BISC220/S14: Mod 1 Lab 3

Purifying and Handling Proteins in the Lab

Solubilization:
The first step in the purification of a specific intracellular protein is extraction of the protein from the cells. Bacterial cells can be broken and their enzymes extracted (solubilized) by a variety of techniques that may involve mechanical techniques such as grinding or chemical lysis of the cells. The objective is to release the desired enzyme from the cells by as gentle a method as possible in order to retain activity of the enzyme.

Isolation and Concentration: There are several methodologies that can be employed for the purification of enzymes. These include:

1. differential solubility
2. ion exchange chromatography
3. affinity chromatography
4. molecular sieve techniques
5. density gradients
6. electrophoresis
7. electrofocusing
   

Most of these methods rely on differences in charge (sum of positive and negative charge) or molecular weights of the enzymes and other proteins. Generally cruder, less time consuming methods, are used in the initial processing of crude extracts. This is necessary not only because of the large quantities of protein to be processed but also because of the complexity of the protein mixture. As noted above some of these purification procedures are discussed in your text.

A classic method for the partial purification of enzymes from crude extracts is based on the differential solubility of proteins. Proteins remain dissolved in solution because of their charged surface residues (amino acid side chains) which interact with the molecules of the solvent (water). If such interactions are prevented, the protein molecules will interact principally with one another forming huge aggregates that precipitate out of solution. A common method of enzyme precipitation is by the addition of inorganic salts such as ammonium sulfate.

Stabilization During Purification: When working with enzymes, it is important to provide environmental conditions that will reduce the amount of enzyme denaturation that occurs during purification procedures. Denaturation results in loss of enzymatic activity. The following are some of the conditions that must be controlled:

1. pH: Enzymes have multiple charges on their surfaces. These charges must be preserved to maintain the native structure and enzymatic activity. Buffers are used to maintain the pH of enzyme solutions at a desired pH. The buffer must be in the appropriate concentration, have the correct pKa, and must not adversely affect the protein. See your general chemistry text for a review of buffers and other inorganic chemistry terms.
2. Ionic Strength : The ionic strength of the buffer solution is usually critical. Many enzymes require a medium that has a greater ionic strength than provided by the buffer alone. Inorganic salts such as potassium chloride or sodium chloride, which increase the ionic strength, are commonly added to increase the stability of enzymes. Adding sucrose or glycerol to the buffer can often stabilize enzymes requiring a more hydrophobic environment. We will be using glycerol to stabilize β-galactosidase during storage.
3. Protection Against Sulfhydryl Oxidation: Enzymes may contain many sulfhydryl groups (SH). One or more may be required for the activity of an enzyme. If these sulfhydryl groups become oxidized, they form intra- or intermolecular disulfide bonds. If necessary, the most effective method of retarding such oxidation is the addition of a reducing agent to the buffer. Dithiothreitol, dithioerythritol and β-mercaptoethanol are among the most effective reducing agents used. β-galactosidase seems to be most stable in a reducing environment, so often β-mercaptoethanol is included in the solutions used to extract and assay the enzyme.
4. Protection Against Heavy Metals: In addition to oxidation, sulfhydryl groups may react with heavy metal ions such as lead, iron or copper. Principal sources of metal ions are the reagents used to make up buffers, substrates, and water itself. Deionized distilled water is used to make up reagents and a chelating agent such as EDTA (ethylenediaminetetraacetic acid) can be added as well.
5. Temperature: A rule of thumb is to keep enzymes as close to 0-4°C as possible during purification procedures. A few enzymes exist, however, that denature or inactivate at cold temperatures.

Basic Rules for Handling Enzymes

The following information is taken from a popular primer, now published by Roche Pharmaceuticals, but first published in the December 1985 issue of BMBiochemica. by Boehringer Mannheim Biochemicals.

1. For best stability, enzymes should be stored in their original commercial form (lyophilized, ammonium sulfate suspension, etc.), undiluted and at the appropriate temperature specified on the label.
2. For enzyme solutions and assay buffers, use the highest purity water available. Glass distilled water is best. Deionized water, especially if passed through an old filter or a reverse osmosis device, may contain traces of organic contaminants which inhibit enzymes.
3. Enzymes should be handled in the cold (0-4°C) Dilute for use with ice-cold buffer or distilled water as appropriate for each enzyme. While using an enzyme solution or suspension at the bench, keep it in on ice.
4. Dilute enzyme solutions are generally unstable. The amount of enzyme required for the experiment should be diluted within 1-2 hours of use. Enzymes should not be diluted for long-term storage.
5. Enzymes, especially those that have been diluted, should be checked for activity periodically to ensure that any slight loss in activity is taken into account when designing an experimental protocol. Expiration dates on vials only refer to enzymes stored in the original form at the correct temperature.
6. Do not shake crystalline suspensions (e.g. ammonium sulfate suspensions) since oxygen tends to denature the enzyme. The material should be resuspended with gentle swirling or by rolling the bottle on the lab bench. Once the enzyme crystals have been uniformly resuspended, remove the amount needed with a pipette. In many cases, the enzyme crystals may be used directly in the assay procedure.
7. Do not freeze crystalline suspensions. Freezing and thawing in the presence of high salt concentrations causes denaturation and loss of activity.
8. Vials containing lyophilized enzymes (as well as cofactors, such as NADH and HADPH) should be warmed to room temperature before opening. This prevents condensation of moisture onto the powder, which can cause loss of activty or degradation. If the reagent is hygroscopic, one such mishandling may well ruin the entire vial.
9. Avoid repeated freeze-thawing of dilute enzymes and lyophilizates in solution. Store in small aliquots. Thaw one portion at a time and store that portion once thawed at 4C. The stability of individual enzymes may vary greatly and often should be determined empirically under your exact conditions.
10. Detergents and preservatives should be used with caution, since they may affect enzyme activity. Sodium azide, for example, inhibits many enzymes which contain heme groups (e.g. peroxidase). Detergents added at concentrations above their critical micellar concentration form micelles which may entrap and denature the enzyme.
11. Enzymes should be handled carefully. To avoid contamination of any kind, use a fresh pipette for each aliquot that is removed from the parent vial. Never return unused material to the parent vial. Wear gloves to prevent contaminating the enzyme with proteases, DNAses, RNAses, and inhibitors often found on fingertips. Never pipette by mouth.
12. Adjust the pH of the enzyme buffer at the temperature at which it will be used. Many common buffers (Tris, glycylglycine, Bes, Aces, Tes, Bicine, Hepes) change rapidly as the temperature changes. For instance, Tris buffer decreases 0.3 units of pH for EVERY 10°C rise in temperature. A solution of Tris, adjusted to pH7.5 at +25°C will have a pH of 8.1 at 4°C or 7.2 at 37°C. The change in pH per 10°C temperature change for other buffers is: Aces, -0.20; Bes, -0.16 ; Bicine,-0.18; glyclyglycine, -0.28; Hepes,-0.14; Tes, -0.20 [Good, NE et al, (1966) Biochemistry 5:4-7.
13. The absorbance at 280nm, widely used to quickly determine the protein concentration of an enzyme solution, actually is due to the presence of tyrosine and tryptophan in the protein. If an enzyme (e.g. superoxide dismutase) contains low amounts of these two amino acids it will not absorb significantly at 280nm.

Detailed information is available on many enzymes. The following are excellent resources.

Resources: Methods in Enzymology, published by Academic Press, Editors in chief: Sidney P Colowick and Nathan O. Kaplan. There are more than 165 volumes in this series, covering an extensive range of topics.

The Enzymes, 3rd edition, edited by Paul D. Boyer. An excellent, broad series which focuses more on the properties of the enzyme and less on methodology than Methods in Enzymology.

Methods in Enzymatic Analysis, 3rd edition. Editor-in-chief: Hans U. Bergmeyer, published in Verlag Chemie. In-depth discussions of techniques of analysis that use enzymes or that assay enzymatic activity.

Affinity Chromatography and Polyacrylamide Gel Electrophoresis

Affinity Chromatography
The use of metal chelate affinity chromatography for protein purification was first reported by Porath and colleagues in 1975. This landmark report applied the knowledge that histidine and cysteine form rather stable complexes with some cations such as zinc and copper ions. We now know that the amino acid tryptophan shares this characteristic with histidine and cysteine and that some proteins have specific binding sites for these metals. Porath et al (1975) devised a method to tightly bind metal ions to a solid matrix. Agarose beads can be used as such a matrix in a column through which protein solutions are passed. Some proteins in these solutions bind to the matrix but can be specifically eluted by a low pH wash solution. Later work (Porath and Olin, 1983; Kagedal, 1985) showed that it is possible to release proteins from such columns by use of a strong soluble chelator such as EDTA or by chemically competing with the binding. For example, histidine binding can be disrupted by including imidazole in the protein elution buffer. The chemical structure of imidazole is similar to the ring structure of histidine, therefore it competes for the binding site on the cation matrix.

In today’s lab, we are using agarose beads chelated with nickel to fractionate the 6xHis-tagged β-galactosidase. Nickel effectively binds histidine, and the combination of 6 histidine residues in a row at the amino terminus of the β-galactosidase molecule should lead to rather tight binding. After washing the nickel chelated agarose to remove any unbound or loosely bound protein, we will use a buffer containing 200 mM imidazole to release the 6xHis tagged protein from the nickel chelated agarose beads.

Protocol for the Purification of 6xHis Tagged β-galactosidase Affinity Column Purification Protocol

To do today: in this laboratory session you will purify β-galactosidase from the cell pellet of the genetically modified E. coli that you induced last week to over express this enzyme.

Using B-Per 6xHis Fusion Protein Purification Kit©, product #78100 by Pierce, Inc.

Microsoft Word File: Media:Purification of 6xHis Tagged β gal Protocol.doc

1. Defrost the frozen pellet of E.coli BL21(pET-14b) cells prepared in the previous lab session and add 10.0 ml of B-Per Protein Extraction Reagent™, a mild anionic detergent (the exact ingredients of which are proprietary), to the centrifuge bottle. Add 20μl of DNAase (to achieve a concentration of 1 unit/ml from the 5000units/ml stock) to the B-Per and cells. Resuspend the cells by vortex mixing and pipetting up and down until the pellet is off the side of the bottle and all clumps are gone. Try to avoid bubbles. This should take about 5 minutes total, of alternately mixing and letting the mixture sit in your ice bucket. Your goal is to dissolve the cell walls and membranes which will lyse the cells and allow degradation of the DNA. Ask your instructor to check your suspension before proceeding to step 2. If you have obvious clumps, the cells are less likely to be lysed sufficiently.
2. Shake the suspension gently for 10 minutes at room temperature using a platform shaker. During this time the lysed cells will release their contents and soluble bacterial proteins will be dissolved in the lysate. Be sure to break up any obvious clumps with gentle vortexing.
3. Carefully pour the entire suspension into a Corex tube fitted with a rubber adapter and centrifuge for 15 minutes at 10,500 rpm in the Sorval refrigerated centrifuge at 4C using an SS-34 rotor. You will need to make a balance tube with water. Be sure and record in your lab notebook the speed as g force (use the chart on the wall near the centrifuges to make the conversion).
4. Pour the supernatant only into a 15 ml graduated conical centrifuge tube. This solution of dissolved proteins in B-Per reagent is your cell free or Crude Extract. The pellet is bacterial cell debris that can be dumped into your waste container. The Corex tubes are NOT disposable. Please rinse them out and leave them on your bench or return them to your instructor.
5. Determine and record the total volume of the crude extract (CE) using the volume markings on the tube. Pipet 600 µl of the CE into a microfuge tube labeled with your team color, lab day, initials and "Crude Extract". Store the aliquot on ice. At the end of lab today, you will give what remains of this aliquot to your instructor to freeze for use next time.
6. Add 1000µl (1ml) of the Nickel-Chelated Agarose to the volume of Crude Extract remaining in the 15ml conical tube. It is essential that the agarose suspension be properly mixed before removing an aliquot, so mix it well by swirling or gentle vortexing just before removing it from the stock.
7. Shake the Agarose-CE mixture gently for 10 minutes at room temperature on the platform shaker. Make sure the cap is secured tightly. Centrifuge for 5 minutes at top speed in a clinical centrifuge.
8. Using a Pasteur pipette, carefully remove and discard the supernatant solution into a waste container provided. Do not try to pour off the supernatant! The 6xHis tagged proteins should now be bound to the nickel- chelated agarose beads at the bottom of the tube.
9. Add 3.0 ml of Wash Buffer #1® to the agarose beads in the conical tube and resuspend using a pipette. Do not vortex. Gently shake the suspension at room temperature for 5 minutes on the platform shaker. Centrifuge for 3 minutes at 2500rpm. Carefully remove supernatant and discard.
10. Add 3.0 ml of Wash Buffer #2® to the centrifuge tube and resuspend the agarose using a pipette. Do not vortex. Gently shake the suspension at room temperature for 5 minutes on the platform shaker. Centrifuge for 3 minutes. Carefully remove supernatant and discard.
11. Add 250µl of Wash Buffer #2® to the agarose, resuspend completely and transfer all of this suspension from the centrifuge tube to a B-PER™ Spin Column that has been placed into one of the special collection tubes provided. To insure that the agarose beads don’t get stuck in the pipet tip of your P1000, cut about a 1/4 inch off the end of it with a single edged razor blade. Centrifuge for 2 minutes at maximum speed in a microcentrifuge. Discard the collection tube with the flow-through.
12. Place the spin column in a new collection tube and add 500µl of Elution Buffer containing imidazole. Mix the agarose and the buffer in the spin column using the NOT DISPOSABLE plastic mixer provided by your instructor. Incubate the spin column at room temperature for 5 minutes. During this time, most of the 6x His tagged protein will be eluted from the nickel-chelated agarose into the buffer. After the 5 minute incubation period, recover the protein by centrifuging for 2 minutes at top speed in a microcentrifuge. Your purified fraction will be the flow-through in the collection tube. If you have considerably less than 0.5ml, respin the column to collect more volume.
13. Take the spin column out of the collection tube and discard the spin column. Do not discard the material in the collection tube. Using a Pasteur pipet, transfer all of the contents of this collection tube containing the purified beta-galactosidase to a new microfuge tube with volume markings. Record the volume in your notebook! Label the tube to indicate that this is the purified β-galactosidase fraction ("PF β-gal") and save it in your ice bucket.
    

DO NOT DISCARD ANY OF THE CRUDE EXTRACT OR PURIFIED FRACTION!