus at a multiplicity of infection (m.o.i) of 5 to 10 (see Hint #1).
3. Incubate the infected culture for 3 days at 26°C.
4. Collect the cells by centrifugation at 1,000 X g (e.g., 2500 rpm in a Sorvall GSA rotor) for 5 min at 4°C.
5. Decant the supernatant and wash the cell pellet with 50 ml of ice-cold PBS (see Hint #2).
6.Centrifuge at 1,000 X g (e.g., 2500 rpm in a GSA rotor) for 5 min at 4°C to pellet the cells.
NOTE: All procedures from this point on should be performed on ice or at 4°C with ice-cold buffers.
7. Decant the PBS and resuspend the cell pellet in 6.5 ml of cold Homogenization Buffer.
8. Homogenize the cells in a Wheaton Dounce Homogenizer using 12 strokes with the A pestle in a 7 ml vessel.
9. Incubate on ice for 15 min.
10. Centrifuge the resulting whole cell lysate at 190,000 X g (e.g., 46,000 rpm in a Beckman Ti70.1 rotor) for 30 min.
11. Collect the supernatant into a 15 ml conical tube and dilute with an equal volume of cold Dilution Buffer.
12. Add 250 μl of a 50% (v/v) slurry of anti-FLAG M2 Affinity Resin (Kodak/IBI) previously equilibrated in Dilution Buffer.
13. Incubate the lysate with the resin for 4 hr at 4°C with gentle mixing.
14. Pellet the resin by centrifugation at 1,000 X g in a 4°C table-top centrifuge.
15. Aspirate the supernatant.
16. Wash the resin in batch 4 times with 10 ml of cold Wash Buffer per wash, pelleting the resin by centrifugation at 1,000 X g in a 4°C table-top centrifuge and aspirating the supernatant after each wash.
17. After the final wash, transfer the resin in 1 ml of cold Wash Buffer to a 1.5 ml microcentrifuge tube.
18. Pellet the resin by centrifugation for 20 seconds in a microcentrifuge, and then aspirate as much of the Wash Buffer as possible from the resin.
19. Elute the purified receptor in batch by incubating the resin in 150 μl of cold Elution Buffer for 10 min.
20. Pellet the resin by centrifugation for 20 seconds and remove the supernatant containing the purified receptor supernatant to a new tube.
21. Repeat the elution one or two more times (Step #19 and #20).
22. Freeze the purified receptor in aliquots in Liquid Nitrogen and store at -80°C. A preparation of estrogen receptor is stable for up to six months when stored at -80°C with limited thawing and refreezing.
23. Check the protein preparation for purity and yield by SDS Polyacrylamide Gel Electrophoresis using Bovine Serum Albumin standards (see Protocol on SDS-PAGE). Typical yields of estrogen receptor are 5 to 10 μg per 500 ml culture.
24. Perform functional tests on the purified protein, e.g. a ligand-binding assay and DNA-binding assays for the estrogen receptor.
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| Elution Buffer |
| 15% (v/v) Glycerol
0.2 mg/ml FLAG peptide (Kodak/IBI)
100 mM NaCl
20 mM Tris-Cl, pH 7.5
2 mM DTT
1 mM PMSF
0.2 mM EDTA
0.5 mg/ml purified recombinant insulin (Boehringer Mannheim)
0.1% (v/v) IGEPAL CA-630
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| Wash Buffer |
| 2 mM DTT
0.2 mM EDTA
1 mM PMSF
0.2% (v/v) IGEPAL CA-630
10% (v/v) Glycerol
150 mM NaCl
1.5 mM MgCl2
20 mM Tris-Cl, pH 7.5
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| Dilution Buffer |
| 1.5 mM MgCl2
0.5% (v/v) IGEPAL CA-630 (Sigma)
20 mM Tris-Cl, pH 7.5
20 μg/ml Aprotinin
1 mM PMSF
20 μg/ml Leupeptin
2 mM DTT
10% (v/v) Glycerol
0.2 mM EDTA
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| Homogenization Buffer |
| 1.5 mM MgCl2
20 mM Tris-Cl, pH 7.5
500 mM NaCl
20% (v/v) Glycerol
20 μg/ml Aprotinin
2 mM DTT
1 mM PMSF
20 μg/ml Leupeptin
0.2 mM EDTA
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| PBS |
| 1.8 mM Potassium Phosphate, Monobasic (K2HPO4)
4.3 mM Sodium Phosphate, Dibasic (Na2HPO4)
pH 7.2
2.7 mM KCl
137 mM NaCl
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| 0.2 M PMSF |
| Prepare in 100% Ethanol
0.2 M Phenylmethylsulfonyl Fluoride (PMSF) (CAUTION! see Hint #3)
Store at -20°C
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| FLAG-Taged |
| Titered recombinant baculovirus constructed to express the epitope-tagged protein of interest
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| TMN-FH Medium |
| 10 μg/ml Penicillin (Gibco/BRL)
10 μg/ml Streptomycin (Gibco/BRL)
3.3 mg/ml Yeastolate Solution (Gibco/BRL)
3.3 mg/ml Lactalbumin Hdrolysate (Gibco/BRL)
Grace's Insect Cell Culture Medium, pH 6.2 with KOH (Gibco/BRL)
Sterile filter with 0.2 μm filter.
Add 10% (v/v) Fetal Bovine Serum (Gibco/BRL)
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Potassium Phosphate, Monobasic
Glycerol
Insulin, Purified Recombinant
Yeastolate
Leupeptin
Lactalbumin Hydrolysate
EDTA
Grace's Insect Cell Culture Medium
IGEPAL CA-630
Magnesium Chloride
Tris
Nitrogen, Liquid
FLAG-Tagged Protein
FLAG Peptide
Sodium Phosphate, Dibasic
Anti-FLAG M2 Affinity Resin
Potassium Chloride
Sodium Chloride
Fetal Bovine Serum (FBS)
Phenylmethylsulfonyl Fluoride (PMSF)
Aprotinin
Streptomycin
Penicillin
DTT
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1.This protocol uses as an example the estrogen receptor as the protein of interest in the baculovirus FLAG-tagged construct. The optimal m.o.i. for expression of your protein of interest must be determined empirically.
2. All volumes listed in this protocol are based on an initial starting culture volume of 500 ml.
3. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
4. The contributors of this protocol have used the baculovirus expression system to produce estrogen receptor, a nuclear hormone receptor. They have found that this protocol yields highly purified (greater than 90% purity) and highly active estrogen receptor, as assessed by ligand-binding, DNA-binding, and transcriptional activity. They have used this protein in chromatin assembly reactions (see Protocol on Chromatin Assembly on Template DNA with Transcription Factors and Drosophila S-190 Chromatin Assembly Extract). Assaying the chromatin assembled in these reactions is useful for studying the role of nuclear hormone receptors as transcriptional activators.
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1. Kraus, W. L. and Kadonaga, J. T. 1999. Ligand- and cofactor-regulated transcription with chromatin templates. In Nuclear Receptors: A Practical Approach (D. Picard, ed) pp. 167-189. Oxford University Press, Oxford/New York.
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