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MOLECULAR BIOLOGY: WORKING WITH DNA

EXPRESSION: FUSION PROTEINS

Purification of Expressed Proteins from Baculovirus-Infected Sf9 Insect Cells Using Metal Chelation Chromatography

Purification of Expressed Proteins from Baculovirus-Infected Sf9 Insect Cells Using Metal Chelation Chromatography
Contributor: The Laboratory of Jim Kadonaga at the University of California, San Diego
 
Overview
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 (FLAG tag) or metal chelation chromatography (His6 tag). This protocol uses metal chelation to purify the protein (see Hint #4).
 
Procedure
1. Plate 2 X 107 Spodoptera frugiperda (Sf9) insect cells from a log phase culture in a 15 cm diameter plate in TMN-FH Medium.

2. After the cells have attached firmly, replace the medium with 25 ml of fresh TMN-FH Medium.

3. Infect the culture with freshly amplified recombinant-His6 baculovirus at a multiplicity of infection (m.o.i) of 5 to 10 (see Hint #1).

4. Incubate the infected culture for 3 days at 26°C.

5. Remove the cells from the plate by pipetting the medium over the loosely attached monolayer. Transfer the cell suspension to a 50 ml conical tube.

6. Collect the cells by centrifugation at 1,000 X g in a table-top centrifuge for 5 min at 4°C.

NOTE: All procedures from this point on should be performed on ice or at 4°C with ice-cold buffers.

7. Resuspend the cell pellet in 1.0 ml of cold PBS and transfer the cells to a 1.5 ml microcentrifuge tube (see Hint #2).

8. Pellet the cells by centrifugation for 20 seconds in a microcentrifuge and aspirate the PBS.

9. Resuspend the cell pellet in 1.0 ml of cold Homogenization Buffer.

10. Homogenize the cells in a Wheaton Dounce Homogenizer with 10 strokes using the A pestle in a 2 ml vessel.

11. Incubate on ice for 15 min.

12. Centrifuge the resulting whole cell lysate in a microcentrifuge for 10 min.

13. Collect the supernatant into a new 1.5 ml microcentrifuge tube.

14. Add 30 μl of a 50% (v/v) slurry of Nickel-NTA Agarose (Qiagen) previously equilibrated in Homogenization Buffer.

15. Incubate the lysate with the resin for 2 hr at 4°C with gentle mixing.

16. Pellet the resin by centrifugation in a microcentrifuge for 20 seconds and aspirate the supernatant.

17. Wash the resin 4 times with 1.0 ml of cold Wash Buffer per wash, pelleting the resin by centrifugation in a microcentrifuge for 20 seconds and aspirating the supernatant after each wash

18. After the final wash, aspirate as much of the wash Buffer as possible from the resin.

19. Elute the purified protein in batch by incubating the resin in 100 μl of cold Elution Buffer for 10 min.

20. Pellet the resin by centrifugation and remove the supernatant containing the purified protein to a new tube. Repeat the elution (Step #19).

21. Freeze the purified protein in aliquots in Liquid Nitrogen and store at -80°C. A preparation of p300 is stable for up to one year when stored at -80°C with limited thawing and refreezing.

22. Check the protein preparation for purity and yield by SDS polyacrylamide gel electrophoresis with Bovine Serum Albumin standards (see Protocol on SDS-PAGE). Typical yields are 20 to 40 μg of purified protein per 15 cm diameter plate.

23. Perform functional tests on the purified protein, e.g. a ligand-binding assay for a receptor fusion protein or a histone acetyltransferase assay for p300.

Solutions
PBS   4.3 mM Sodium Phosphate, Dibasic (Na2HPO4)
pH 7.2
2.7 mM KCl
1.8 mM Potassium Phosphate, Monobasic (KH2PO4)
137 mM NaCl
TMN-FH Medium   10 μg/ml Penicillin (Gibco/BRL)
10 μg/ml Streptomycin (Gibco/BRL)
Add 10% (v/v) Fetal Bovine Serum (Gibco/BRL).
3.3 mg/ml Lactalbumin Hydrolysate (Gibco/BRL)
3.3 mg/ml Yeastolate Solution (Gibco/BRL)
Sterile filter with a 0.2 μm filter.
Grace's Insect Cell Culture Medium, pH 6.2 with KOH (Gibco/BRL)
Elution Buffer   100 mM NaCl
10 mM Tris-Cl, pH 7.5
0.1% (v/v) Igepal CA-630
10% (v/v) Glycerol
2 mM PMSF
250 mM Imidazole
2 mM 2-Mercaptoethanol
Wash Buffer   0.2% (v/v) Igepal CA-630
200 mM NaCl
15 mM Imidazole
10 mM Tris-Cl, pH 7.5
10% (v/v) Glycerol
2 mM PMSF
2 mM 2-Mercaptoethanol
Homogenization Buffer   2 mM 2-Mercaptoethanol
2 mM PMSF
10 mM Tris-HCl, pH 7.5
0.1% (v/v) Igepal CA-630 (Sigma)
500 mM NaCl
20 μg/ml Aprotinin
20 μg/ml Leupeptin
15 mM Imidazole
10% (v/v) Glycerol
0.2 M PMSF   Prepare in 100% Ethanol
0.2 M Phenylmethylsulfonyl Fluoride (PMSF) (CAUTION! see Hint #3)
Store at -20°C
 
BioReagents and Chemicals
2-Mercaptoethanol
Leupeptin
Glycerol
IGEPAL CA-630
Yeastolate
Lactalbumin Hydrolysate
Grace's Insect Cell Culture Medium
Streptomycin
Penicillin
Sodium Phosphate, Dibasic
Tris
Potassium Chloride
Sodium Chloride
His6-tagged Protein of Interest
Aprotinin
Fetal Bovine Serum (FBS)
Nickel-NTA Agarose
Nitrogen, Liquid
Imidazole
Potassium Phosphate, Monobasic
Phenylmethylsulfonyl Fluoride (PMSF)
 
Protocol Hints
1. This protocol uses as an example p300 as the protein of interest in the baculovirus His6-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 initial starting material from one 15 cm diameter plate. Human p300 is synthesized particularly well in Sf9 cells. However, for other proteins that are not synthesized as well, this preparation could be scaled up to more plates or to a large volume suspension culture.

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 p300, a transcriptional co-activator with intrinsic histone acetyltransferase activity. They have used this protein in chromatin assembly reactions (see Protocol on Assembly of Chromatin with Drosophila S-190 Chromatin Assembly Extract or Protocol on Chromatin Assembly on Template DNA with Transcription Factors and Drosophila S-190 Chromatin Assembly Extract) and in transcriptional assays of in vitro assembled chromatin (see Protocol on Transcription of Chromatin Reconstituted with Drosophila S-190 Chromatin Assembly Extract and Primer Extension Analysis or Protocol on Assay for a Single Round of in vitro Transcription from Assembled Chromatin Templates Using a HeLa Cell Nuclear Extract).

 
Citation and/or Web Resources
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.