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MOLECULAR BIOLOGY: WORKING WITH DNA

EXPRESSION: FUSION PROTEINS

Purification of Maltose Binding Protein-Fusion Proteins from E. coli

Purification of Maltose Binding Protein-Fusion Proteins from E. coli
Contributor: The Laboratory of Donald Rio at the University of California, Berkeley
 
Overview
This protocol is a general outline for purification of MBP-fusion proteins expressed in E. coli. After cloning the gene into the expression vector pMAL-c2, be sure to check 2 to 5 clones for expression of the fusion protein at both 30°C and 37°C in NB42. One method for purifying a heterologously expressed protein of interest is to clone the gene in frame with the Maltose Binding Protein (MBP) coding sequence and introduce the resulting construct into E. coli.. The MBP-fusion protein is purified from a bacterial lysate by binding to an amylose resin. After washing the resin of bacterial impurities, the fusion protein is eluted off the amylose resin with maltose.
 
Procedure
A. Cell preparation (see Hint #2)

1. Inoculate 500 ml of Rich Media with 5 ml of an overnight culture of strain expressing the MBP-fusion protein. Grow at the appropriate temperature to an optical density of 0.5 at 600 nm (OD600 of 0.5).

2. Optional: Collect 1 ml of uninduced cells and centrifuge in a microcentrifuge for 2 min. Discard the supernatant and resuspend by vortexing in 50 μl of 1X SDS-PAGE Sample Buffer. Store on ice.

3. Induce the bacteria with 1.5 ml of 0.1 M IPTG for 2 hr at 30°C (see Hint #3).

4. Optional: Collect 0.5 ml sample of induced cells and centrifuge in a microcentrifuge for 2 min. Discard the supernatant and resuspend by vortexing in 100 μl of 1X SDS-PAGE Sample Buffer. Store on ice.

5.Centrifuge the culture for 10 min in a GSA rotor at 6,000 rpm (5,900 X g) to pellet the cells.

6. Remove the supernatant and resuspend the cell pellet in 10 ml of ice-cold Lysis Buffer.

7. Transfer the cell suspension to Ti70 tubes. Snap freeze the cells in Liquid Nitrogen or a dry ice-Ethanol bath. Or store the cells at -20°C until you are ready to proceed.

8. Thaw frozen cells in cold water.

9. Sonicate, using 15 second bursts at a setting of 4 with the tip of a cell sonicator probe. Be sure to minimize foaming, keep sample in ice water bath during sonication, and cool on ice between each burst. Repeat sonication bursts until no more protein is released. Monitor protein release of 10 μl aliquots of the lysate in the Bradford Assay (see Protocol on Protein Concentration Determination Using the Bradford Assay). The suspension should be partially clear when finished.

10. Centrifuge at 4°C for 30 min at 9,000 X g.

11. Transfer the supernatant to a 15 ml conical tube. The crude extract can be frozen in Liquid Nitrogen or placed immediately into an Amylose column.

12. Optional: Resuspend the pellet in 10 ml Lysis Buffer. Take a 5 μl aliquot of the resuspended pellet and add 5 μl of 2X SDS-PAGE Sample Buffer. Store on ice. Take 5 μl of the crude extract and add 5 μl of 2X SDS-PAGE Sample Buffer. Store on ice.

B. Fusion Protein Purification on Amylose Agarose (See Hint #4)

1. Pour a 1 ml Amylose resin column. The capacity is 3 mg of Maltose-binding protein/ml resin.

2. Attach the column to the FPLC, and wash with 5 to 10 column volumes of Wash Buffer.

3. The FPLC parameters should be as follows: 0.2 ml/min flow rate, 0.5 cm/ml chart speed, 1 AU absorbance, 1 ml fraction size.

4. Load the sample (see Hint #5). Collect the flow-through in a 50 ml conical tube.

5. Once loaded, wash the column with 10 column volumes of Wash Buffer.

6. Elute the MBP-fusion protein with 15 column volumes of Elution Buffer.

7. Pool the MBP-fusion protein containing fractions. Store at -70°C. If necessary the protein can be concentrated in a Centricon or Centriprep concentrator (Amicon).

8. Optional: Take 10 μl of eluted fusion protein and add 10 μl of 2X SDS-PAGE Sample Buffer. Boil all the SDS-PAGE samples for 3 minutes. To analyze induction, yield, and purification of the fusion protein, load samples onto a 10% SDS-polyacrylamide gel (see Protocol on SDS-PAGE). After gel has run, stain with Coomassie Blue to visualize the proteins or prepare the gel for Western blotting and blot with anti-MBP antibody (see Protocol on Western Blotting).

Solutions
2X SDS-PAGE Sample Buffer   4% SDS
0.005% Bromophenol Blue
5% 2-Mercaptoethanol
20% Glycerol
125 mM Tris-Cl, pH 6.8
0.1 M Isopropyl β-D-Thiogalactopyranoside (IPTG)   Sterile filter through 0.22 μm filter
0.1 M IPTG (CAUTION! see Hint #1)
Elution Buffer   1 mM EGTA, pH 8.0
Filter through a 0.22 μm filter before use.
Add the DTT, PMSF, and Na2S2O5 fresh before use.
200 mM KCl
50 mM Tris-HCl, pH 8.0
1 mM EDTA, pH 8.0
2 mM Na2S2O5
10 mM Maltose
0.5 mM DTT
1 mM PMSF
10% (v/v) Glycerol
Wash Buffer   1 mM EGTA, pH 8.0
Filter through a 0.22 μm filter before use.
Add the DTT, PMSF, and Na2S2O5 fresh before use.
200 mM KCl
50 mM Tris-HCl, pH 8.0
1 mM EDTA, pH 8.0
2 mM Sodium Metabisulfite (Na2S2O5)
0.5 mM DTT
1 mM PMSF
10% (v/v) Glycerol
Protease Inhibitor Cocktail (1000X) (CAUTION! see Hint #1)   0.5 mg/ml Leupeptin
0.5 mg/ml Aprotinin
in ddH2O
0.5 mg/ml Chymostatin
0.5 mg/ml Antipain
0.5 mg/ml Pepstatin
Lysis Buffer   1 mM EGTA, pH 8.0
100 mM KCl
50 mM Tris-HCl, pH 8.0
1 mM EDTA, pH 8.0
Add the DTT, PMSF, and Protease Inhibitor Cocktail fresh before use.
1X Protease Inhibitor Cocktail
0.5 mM DTT
1 mM Phenylmethylsulfonyl Fluoride (PMSF) (CAUTION! see Hint #1)
1 M NaCl
Rich Media   5 g/liter NaCl
5 g/liter Yeast Extract
When cooled, add Ampicillin to 100 μg/ml
Autoclave
2 g/liter Glucose
10g/liter Tryptone
 
BioReagents and Chemicals
Glycerol
Dry Ice
Sodium Metabisulfite
Leupeptin
EDTA
PMSF
Yeast Extract
Tryptone
Tris-HCl
2-Mercaptoethanol
Potassium Chloride
Sodium Chloride
Aprotinin
SDS
DTT
EGTA
Glucose
Maltose
Antipain
IPTG
Ampicillin
Chymostatin
Pepstatin
Liquid nitrogen
 
Protocol Hints
1. This substance is a biohazard. Please consult with this agent's MSDS for proper handling instructions.

2. If expression or yield is poor in the host bacterial strain, try introducing the fusion plasmid into a protease deficient lon- and/or rpoH- host strain. lon- strains lack the activity responsible for degrading aberrant proteins. rpoH- strains lack a heat shock transcription factor that activates some stress-induced proteases. CAG629 is both lon- and rpoH-, but is also a difficult strain to work with. ER2508 and CAG597 are good alternatives; ER2508 is lon- and CAG597 is rpoH-. The CAG strains are difficult to transform by heat shock of competent cells. Often the strains must be transformed by electroporation. These strains are available from New England Biolabs.

3. The induction can be carried out at 37°C; however, lower temperatures favor increased fusion protein stability. Incubations of 1 hour or 3 hour might give higher yields.

4. Outlined here is the procedure for FPLC purification on Amylose beads. It is easy to purify MBP-fusion protein by FPLC, however batch elution can also be employed.

5. You do not need to dilute the sample in order to reduce the NaCl concentration. The fusion protein will bind just fine in 1 M NaCl.

 
Citation and/or Web Resources
2. di Guan C, Li P, Riggs PD, Inouye H. Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. Gene 1988; 67:21-30
1. Maina CV, Riggs PD, Grandea AG 3d, Slatko BE, Moran LS, Tagliamonte JA, McReynolds LA, Guan CD. An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. Gene 1988; 74:365-73