Purification of GFP by Column Chromatography

 

Purpose:  To use hydrophobic interaction chromatography to extract and purify Green Fluorescent Protein (GFP) from E. ColiMM294 transformed by a plasmid carrying GFP (E.coli/pGREEN).

 

Credits:  DNA Learning Center, David Micklos et al. and occc program student Travis Wright.

 

Day 0:  Overnight culture of GFP-transformed E.coli.

 

1.  Using a sterile inoculating loop remove one of the transformed E.coli colonies from

     the agar plate.

2.  Inoculate into a culturing flask containing LB+AMP broth.  Gently mix to disperse clump of cells throughout broth.

3.  Seal flask and place into a 37ºC shaking water bath or incubator overnight.  E.coli

     cells must receive good aeration in order to fully express the GFP gene.

 

Day 1

 

Equipment and Supplies:

Microcentrifuge                                   

4 mL overnight culture of E.coliMM294/pGREEN grown on shaker for good aeration in 50 mL tube containing LB/amp media.

TE buffer

Lysozyme (50 mg/mL)

37o C water bath

 

Procedure:

  1. Transfer 1 mL of overnight culture into 1.5 or 2.0 mL sterile culture tube and label.
  2. Place in microcentrifuge in balanced configuration and spin at 6000 x g for 5 minutes.  Remove and discard supernatant; take care not to disturb cell pellet.
  3. From same overnight culture tube, transfer another 1 mL into same 1.5 mL tube and repeat steps one and two.  Repeat till all 4 mL of the overnight culture has been pelleted in the same tube.  Discard supernatant at each step, taking care not to disturb pellet.
  4. To cell pellet, add 250 ml of TE buffer.  Suspend cells by pipetting fluid up and down in tube till pellet is gone and cells are dispersed in buffer.
  5. Add 100 ml of lysozyme to culture tube; mix well by inverting tube 8-10 times.  Place in 37oC water bath for 60 minutes incubation for enzyme lysozyme to work to break open cells.
  6. Place culture tube in -20oC freezer overnight.  Ice crystal formation helps disrupt cell walls, membranes, and intracellular vesicles.  If short on time, you can omit overnight freezing and go on to next step and still get adequate results.

 

 

 

Day 2

Sample tube from freezer overnight

Microcentrifuge

Binding Buffer (4 M ammonium sulfate in TE pH 8)

Wash Buffer (1.3 M ammonium sulfate in TE pH 8)

Equilibration buffer (2M ammonium sulfate in TE pH 8)

TE buffer pH 8

Hydrophobic Bead Resin:  Macroprep Methyl HIC BioRad #158-0080

Black light source

The column and accessories can be obtained from BioRad with the following catalog numbers.

                        Column            cat #  737-0711

                        stopcock          cat#   7328102

                        drop forming     cat#   7318131

                        female luer        cat#   7318222

                        HIC resin         cat#   158-0080

 

Procedure:

 

HIC purification works quite simply by taking advantage of the properties of the hydrophobic (water hating) functional groups of the GFP.

 

1.  Following freezing, thaw cell suspension by holding in hand until thawed.

2.  Microfuge solution for 5 minutes at 6000 x g.  This step will pellet the larger pieces of

     cellular debris while leaving the protein of interest (GFP) in the supernatant (liquid).

3.  Remove 250 μL of supernatant and place into a clean centrifuge tube.

4.  Add  250 μL of binding buffer.  Binding buffer is the most concentrated (high salt (content)  of the buffers.  When added to the supernatant containing GFP, the binding

 buffer causes the GFP to undergo conformational changes that expose its hydrophobic functional groups.  During this process the 3-D shape of the GFP molecule is altered allowing the hydrophobic functional groups to move to the exterior of the molecule  which will in turn allow the GFP to bind to the HIC resin in the column.

 

5.  Packing the column

     a.  Attach the column to a ring stand high enough to allow for the placement of a

          test tube rack underneath the column.  The column needs to be perpendicular to

          the work bench.   Attached to the column will be a 2-way stopcock, a female luer,

          and a drop former.  The column needs be 10 cm tall, with a diameter or 0.7 cm,

          holding a bed volume of 4.0 mL.  Check to be sure the column is not clogged by             running water through the empty column.  If water does not flow through, rinse      the column with acetone or alcohol, and then distilled water.

     b.   Place 1 mL of equilibration buffer into the column with stopcock closed.

     c.   Remove 25 mL of ethanol from HIC resin container without disturbing resin.

           Replace with 25 mL of 2 M ammonium sulfate in TE buffer.  (Follow calculations

           from page 2 to make 2M ammonium sulfate).   Shake resin vigorously to suspend

           resin in the ammonium sulfate.  Add 3 mL of HIC resin to column slowly by letting the resin drizzle down the side of the column.  At this time HIC resin is in solution and column bed is not yet formed.

     d.   Place a test tube under the column to collect buffer.  Open the stopcock to allow

           the equilibration  buffer to run out of column.  This will begin the packing process.

     e.   While still running the equilibration buffer through the column, add 3 mL of

           equilibration buffer to the column by drizzling the buffer down the side of the

           column.  This will prevent the disturbing of the column bed.

     f.    Allow buffer to run through column until the meniscus reaches the top of the bed.

           Close the stopcock.  The column is now packed.  The column should be about 2 cm tall and as level as possible.  If top of column is slightly askew it will not effect purification. 

        

6.   Place a new test tube under the column.  Place 400 μL of supernatant/binding buffer

      solution (step 4) into the column by again letting solution slowly run down the side of   

      the column.  It is imperative to the success of purification that the column bed is

      disturbed as little as possible. It is also crucial to keep the column wet with liquid    above it.

7.   Open the stopcock and let the solution run through until the meniscus reaches the top

      of the column bed.  Do not let the resin bed become dry at any time. The GFP is now

      bonded to the resin in the column bed.  At this time there will be a visible green band

      located at the top of the column bed.  The band should be visible to the naked eye but,

      the use of a ‘black’ light will increase visibility and demonstrate the bonding of

      the GFP to the resin. 

8.   Without changing test tubes, add 1000 μL of wash buffer to the column as before

       without disturbing the column bed. 

9.   Open the stopcock and drain solution until the meniscus reaches the top of the

      column bed.  Do not let the column bed run dry.  Close the stopcock. This step has

      two functions.  First the wash buffer will remove any cell debris that was not removed   

      in step 2 and also remove any undesired proteins.  The  wash buffer is also a lower

      salt (ammonium sulfate) concentration than the binding buffer.  This low salt

      concentration will begin to reverse the changes to the 3-D shape of the GFP molecule

      that occurred in step 4.  The GFP is still bound to the resin, though not as strongly as

      before.

10. Without changing test tubes, add 1000 μL of TE buffer to the tube as before.  Do not

      disturb column bed.  Open the stopcock and allow solution to run through column.

      As the TE buffer enters and leaves the column, the band of GFP at the top of the bed

      will begin to migrate towards the bottom of the bed.  This can be seen with the naked

      eye.  However use of black light further demonstrates this movement.   TE

      buffer contains no salt (ammonium sulfate)  and allows the GFP to return to its

      normal 3-D shape.  In its normal shape,  GFP’s hydrophobic functional groups are positioned toward the center of the molecule.   Once this occurs, the GFP can no longer interact with the hydrophobic resin. 

11. Pay particular attention to the green band as it migrates through the column.  Once

      the green band nears the bottom of the bed, close the stopcock.  This should coincide

      with the meniscus of the buffer reaching the top of the bed. If you do not see green band, watch the drops from the column till you see green color coming off.

12. Change tubes.  Add another 1000 μL of TE buffer as before without disturbing column

      bed.  Open the stopcock.  At this time the GFP will begin to elute from the column

      bed.  The drops of solution that form from the drop former will be visibly green. 

      Allow the GFP to elute through until column until bed is dry OR until the drops at the

      drop former are no longer green. 

13. The tube should contain about 0.5 mL of purified GFP.  Solution needs to be

      viewed under a black light to see GFP.  Solution will have a distinct greenish glow in a dark room that proves presence of GFP.

14. HIC matrix material is very expensive and we need to regenerate it so we can use it again. To regenerate the column after this experiment, to reuse for another group of students, add about 2-3 column volumes (~6 cm liquid for every 2 cm column bed) of 1.3M Wash Buffer. Follow with 3-5 CV (column volumes - abut 6-10 cm liquid) of distilled water. Now column is ready for reuse by another class of students.

 

TEACHER: when all your classes have finished with the columns, recover the HIC matrix for return in the following way: take Equilibration buffer (2 M ammonium sulfate in TE pH8) into a transfer pipette, squirt it into column bed and stir up the matrix with the Equilibration buffer. Transfer all the resuspended column matrix into a container such as a 50 mL centrifuge tube, label, and return matrix with rest of module equipment. Rinse the columns with water and airdry.