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MOLECULAR BIOLOGY: WORKING WITH DNA

EXPRESSION: FUSION PROTEINS

Preparation and Use of a Glutathione S-Transferase Column - A General Method

Preparation and Use of a Glutathione S-Transferase Column - A General Method
Contributor: The Laboratory of Jasper Rine at the University of California, Berkeley
 
Overview
The protocol is a general method for the preparation, use and storage of a Sepharose Glutathione-S-Transferase column. A number of Sepharose GST columns are also available commercially. When selecting the Sepharose resin (such as Sepharose 4B) make sure that the exclusion limit and particle size range match the recombinant GST protein of interest.
 
Procedure
A. Column Preparation

1. At 4°C add greater than 2 volumes of ddH2O to the Sepharose GST resin and allow beads to "swell" overnight (see Hint #1).

2. Degas the swollen beads under vacuum for approximately 30 minutes. Occasionally tap the sides of the vacuum flask to release air bubbles caught on the sides of the glass.

3. Place filter paper at the bottom of a column (see Hint #2).

4. Gently layer the Sepharose beads into the column using a wide-bore transfer pipette (see Hint #3).

5. Do not completely fill the column with beads; always leave approximately 1 to 2 cm of space between the bead bed and the top of the column.

6. The remainder of this protocol should be carried out at 4°C either in a cold room or in a column refrigerator.

6. Wash the column with ice-cold Column Buffer (see Hint #4).

7. While washing the column adjust the height of the Column Buffer to adjust the flow rate of the eluate (see Hint #5).

8. To store the column overnight or for a couple of days wash the column with 5 column volumes of Column Buffer with NaN3. Always keep the prepared column at 4°C.

B. Eluting Unwanted Protein From the Column

1. If the column was stored in NaN3 wash out the NaN3 with 10 column volumes of Column Buffer.

2. Remove the cap off of the column and carefully layer the supernatant onto the top of the beads without disturbing the bead bed.

3. Replace the column top and allow the column to flow for approximately 1 to 5 column volumes.

4. Repeat Steps #B2 and #B3 until all of the lysis supernatant has been loaded onto the column. (see Hint #6).

5. Wash the column with Column Buffer until no protein is detected in the eluate (approximately 10 to 15 column volumes, see Hint #7).

C. Eluting Protein of Interest from the Column

1. Eluate the protein of interest from the column by adding reduced Glutathione to the Column Buffer.

2. Wash the column with approximately 10 to 15 column volumes of Column Elution Buffer.

3. Save all fractions until no protein is detected in the eluate.

4. Either analyze each fraction by gel electrophoresis (a very sensitive analysis, see protocol on Gel Electrophoresis) or by protein determination (not as sensitive, see protocol on Protein Quantification).

5. Pool collected fractions of interest.

6. Dialyze the pooled fractions to remove any excess Glutathione (depending on your needs).

Solutions
Column Buffer   Use the lysis buffer of your choice (see Hint #9)
Column Elution Buffer   The concentration of reduced Glutathione should be determined empirically
Column Buffer with Reduced Glutathione
Column Buffer with NaN3   0.02% (w/v) Sodium Azide (NaN3, CAUTION! see Hint #8)
 
BioReagents and Chemicals
Resin
Sodium Azide
Glutathione
 
Protocol Hints
1. You will want to make sure that you prepare enough swollen beads to fill the column you will be using. Columns can range in size depending on the amount of protein need to be purified; however a range of 5 to 10 ml is a good starting point.

2. Cut the filter to the correct diameter of the column and place it snuggly at the bottom of the column. Add ddH2O to the column and make sure that the filter does not impede the flow. Allow the ddH2O to flow out of the column. Depending on your column use the specific filter as per the manufacturer's instructions. Also, the column size can vary greatly depending on the amount of staring material from a Pasteur pipette to a large "batch" column.

3. Do this slowly and methodically so that there are no pockets of air trapped inside the column and you do not cause damage to the beads.

4. The column should be washed using 10 to 15 column volumes of the Column Buffer. Place the buffer above the column and run tubing from the bottom of the buffer to the inlet on the column head.

5. A flow rate of 1 to 2 ml per minute is a good starting point.

6. Be sure to not overload the column with recombinant GST protein. This is tricky to determine but with some practice and understanding of how much protein to expect from how many collected cells will help in the determination. The amount loaded needs to the determined empirically.

7. Use any generalized protein quantification protocol, such as Bradford (See protocol on Protein Quantification). An easy trick is to collect a small volume of eluate onto parafilm. Pipette 5 μl of eluate and combine with 50μl of Bradford and check the absorbance.

8. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

9. Typically use a buffer that is identical to the buffer used to lysis the cells when collecting the recombinant GST protein

 
Citation and/or Web Resources
1. Smith, D.B. and Johnson, K.S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 1988;67:31-40.
2. Tudyka, T. and Skerra. A. Glutathione S-transferase can be used as a C-terminal, enzymatically active dimerization module for a recombinant protease inhibitor, and functionally secreted into the periplasm of Escherichia coli. Protein Science 1997:6:2180-2187.