UNIT 1



UNIT 2

EXTRACTION OF PROTEIN FROM CELLS

Introduction:

Protein Production: An Industry Overview

Protein biochemists in the biotechnology industry like to point out that while the molecular biologists can be credited with keeping new product lines in the pipelines with their gene discovery and manipulations, it’s the protein biochemists who are paying the bills. What are they referring to? Most production facilities in the biotechnology industry are producing some sort of protein product. So, the teams of research and development scientists and engineers working for years to develop a product are usually working towards a protein production process. It is estimated that the total worldwide sales of protein products exceeds $60 billion in sales in an industry that continues to expand every year. What are these protein products? A wide variety of proteins find industrial application. These include enzymes, antibodies, hormones, blood factors, growth factors and diagnostics. The protein products used for medical diagnosis or therapies are the high dollar products. Some examples are listed below.

Table 2.1 Biopharmaceutical protein products approved for general medical use in the EU and/or USA by 2002

| | |Number of approved products |

|Product type |Examples | |

|Blood factors |Factors VIII and IX (for treating hemophilia) |7 |

|Thrombolytic agents |Tissue plasminogen activator or tPA (for treating heart attacks and |6 |

| |strokes) | |

|Hormones |Insulin (for treating diabetes mellitis), growth hormones (for treating|28 |

| |cancer and AIDS) | |

|Hemapoietic growth factors |Erythropoietin (for treating anemias), colony stimulating factors (for |7 |

| |treating immunosuppression) | |

|Interferons |Interferons-(,-(,-( (for treating cancer, AIDS, allergies, asthma, |15 |

| |arthritis and infectious diseases | |

|Interleukin-based products |Interleukin-2 (for treatment of cancer, AIDS, and bone marrow |3 |

| |suppression) | |

|Vaccines |Hepatitis B surface antigen, herpes surface antigen |20 |

|Monoclonal antibodies |Various uses. Treatment of cancer and rheumatoid arthritis. Used for |20 |

| |diagnostic and research purposes. | |

|Additional products |Tumor necrosis factor, therapeutic enzymes |14 |

The size of the biopharmaceutical market is sizeable. Some of the leading approved biopharmaceutical products are listed below.

Table 2.2 Approximate annual market values of approved biopharaceutical products.

| | |Annual sales value (US $, |

|Product and (company) |Product description and (use) |billions) |

|Procrit (Amgen/Johnson & Johnson) |Erythropoietin (treatment of anemia) |2.7 |

|Epogen (Amgen) |Erythropoietin (treatment of anemia) |2.0 |

|Intron A (Schering Plough) |Interferon-( (treatment of leukemia) |1.4 |

|Neupogen (Amgen) |Colony stimulating factor (treatment of neutropenia) |1.2 |

|Avonex (Biogen) |Interferon-( (treatment of multiple sclerosis) |0.8 |

|Embrel (immunex) |Monoclonal antibody (treatment of rheumatoid arthritis) |0.7 |

|Betasteron (Chiron/Schering Plough) |Interferon-( (treatment of multiple sclerosis) |0.6 |

|Cerezyme (Genzyme) |Glucocerebrosidase (treatment of Gaucher’s disease) |0.5 |

At the low-dollar end, proteins are produced in bulk quantities for the food, chemical, and pharmaceutical industries. Unlike the biopharmaceutical proteins, these industrial enzymes do not require rigorous purification and can be produced in larger and less expensive processes. Bulk enzymes have a billion-dollar annual market, by far due to proteases used in detergents. Some examples of these types of protein products are listed below.

Table 2.3. Some enzyme products for industrial applications

|Enzyme |Industrial application |

|Proteases |Inclusion in detergent preparations |

| |Cheese- making |

| |Brewing/baking industries |

| |Meat/leather industries |

| |Animal/human digestive aids |

|Amylases |Starch processing industries |

| |Fermentation/ethanol production industries |

|Cellulases/hemicellulases |Brewing industry |

| |Fruit juice production |

| |Animal feed industry |

|Pectinases |Fruit juice/fruit processing industry |

|Glucose isomerase |Production of high-fructose syrups |

|Lipases |Dairy industry |

| |Vegetable oil industry |

| |Chemical industry |

|Cyclodextrin glycosyltransferase |Productions of cyclodextrins for the pharmaceutical and other |

| |industries |

|Penicillin acylase |Production of semisynthetic penicillins |

Sources of Protein Products

While bulk enzymes produced for the food and chemical industries are most often isolated directly from microbial or plant sources, biopharmaceuticals are more often isolated from recombinant organisms. Although biopharmaceutical protein products such as insulin were originally isolated from human and animal tissues, they are not likely to found in these natural sources in high concentrations, making the extraction and purification of these proteins prohibitively expensive. Also, contaminating residuals from natural sources can be unsafe, whether due to allergic responses in patients or due to contaminating viruses or prions.

These disadvantages can be overcome by using a recombinant productions system. By isolating the gene coding for a specific protein and cloning it into a high-expression vector in a recombinant host, the possibility of contaminating viruses and prions can be eliminated. The higher level of expression of the protein in a recombinant host can greatly reduce purification costs, and protein engineering can be used to design improvements in stability or effectiveness of a protein product.

The table below lists some expression levels of biopharmaceutical proteins in a bacterial expression host, Escherichia coli. While these proteins isolated from human and animal tissue sources might be at levels nearly indetectable, they can become the dominant protein expressed in a recombinant organism.

Table 2.4. Heterologous protein expression in E. coli

| |Expression level |

|Protein |(% of total protein) |

|Insulin |20 |

|Bovine growth hormone |5 |

|Interleukin 2 |10 |

|Human tumor necrosis factor |15 |

|interferon ( |25 |

E. coli was the first host used for production of recombinant proteins because it was well understood genetically and was very amenable to transformation and expression of recombinant genes. Its fermentation characteristics were also well understood. Not all proteins are expressed well in E. coli, however, in part due to the bacterial host’s inability to perform necessary post-translational modifications to recombinant proteins. Also, E. coli produces an endotoxin that acts as a pyrogen when injected, and this endotoxin is very difficult to purify from an E. coli fermentation.

In more recent years, however, the biotechnology industry has turned to alternative hosts for recombinant hosts in productions systems: fungal, plant, and animal tissue culture. Below is a table outlining some examples of production hosts for recombinant proteins, giving some examples of recombinant therapeutic proteins approved for general medical use that are produce in them, along with some advantages that these hosts present.

Table 2.5. Recombinant hosts used for protein production

| |Some approved therapeutic proteins in | |

|Recombinant host |production |Advantages of host |

|Saccharomyces cerevisiae (yeast) |Novolog (engineered insulin) |Well characterized genetics & fermentation |

| |Leukine (colony stimulating |GRAS (“generally regarded as safe”) by regulators |

| |factor) |Rapid and inexpensive fermentations |

| |Recombinvax, Comvax, Infanrix, Twinrix, |Can carry out some post-translational |

| |Primavex, Hexavax (subunit vaccines) |modifications of proteins |

| |Regranex (platelet-derived | |

| |growth factor) | |

|Insect cells |Bayovac CSF E2 and Porcilis Pesti (swine |High-level recombinant protein expression |

| |flu subunit vaccines) |Performs post-translational modifications |

| | |Can be engineered to secrete recombinant |

| | |Proteins |

| | |Human pathogen-free |

| | |Cheaper to culture than mammalian cells |

|Mammalian cells |Insulins |Ability to carry out necessary post- |

| |Tissue plasminogen activator |translational modifications |

| |Follicle-stimulating hormone |protein glycosylation patterns most closely |

| |Interferon-( |mirrors that found in humans |

| |Erythropoietin | |

| |Glucocerebrosidease | |

| |Factor VIIa | |

| |Vaccines | |

Downstream Processing of a Protein Product

There is no single best way to purify a given protein. The optimal protein purification strategy depends on the properties of the protein being purified, the starting concentration of the protein being purified, and the types of contaminating materials that it is being purified from. Most proteins produced commercially rely on fermentation by microbial or animal cell culture. The process of harvesting and purifying a protein being produced in an industrial setting is referred as “downstream processing.” It includes all steps of production downstream of the fermentation step. Since downstream processing of a protein can often exceed all other costs of production combined, it must be a carefully designed strategy, often requiring extensive development by scientists and engineers. An optimal purification scheme results in a maximal yield with the fewest and least expensive of steps of purification.

The following outlines the general steps that are part of downstream processing of proteins. Each step will be discussed in greater detail later in this lab manual.

1. Since most proteins are not secreted from cells, the first step of downstream processing generally consists of a cell disruption step, followed by removal of unbroken cells and cell debris. Cell disruption can be done by a relatively mild treatment with chemicals, or a more rigorous physical disruption by sonication or homogenization. Clearing the lysate of insoluble debris can be done by centrifugation or by filtration. Partitioning between two immiscible liquid phases can also be used in some cases.

2. Since processing of large volumes is expensive, the first purification step usually includes concentrating the protein extract to a smaller volume. This can be done by precipitating the protein, by adsorbing the protein to a column such as ion exchange, or by ultrafiltration through a membrane of a pore size that does not allow the protein to pass through.

3. Once the protein solution volume has been reduced to a more manageable size, purification can proceed by a number of techniques. Chromatography offers the highest resolution, but generally speaking one chromatographic step is not sufficient to purify the protein to homogeneity. Some types of chromatography that can be used include:

a. size exclusion chromatography (molecular sieving)

b. ion-exchange chromatography

c. hydrophobic interaction chromatography

d. affinity chromatography

e. adsorption chromatography on hydroxyapatite

4. For biopharmaceutical products that require higher levels of purity and can command a higher price in the marketplace, some more sophisticated techniques can be used to purify a protein. These include immunoaffinity techniques and high performance liquid chromatography (HPLC).

5. When the protein has been purified sufficiently, it is either dried by lyophilization or freeze-drying techniques, or it is formulated into a solution that stabilizes its activity and integrity.

Green Fluorescent Protein

In this lab module, we will purify the green fluorescent protein (GFP), a fluorescent protein naturally occurring in the Pacific jellyfish Aequoria victoria that has been successfully cloned into a number of organisms from bacteria to mice. Although originally chosen for its novelty of causing the transgenic organisms to glow green, GFP has been successfully used as a marker for transformation. Recent studies have created gene fusion in which the GFP gene is fused to genes of target markers on either the N- or C-terminus of the protein that they encode. The GFP becomes a marker for the intracellular location of the target gene product, tracking its migration by fluorescence microscopy into the nucleus, mitochondria, secretory pathway, plasma membrane or cytoskeleton. GFP can also be used as a reporter of gene expression levels as well as a measure of protein-protein interactions. Therefore, GFP is a very useful tool for both geneticists and for cell biologists.

The green fluorescent protein is a medium-sized protein of 238 amino acids and a molar mass of 27,000 daltons. In spectrophotometry it shows a major absorption peak at 395 nm and a minor absorption peak at 475 nm. The characterizing molar extinction coefficients are 30,000 and 7,000 M-1cm-1 respectfully. Fluorescence at 508 nm is not energy requiring and depends on the amino acids serine-65, tyrosine-77, and glycine-67. This trimer forms a fluorescent chromophore after translation by cyclization and oxidation reactions.

Once isolated, the GFP is stable across a wide range of temperatures and pH. It is very resistant to denaturation, requiring treatment with 6 M guanidine hydrochloride at 90oC or pH of 12.0. Furthermore, it is able to renature completely within minutes following many denaturing protocols, including sulfhydryl reagents such as 2-mercaptoethanol.

GFP consists of a dimer, each made of a barrel-shaped cylinder made primarily of ( pleated sheets on the outside and (-helices on the inside, a structure that is unique among proteins. This structure produces a compact domain that surrounds and protects the fluorophore located at the center of each cylinder as shown in Fig. 2.1. The N-terminal region of the protein acts as a “cap” on the end of the protein, further protecting the core fluorophore. When this cap is disrupted, the fluorescence may be easily quenched. The dimers are probably held together with the hydrophilic interactions of the pleated sheets on the outside of the cylinders.

[pic]

Fig 2.1: Overall Shape of GFP Monomer (from Carson, M, 1987. J. Mol. Graphics 5:103-106.)

In this module, we will extract GFP from transformed yeast cells by sonication, three-phase extraction, and homogenization by glass beads. In a later lab exercise, we will concentrate the GFP by precipitating it with ammonium sulfate and purify it by column chromatography. We will then check the purity of this isolated GFP by SDS-PAGE electrophoresis.

These techniques of cell disruption, protein extraction, protein precipitation, column chromatography and electrophoresis are basic techniques used in labs for isolating and characterizing many different types of proteins including enzymes. We will be using GFP as the protein of choice because it glows green under UV light and therefore readily visualized.

Lab 2-A:

Preparation of Reagents

Introduction:

The ability to make reagents is an essential skill for any biotechnicians. The accuracy of calculation and of measurement is critical to the outcome of any experiment, whether it be one you do yourself or one in which you prep for someone else. There are several critical aspects to making solutions that should be followed at all times.

➢ Check and recheck each calculation. It is best if two people make a calculation independently and then cross check their answers.

➢ Read each reagent bottle twice, once before using and once afterwards. This helps ensure that the right reagent is used.

➢ Complete a media prep form for every solution you prepare. This should include the formula, with the supplier and catalog number if available as well as the concentration and the amount weighed out for each reagent. Some media prep forms will also have space to include the balance number, pH meter number and other pieces of important information.

➢ Label each bottle before filling. Write down the name of the solution, your initials and the date. Some industries have special blank labels to be used for each reagent. Others use tape and a permanent marker.

➢ Record any changes observed, no matter how trivial. This record can be used to trace back a problem to its source quickly and easily or to confirm that a problem does not lie in the reagents or their preparation.

Review of calculations for making solutions:

A. Making Molar Solutions

The formula for making molar solutions is:

g needed = formula weight x molarity x liters

g needed = g/mole x mole/liter x liter

where the formula weight, also called the molecular weight is given as gram/mole. The formula weight is usually listed as F.W. on the reagent bottle. The molarity is the number of moles/liter and is abbreviated as M. The volume of the solution is listed in liters.

Example 1: Make 1 liter of 0.5 M solution of NaCl (F.W. = 58)

To get the grams of NaCl needed, first convert each of the values to the standard. That is, 58 becomes 58 g/mole, 0.5 M becomes 0.5 mole/L and 1 liter stays at 1L. By doing this step first, you will be able to cancel factors and make sure that your answer is correct. Then, plug the values into the equation and solve:

g needed = 58 g/mole x 0.5 mole/L x 1 L

The moles and the liters cancel out and

g needed = 29 g

You would weigh out 29 g NaCl and place it in something less than 1 liter of water. When the NaCl is dissolved, you would bring it to volume (BTV) of 1 liter. By dissolving the reagent in less than the final volume and then BTV, you make sure that you do not make too dilute a solution. Note that pH is adjusted before BTV, and then quickly checked afterwards to confirm it has not changed with the addition of the slight amount of water.

In this and in most labs, you will use distilled or deionized water to make all solutions. Never use tap water unless specifically indicated

Example 2: Make 100 mL 25 mM Tris (FW 121.1), pH 7.5

As in example 1, first convert each of the values to the standard. Therefore the formula weight becomes 121.1 g/mole, 25 mM becomes 0.025 mole/liter (to go from mM to M divide by 1000) and 100 mL becomes 0.1 L.

Plug these values into the formula:

g needed = 121.1 g/mole x 0.025 mole/L x 0.1 L

The moles and the liters cancel out (but only if you have made the conversions beforehand) and

g needed = 0.30275 g

This needs to be rounded off to 0.30 g since balances will not measure this precisely.

You would weight out 0.30 g Tris and place it in about 80 mL distilled or deionized water. Then a adjust the pH to 7.5 with acid, usually HCl and BTV 100 mL with water.

B. Making Percent Solutions

The sales tax in this state is 8.25%. That means that we pay $8.25 for every $100 worth of merchandize. Percent solutions work the same way, except that instead of dollars, grams and mL are used instead. Thus, a 5% solution means 5 g solid dissolved in 100 mL water or 5 mL liquid dissolved to 100 mL water.

Example 1: Make 100 mL 2% (w/v) tryptone.

In this simple solution, you would place 2 g tryptone into about 80 mL water. Once the tryptone is dissolved, BTV 100 mL. Note that moles and molarity are never needed in making percent solutions.

Sometimes the percent solution will be designated as (w/v) or (v/v) as in the protocol below. (W/v) means weight to volume so in a 2% (w/v) you would weigh out 2 g reagent per 100 mL water. The term (v/v) refers to a liquid reagent. For a 100 mL of 5% (v/v) glycerol you would measure out 5 mL glycerol to be added to 95 mL water. Note that you need to subtract the volume of the glycerol from the water in order to get the correct final volume.

Example 2: Make 500 mL of 50% (v/v) glycerol.

For 100 mL 50% (v/v) glycerol you would combine 50 mL glycerol and 50 mL water. Since you need five times that amount you would combine 250 mL glycerol with 250 mL water.

C. Combined Molarity and Percent Solutions

Several of the solutions in this lab are a combination. Some of the reagents are given as molar solutions and some given as percents. This is sometimes done when making media for bacteria and other cells. Treat each ingredient individually, added them to the water and allowing them to dissolve before bringing to volume.

Review of calculations for making dilutions from stock solutions:

A. From a concentrated stock

We frequently make up a stock solution that is more concentrated than the working solution. That way we can keep the stock on our bench and dilute it when necessary. The formula for diluting from a stock solution is:

| |

|C1V1 = C2V2 |

| |

|Where C1 is the concentration of the stock solution, V1 is the volume needed (this is usually the unknown), C2 is the final concentration of |

|the solution and V2 is the final volume. |

Example 1: Make 10 mL 20 mM solution from a stock of 100 mM.

The most difficult part of these problems is deciding what value is what. One way to solve this is to write over each value C1, V2, etc. Thus, the problem would look like this:

V2 C2 C1

Make 10 mL of 20 mM solution from a stock of 100 mM

Plug these values into the formula and solve for V1.

100 mM x ? mL = 20 mM x 10 mL

? mL = 20 mM x 10 mL

100 mM

= 2 mL

Therefore you would take 2 mL of the stock solution and add to 8 mL (10 mL – 2 mL) water to get the desired final concentration.

Note that although molarity is used, you do not need to know the formula weight of the reagent in the solution. Furthermore, you do not need to covert to liters and moles/liter as you had to do when dealing with molar solutions. The only caveat is to make sure that the units on each side of the equation are the same. In this case we have mM and mL on both sides of the equation and so are all set. However, it you had mM and μM, then you would have to convert one to the other.

B. Dilution from a “times” stock

Sometimes stock solutions are given as a “times” stock such as 10X. (A 10X stock is usually read as “ten X”.) This means that the stock is ten times as concentration as the final solution. In order to dilute a “times” stock, follow the same dilution formula as above.

Example 1: Make 50 mL working solution from a 10X stock solution.

In this case, we can do the same as we did above:

V2 C2 C1

Make 50 mL working solution from a 10X stock.

The implication is that the working solution, C2, is 1X. Therefore when we plug in the values

10X x ? mL = 1X x 50 mL

Solving as above gives us 5 mL of the 10X stock solution added to 45 mL (50 mL – 5 mL) water to make the 1X working solution.

In many biotechnology laboratories, strict records are maintained on every reagent that is made. This documentation allows you or anyone else to trace the history of a project thought a clear paper trail. This paper trail is extremely valuable for both scientific and for legal reasons. Scientifically, a paper trail permits the documentation of the most efficient and successful protocols, thereby increasing the probability for success. Legally, the paper trail supports any patent claims as well as any legal disputes on ownership.

Documentation takes several forms. Nearly all labs require the use of the lab notebook as has been discussed in Unit 1. Many labs also require the use of Media Prep forms to document who made what solution from which stocks at what time and under what conditions. This form should also document how the media is to be stored. We will use a Media Prep form whenever we make a solution. These are kept with your lab notebook and handed in with each lab report.

| |

|Safety Considerations: |

|Wear closed toed shoes whenever you are in lab. |

|Wear gloves while handling the acid to adjust the pH. |

|Wear gloves when handling the PMSF and dispose in hazardous waste bin. |

Protocol:

1. Each group is to make ONE of the following reagents for use by the rest of the class. Make sure you double check your calculations with your partners. When finished, divide the solution into 5 bottles and label each with the reagent, your initials and the date. Make out a Media Prep form.

|Solution |Final concentration |Final volume |Comments |

|EDTA in purified H2O, pH 8.0 |0.50 M |20 mL |The disodium salt of EDTA is not very soluble until the pH has be |

| | | |adjusted to 8.0 with NaOH. So, add the correct weight of EDTA to a |

| | | |beaker along with about half the required water. Adjust the pH with|

| | | |stirring to pH 8.0 with 6 M NaOH. When the solution has dissolved, |

| | | |adjust the pH to 8.0. Store at room temperature. |

|Sodium phosphate, monobasic |1.0 M |20 mL |Store in the refrigerator |

|Sodium phosphate, dibasic |1.0 M |20 mL |Store in the refrigerator |

|PMSF in methanol |0.20 M |1.0 mL |Make this solution up in methanol. (The aqueous solution is very |

| | | |unstable.) Store in a microcentrifuge tube in the freezer. |

MEDIA PREP FORM Control #

Name of Solution/Media:

Amount prepared: Preparation Date:

Preparer(s):

|Component |Brand/lot # |Storage conditions/ |FW or initial |Amount used |Final concentration |

| |(Vendor) |date received |concentration | | |

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|Balance used |Calibration status |

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|pH meter used |Calibration status |

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|Initial pH |Final pH |Adjusted pH with |

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|Prep temperature |Sterilization procedure/ sterility |Storage conditions |

| |testing | |

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Calculations/Comments:

MEDIA PREP FORM Control #

Name of Solution/Media:

Amount prepared: Preparation Date:

Preparer(s):

|Component |Brand/lot # |Storage conditions/ |FW or initial |Amount used |Final concentration |

| |(Vendor) |date received |concentration | | |

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|Balance used |Calibration status |

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|pH meter used |Calibration status |

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|Initial pH |Final pH |Adjusted pH with |

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|Prep temperature |Sterilization procedure/ sterility |Storage conditions |

| |testing | |

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Calculations/Comments:

Lab 2-B

Extraction of GFP

Introduction:

We discussed in the Introduction to this module how green fluorescent protein (GFP) has become widely used in biotechnology as a marker for transformation. This is because GFP is an easily visualized and stable protein. Furthermore there is only one gene involved in making GFP and the resulting protein is not modified after translation. This means that GFP is easily transformed and expressed in a wide variety of prokaryotic and eukaryotic organisms.

In this lab we will be using transformed S. cerevisiae as the source of GFP. The protein will be extracted by using 2-mercaptoethanol in a Tris buffer. After centrifuging the rest of the cell components, the supernatant containing the GFP and other proteins is treated with ammonium sulfate. This precipitates the proteins, which are then collected by high-speed centrifugation.

Protein Extractions

The first step in the purification of an intracellular protein is the disruption of the cell structure, allowing the release of proteins. There are many methods for cell disruption, listed below. When choosing a method for cell disruption, the goal is to find the gentlest method that will do the job and that gives the highest yield of protein. Protein yield is a question of how much protein is released, or how effective the disruption technique was. It is also a question, however, of the amount of damage the extraction method wreaks on the proteins being released. In general, it is best to search for the mildest effective treatment in order to avoid damaging the proteins being isolated. Some proteins are susceptible to the shear forces required to break down cell walls, so in these cases, milder techniques should be tried. More often, proteins are susceptible to damage by exposure to air, so it is best to avoid introducing air into the cell suspension during the treatment and to keep the time required for cell disruption to a minimum. The cell suspension should be kept cold at all times. Although organic solvents are effective towards cell disruption, most proteins are not stable towards this treatment, but it a protein is stable towards organic solvents, this is the treatment of choice. In such a case, not only is the protein effectively released, it is also simultaneously purified from less stable proteins.

The buffering conditions of a cell lysate are also very important in the stability of proteins. The pH can change when cytoplasmic contents are released from a cell, so a buffer must be used to maintain a constant pH. The selection of a buffer should be based on its pKa: it should be within 1 unit of the pH that is to be maintained. The best pH to buffer at is often dependent on the protein being isolated. Most proteins are most stable around neutral pH or under slightly alkaline conditions. If the protein of choice is found to be stable at extremes of pH, however, buffering at these pH’s can help to extract the protein more selectively. The less stable proteins will denature and will be removed by the centrifugation step. This effectively purifies the protein by selective denaturation and removal of other proteins.

Most proteins are susceptible to oxidative damage, so can often be stabilized by adding a reducing agent. Dithiothreitol (DTT) and 2-mercaptoethanol are often used as reducing agents during protein isolations. These sulfhydryl reagents also protect susceptible proteins from damage by metals that may be present in buffering solutions. DDT is less volatile than is 2-mercaptoethanol, so is usually the preferred reagent used to maintain a reducing environment in a cell lysate. Since it has two thiol functional groups instead of one as in the case of 2-mercaptoethanol, DTT may be used at lower concentrations.

Table 2.6. Some methods of cell disruption

| | |

|Method |Underlying basis |

|Gentle |Cell lysis |Osmotic shock: rapid immersion in hypotonic solution |

| |Enzyme digest/cell lysis |Digestion of cell wall; contents release following osmotic shock |

| |Potter-Elvehjem homogenizer |Cells forced through a narrow gap, cell membranes disrupted by shear |

| | |forces |

|Moderately harsh |Freeze-thaw |Slow freeze-thaw cycles break cell walls by ice crystal formation and |

| | |growth |

| |Waring blender |Cells broken by shear forces of rotating blades |

| | | |

| |Organic extraction |Mixing with an immiscible organic solvent can weaken cell walls and |

| | |dissolve biological membranes |

| |Grinding |Abrasive grinding with glass beads or a mortar and pestle, usually |

| | |with frozen cells and sand or alumina |

|Vigorous |French press cell |Cells forced through small orifice at high pressure and disrupted by |

| | |rapid pressure drop and high shear forces |

| |Explosive decompression |Cells equilibrated with an inert gas (e.g. N2) at high pressure, |

| | |rapidly decompressed to 1 atm |

| |Bead mill |Rapid vibrations with glass beads grind cell walls |

| | | |

| |Ultrasonication |High-pressure sound waves cause cell rupture by cavitation and shear |

| | |forces |

Proteins must also be protected from degradation by proteases during isolation steps. Cell disruption tends to release lytic enzymes, especially from cell rich in lysosomes. To reduce proteolytic damage to proteins, protease inhibitors are often added to a lysis buffer. The protease inhibitors that can be used are listed below in Table 2.7. Protease inhibitors often have low solubility in water, so stock solutions must be made up in organic solvents. Phenylmethylsulfonyl fluoride is the inhibitor most often used, but must be prepared fresh due to its instability in water (t1/2 = 30 minutes at 25oC and pH 7.0). Stocks are made up in methanol and stored in the freezer.

Table 2.7. Some inhibitors used to control proteolysis

|Type of tissue being disrupted |Inhibitors added (final concentration) |Stock solutions |

|Animal tissues |PMSF (1 mM) |0.2 M in methanol |

| |Benzamidine (1 mM) |M in H20 |

| |Leupeptin (10(g/mL) |mg/mL in H2O |

| |Pepstatin (10 (g/mL) |5 mg/mL in methanol |

| |Aprotinin (1 (g/mL) |mg/mL in H2O |

| |Antipain (0.1 mM) |10 mM in H2O |

|Plant tissues |PMSF (1 mM) |M in methanol |

| |Chmostatin (20 (g/mL) |1 mg/mL in DMSO |

| |EDTA (1 mM) |0.1 M in H2O |

|Yeasts, fungi |PMSF (1 mM) |0.2 M in methanol |

| |Pepstatin (15 (g/mL) |5 mg/mL in methanol |

| |1,10-phenanthroline (5 mM) |1 M in ethanol |

|Bacteria |PMSF (1 mM) |M in methanol |

| |EDTA (1 mM) |M in H2O |

Once cells have been disrupted, the cell lysate must be clarified, removing cell debris and unbroken cells either by filtration or by centrifugation. Since cellular materials are highly compressible, they rapidly clog pores of filters, so centrifugation is usually the method used to remove particulate matter when the volumes are small.

It is usually impossible to know at the outset which extraction technique will work best for a particular protein. To find the best method, several methods are tried and compared. In this exercise, we will break up into groups of 2-3 students and each group will be given a different technique for cell disruption. The yield of GFP by each group will be determined by the intensity of fluorescence of the isolated protein under a long wavelength ultraviolet light. To determine how selective the extraction method was in release the GFP, the total amount of protein will be determined by measuring absorbance at 280 nm.

Safety: Goggles and gloves should be worn throughout. Special care should be used when extracting the proteins since 2-mercaptoethanol is a suspected carcinogen.

Part I: Preparation of Extraction Buffer and Cell Suspension

|Protocol |Comments, Observations, Calculations |

| | |

|Calculate the correct dilutions to prepare 15 mL total volume for the |The extraction buffer contains an unstable component (PMSF), so should|

|entire class. Your final concentrations should be: |be made immediately before use. |

|Sodium phosphate, pH7.5 10 mM | |

|EDTA pH 8.0 1 mM | |

|PMSF 0.5 mM | |

| | |

| | |

|Check your calculations with your instructor and make the solution | |

|using pipets and micropipetters and store in a 15 mL conical | |

|centrifuge tube on ice. | |

| | |

| | |

|Add the extraction buffer to a 50 mL centrifuge tube containing a cell| |

|pellet of transformed S. cerevisiae cells expressing GFP. Pipette up | |

|and down to suspend the cells. | |

| | |

| | |

|Disrupt the cells by 3 cycles of freezing and thawing. The freezing | |

|and thawing should be slow to maximize the damage to the cell by ice | |

|crystals. Freeze by simply placing the cells in a –20oC freezer for | |

|30 minutes. Thaw by simply placing cells at room temperature. | |

| | |

| | |

|Transfer 15 (L to a microfuge tube. Label and freeze for later | |

|analysis by SDS-PAGE electrophoresis. Save the rest of your cell | |

|suspension for Part II. | |

Part II: Extraction of protein from cells

We will compare the following 3 extraction procedures to evaluate their relative effectiveness for release of GFP relative to release of contaminating proteins. Select a lab partner and decide which extraction technique you will perform. After every group has completed an extraction, we will compare results.

|Protocol |Comments, Observations, Calculations |

| | |

|Extraction Protocol 1: glass beads | |

| | |

|Measure approximately 0.20 mL of 0.45 (m diameter glass beads to each | |

|of 2 microcentrifuge tubes. (Select tubes that have calibration | |

|marks.) | |

| | |

| | |

|Resuspend the cell suspension and transfer 300 (L to each | |

|microcentrifuge tube. |Label each tube with its contents, your names, and the date. |

| | |

|Vortex for 30 seconds, place on ice for one minute. Repeat 5 times | |

|(six rounds of vortex, cooling.). | |

| | |

|Transfer the cell debris and buffer by micropipeter to a labeled | |

|microcentrifuge tube and store on ice. | |

| | |

|Add 0.20 mL of lysis buffer to the beads, vortex briefly to rinse the | |

|glass beads, and transfer the supernatant to the microcentrifuge tube | |

|with the cell debris and buffer. | |

| | |

|Balance the microfuge tubes with another group or using a blank. | |

|Centrifuge in the microfuge at 10,000xg for 15 minutes at 4oC to | |

|pellet the cell debris. | |

| | |

|Examine the pellet and supernatant formed under a UV lamp and record | |

|your observations. Where is the most GFP located? | |

| | |

|Transfer the supernatant to clean microcentrifuge tubes and store on | |

|ice. | |

| | |

|Transfer 15 (L of the supernatant to microfuge tubes. Label and | |

|freeze for later analysis by SDS-PAGE electrophoresis. | |

| | |

|Continue with the analysis protocol, below. | |

| | |

|Extraction Protocol 2: sonication | |

| | |

|Transfer 6.0 mL of freeze-thawed cell suspension to a 15 mL conical |Label each tube with its contents, your names, and the date. |

|centrifuge tube. Store on ice. | |

| | |

| | |

|Following instructions in the operator manual, set up the sonicator | |

|with a 3 mm microtip. | |

| | |

| | |

|Insert the microtip in the cell suspension and sonicate with a cycle | |

|of 6 seconds on and 1.0 seconds off over a 60 second interval. Allow| |

|the cell suspension to cool for 2 minutes | |

| | |

| | |

|Repeat the sonication/cooling procedure for a total of 8 times. | |

| | |

|Transfer the sonicated cell suspension to microfuge tubes, balance the| |

|microfuge tubes, and centrifuge in the microfuge at 10,000xg for 15 | |

|minutes at 4oC to pellet the cell debris. | |

| | |

|Examine the pellet and supernatant formed under a UV lamp and record | |

|your observations. Where is the most GFP located? | |

| | |

|Adjust the volume of the supernatant to correspond with the final | |

|volumes of the other extraction protocols: for every 0.3 mL of | |

|supernatant, add 0.2 mL of extraction buffer. Label and store on ice.| |

| | |

| | |

|Transfer a 15 (L sample of the diluted supernatant to a clean | |

|microcentrifuge tube. Label and freeze for later analysis by SDS-PAGE| |

|electrophoresis. | |

| | |

|Continue with the analysis protocol, below. | |

Part III: Analysis Protocol, GFP compared to total protein

|Protocol |Comments, Observations, Calculations |

| | |

|You may monitor cell lysis by centrifugation and visualizing the | |

|relative fluorescence of the cell pellet and the soluble fraction in | |

|the supernatant. Microfuge 1.5 mL of each of the 3 extracts that you| |

|have prepared and illuminate them simultaneously in a dark room with a| |

|long wavelength UV lamp. Observe the relative fluorescence of the | |

|different fractions and record your results. | |

| | |

|To get a quantitative estimate of your yields of extracted GFP, | |

|transfer 1.0 mL to a UV-transparent micropipet and measure the | |

|absorbance in a fluorimeter. Follow the operator manual for the | |

|fluorimeter to adjust the emission wavelength and the absorption | |

|wavelength to settings appropriate for GFP. | |

| | |

|Compare the total amount of protein in resulting from each extraction | |

|protocol by measuring its absorbance at 280 nm. Follow the | |

|description for measuring absorbance in the Seidman texbook: Basic | |

|Laboratory Methods for Biotechnology. You will need to zero the | |

|spectrophotometer against extraction buffer. Record your results in a| |

|table. | |

| | |

|Freeze your isolated protein at –20oC until the next class. | |

Questions for Unit 2

Lab 2-A:

1. Make a media prep form for each of the reagents you and your lab partner(s) made. Hand in with your lab report. These should have been completed at the time the reagents were made.

Lab 2-B:

1. Make a flow chart comparing the different extractions protocols used. At each step, identify whether the protein is in the supernatant or the pellet.

2. What is the purpose of taking a small sample saved after the freeze-thaw cycles and at the end of each extraction?

3. Make a table that lists the fluorescence and the UV absorbance of each of your cell lysates. In a separate column, divide the fluorescence value from the UV absorbance value.

a. What can you say about the relative effectiveness of the different extraction procedures for releasing GFP?

b. What can you say about the relative purity of the GFP released by the different extraction procedures?

4. Which of the extraction protocols would you expect to be the hardest to scale up to an industrial application? Explain your reasoning.

5. Which of the extraction protocols would you expect to work best in a large scale industrial application? Explain your reasoning.

6. Read Chapter 23 “Laboratory Solutions to Support the Activity of Biological Macromolecules and Intact Cells” in Basic Laboratory Methods for Biotechnology: Textbook and Laboratory Reference by Seidman & Moore (pp483-508). Answer the following questions.

a. What are some treatments that can cause proteins to denature?

b. Give a molecular description for why a protein may need to be protected from oxidation. Do you think that all proteins need this protection?

c. How do chelating agents prevent protein losses during extraction procedures?

d. What are two methods that we used to prevent protein losses due to protease activities?

e. How are protein losses from adsorption to surfaces prevented?

References

Chalfie, Martin and Steven Kain, Green Fluorescent Protein, Properties, Applications and Protocols Wiley-Liss, 1998.

Gerhardt, P. (Ed.), Manual of Methods for General Bacteriology, American Society for Microbiologistsa, 1981.

Penna, Thereza Christina Vessoni and Marina Ishii. Selective Permeation and Organic Extraction of Recombinant Green Fluorescent Protein (gfpUV) from Escherichia coli. BMC Biotechnology. 2:7 (2002). Available from 1472-6740/2/7

Price, N,C, (Ed.) Proteins Labfax. Academic Press. (1996)

Walmsley, R.M., N. Billinton, and W.-D. Heyer. Yeast functional analysis report: green fluorescent protein as a reporter for the DNA damage-induced gene RAD54 in Saccharomyces cerevisiae. Yeast 13:1535-1545 (1997)

Walsh, Gary. Biopharmaceuticals: Biochemistry & Biotechnology. (2nd ed). John Wiley & Sons (2003)

Walsh, Gary, and Denis Headon. Protein Biotechnology. John Wiley & Sons. 1994

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