Exercise 9. Affinity Chromatography

Exercise 9. Affinity Chromatography

Objectives

Pure proteins are required for a wide variety of applications. One method to purify a protein is through the introduction of an “affinity tag” that binds to a chromatography medium/column. In this lab you will perform affinity chromatography on His-tagged GFP and will subsequently assess the purity of your purified GFP.

Prelab Questions:

1.  Do you think the method that we are using (His-tagged protein with Affinity Chromatography) of purification would give higher or lower protein purity than other methods of purification, such as Size Exclusion Chromatography or Anion Exchange Chromatography? Why?

2.  Using my calibration curve (given in the write-up), how much of your purified protein product do you need to load onto the gel if your absorbance using the Bradford Reagent at 595nm is 0.55 ? How much water should you add to equal the correct final volume without loading buffer?

3.  The Simply Blue stain that we use to stain the gel has a Coomassie Blue Stain that stains the proteins blue. We use coomassie in another section of Lab 9 (it is present in another solution). Can you think of what other solution / reagent coomassie may be present in?

Background

Large quantities of pure and homogeneous proteins are essential to a number of applications, from scientific studies of protein function to pharmaceutical protein production by the biotechnology industry. In the former example, impurities can complicate or even invalidate the results of scientific experiments. In the latter, however, contaminants in a pharmaceutical protein product can cause side effects that can potentially endanger a patient.

As you learn in separations courses, compounds are separated and purified based on differences in physical properties, and this same principle applies in biological systems. Proteins can be purified on the basis of their size, solubility, density, charge, or ability to bind to a compound. This lab will focus on the last of these approaches, purification of a protein based on its affinity for a small compound, or ligand.

In a previous lab, you inserted the green fluorescent protein into a new vector (pBAD). Similarly, the GSI’s have inserted GFP into a vector (pET19) that contains a recombinant protein tag (His tag). In doing so, this tag has been fused onto the beginning of GFP, so that when GFP is expressed from this vector, six histidine residues are added to GFP’s amino terminus. This His tag binds to the metal nickel with high specificity and affinity. Nickel immobilized on small beads is commercially available and can be loaded onto a column. A crude solution containing GFP is then passed over the column, the column is extensively washed, and the GFP is stripped from the column for recovery by the addition of a histidine analog.

The second phase of this lab is to assess the purity of your sample through SDS-PAGE electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a technique for the separation of polypeptide subunits according to their molecular weight. The protocol involves denaturing the protein sample by heating it in the presence of SDS and a reducing agent (DTT). SDS will bind to the protein causing it to unfold, whereas the reducing agent will reduce the intramolecular and intermolecular disulfide bonds. The binding of SDS by the protein confers a net negative charge and the denatured polypeptide will migrate through a gel of known percent acrylamide in the presence of an applied electric field towards the positive electrode (anode). After the electrophoresis is complete, the gel is stained with a Coomassie Blue Stain to visualize the polypeptide bands. The molecular weight of the polypeptide is inversely proportional to its mobility. The molecular weight of our GFP is roughly 30 kDa and can be verified by a standard protein ladder of known MW species. If your sample is pure, the band associated with the molecular weight of GFP will be the only band present on the gel.

Procedures

Protein Purification (Day One)

I.  Preparing The Column

You are given a syringe column packed with chelating sepharose precharged with bound Nickel. You must wash and equilibrate the column (our column = 1 cm column volume).

1.  Fill the syringe with distilled water. Remove the stopper and connect the column to the syringe (use the adapter provided), to avoid introducing air into the system.

2.  Remove the snap-off end at the column outlet.

3.  Wash the column with 3-5 column volumes (CV) of distilled water by slowly pushing water through the column. Try to push the water out at a steady rate by watching the volume in the syringe go down. Ideal flow rate is 1 mL/min. You may discard the water.

4.  Equilibrate the column with at least 5 CV’s of binding buffer. Practice maintaining constant flow by collecting 1 mL as your partner times you.

II.  Binding, Purifying and Eluting GFP

5.  Apply cell lysate containing impure GFP (supplied by GSIs) to the column. Collect 500uL fractions of flow through (this means that each 500 uL of flow through must be collected into a separate centrifuge tube and labeled).

6.  Continue loading the column (and collecting fractions) until the flow through is a constant color of green (approximately 10 mL lysate). This indicates that you have reached the maximum binding capabilities of the column. You may use the UV source to help decide when you are finished loading.

7.  Wash away impurities using binding buffer. Flow 10-15 CVs buffer through the column to purify GFP. Collect 1 mL fractions of flow through for this step.

8.  When you are confident that all impurities are washed away, elute GFP from the column using elution buffer (0.2M Imidazole). 5 CVs of elution buffer is usually sufficient, but continue eluting until you can no longer see GFP being removed from column. Collect 500 uL fractions of pure product.

9.  Wash column with 5 CVs nanopure water and dispose of water flow through to clean the column before storage.

III.  Quantifying the Flow through and Amount of Purified Protein

You must quantify the amount of protein that you have in order to know how much to load on a protein gel and to calculate a breakthrough curve. You can do this using a Bradford Reagent which binds to the basic sidechains of amino acids and changes color upon binding. The change in absorbance at 595nm detects this change.

7.  You will quantify all fractions collected (from binding, purifying, and eluting steps). Two blanks should be prepared with (1) binding buffer and (2) elution buffer. These will be used to calibrate the instrument. Label cuvettes and centrifuge tubes accordingly.

8.  Place 780ul sterilized water in all centrifuge tubes.

9.  Add 20 ul binding buffer to Blank1 and 10uL elution buffer to Blank2.

10.  Add 20ul fraction sample to others.

11.  Add 200ul Bradford Reagent to each tube and mix the tubes thoroughly.

12.  Set a timer for 10 minutes.

13.  After 10 minutes, transfer each solution to their respective cuvettes.

14.  Blank / calibrate the spectrophotometer at 595nm with Blank1. Read the absorbance of your binding and purifying fractions.

15.  Blank spectrophotometer with Blank2. Read absorbance of eluted fractions. These should be your pure GFP samples.

16.  If absorbance is greater than 1.0, prepare a second sample of that fraction using only 5 uL + 15 uL buffer to assure that measurement is accurate.

17.  Plug the absorbance that you obtained for your sample into the following equation to obtain a rough estimate of your protein content. (note: the below equation is the GSI’s calibration curve, and will vary depending on technique of the experimentalist). This equation will give you the ug of protein in the cuvette. Divide that by 20ul (the amount that you put into the cuvette) and you obtain a concentration that should be representative of the concentration of your collected fraction.

(Absorbance at 595nm) = (ug protein present in cuvette)*0.0448

18.  Save the elution fraction containing the most protein. Label with initials and concentration. Record the concentration of this fraction in your lab notebook. You will need this number when you run your protein gel.

19.  Place this fraction (your purified GFP product) in the refrigerator until we are ready to run our protein gel.

SDS-PAGE Electrophoresis (Day Two)

I.  Preparing your sample

1.  Calculate the amount of protein you will need to load onto the gel. Ideally you want to load 5ug of protein. The total volume you have to work with is 15ul.

2.  In a fresh centrifuge tube, dilute your sample accordingly with sterilized water. (example: for a 1mg/ml sample you would add to your vial 5ul GFP product and 10ul water.) Your total volume should be 15ul.

3.  Add 5ul loading buffer. Make sure this is solution is mixed (can do this with your pipette).

4.  Place centrifuge tube in the heating block for 2-3 minutes at 90oC.

5.  Remove from heating block. You are now ready to load your sample into the gel.

II.  Preparing the gel (This will be done by the GSI)

6.  Into gel box, add 80ml 10X running buffer to 720ml water. Make sure this is well mixed. (this mixes well if you add the water last)

7.  Assemble gel box.

8.  Remove protein gel from plastic and peel off the white tape allowing charge to reach the gel.

9.  Place gel in gel box and remove the comb from the top.

III. Loading the gel

10.  Each lane holds 20ul sample. To assure that no sample flows into nearby lanes, it is better to load only 18ul into each lane. Only 5ul of protein ladder is loaded.

11.  Using 1ul pipette tips, pipette 18ul sample into your pipette.

12.  Keeping the pipette in a vertical position, carefully insert tip into the gel lane that you wish to load into. Be very careful that you don’t scratch the gel in any way!

13.  Release the sample into the lane SLOWLY so that it doesn’t flow out of the lane.

14.  Once sample is in the lane, remove the pipette very carefully, maintaining the vertical position.

15.  If you feel that another sample has contaminated your lane, you may pipette water into it to wash it prior to loading your sample

IV. Running / staining the gel (This will be done by the GSI)

16.  Place the hat on the gel box and plug it into the voltage source.

17.  Gel is run at 125V for 1.5 hours.

18.  When gel is finished running, it is removed from the gel box and the plastic around the gel is cracked open exposing the actual gel. (prior to this, the gel is in a plastic cassette).

19.  Gently remove gel from plastic cassette and place it in a staining box. Wash twice with sterilized water.

20.  Remove water from staining box and fill with ~100ml of Simply Blue.

21.  Gel will now stain for 1 hour on a shaker.

22.  Dump out stain and fill staining box with water. Leave overnight to allow the gel to destain. Gel can then be scanned on a scanner.

Guidelines for Analysis & Conclusions Section

(Remember, these are points you should consider and include in your analysis. This section, however, need not be limited to these specific guidelines.)

1.  For each set of fractions, plot protein concentration vs. elution volume. How do these plots compare to what you expected?

2.  Using the data from the binding step, along with following information about the column, create a dimensionless breakthrough curve. Calculate the number of transfer units Nt. What is the rate limiting step of this chromatography process?

avg particle diameter: 90 μm

void fraction ε : 20% of column volume

column volume : 1mL

ρB = 1.1g/cm3

3.  Use the SDS PAGE gel from this lab to quantify the purity of your pure GFP and of the unpurified GFP (which will be run on the gel as well by the GSI). You can get a rough idea of the purity by eyeballing it, but to get an accurate % purity you must use a program which quantifies the color contrast on the gel. I recommend using Scion Image to obtain a plot. Scion Image will then compute for you the area under each curve which you can use to compute %purity. The website to download Scion Image is: http://www.scioncorp.com/pages/scion_image_windows.htm

4.  Considering the chemical structure of histidine, why would you think it would bind to nickel? When might it be detrimental to add a His-tag onto a protein?

5.  GFP is often used as a “reporter gene,” or a gene whose presence is easily detected or “reported” in cells. How useful do you think GFP is as a reporter gene, considering issues of fluorescence quantification and tracking of fusion proteins?

6.  For production of pharmaceutical proteins, large quantities of product are required. How easy do you think it would be to scale up affinity chromatography?

REFERENCES

The QIAexpressionist: A handbook for high-level expression and purification of 6xHis-tagged proteins (1997) Qiagen.

Scopes, R.K. (1994) Protein Purification: Principles and Practice, Springer-Verlag, New York.

Voet, D., Voet, J.G. (1990) Biochemistry, pp. 824-829, John Wiley and Sons, New York.