17
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
7
Product type
Blood factors
Examples
Factors VIII and IX (for treating hemophilia)
Thrombolytic agents
Tissue plasminogen activator or tPA (for treating
heart attacks and strokes)
Insulin (for treating diabetes mellitis), growth
hormones (for treating cancer and AIDS)
Erythropoietin (for treating anemias), colony
stimulating factors (for treating immunosuppression)
Interferons-,-,- (for treating cancer, AIDS,
allergies, asthma, arthritis and infectious diseases
Interleukin-2 (for treatment of cancer, AIDS, and
bone marrow suppression)
Hepatitis B surface antigen, herpes surface antigen
6
Various uses. Treatment of cancer and rheumatoid
arthritis. Used for diagnostic and research
purposes.
Tumor necrosis factor, therapeutic enzymes
20
Hormones
Hemapoietic growth factors
Interferons
Interleukin-based products
Vaccines
Monoclonal antibodies
Additional products
28
7
15
3
20
14
The size of the biopharmaceutical market is sizeable. Some of the leading approved biopharmaceutical
products are listed below.
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007
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Table 2.2 Approximate annual market values of approved biopharaceutical products.
Product and (company)
Procrit (Amgen/Johnson &
Johnson)
Epogen (Amgen)
Product description and (use)
Erythropoietin (treatment of anemia)
Annual sales value
(US $, billions)
2.7
Erythropoietin (treatment of anemia)
2.0
Intron A (Schering Plough)
Interferon- (treatment of leukemia)
1.4
Neupogen (Amgen)
Colony stimulating factor (treatment of
neutropenia)
Interferon- (treatment of multiple sclerosis)
Monoclonal antibody (treatment of rheumatoid
arthritis)
Interferon- (treatment of multiple sclerosis)
1.2
Glucocerebrosidase (treatment of Gaucher’s
disease)
0.5
Avonex (Biogen)
Embrel (immunex)
Betasteron
(Chiron/Schering Plough)
Cerezyme (Genzyme)
0.8
0.7
0.6
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 billiondollar 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
Proteases
Industrial application
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
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007
19
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
Protein
Insulin
Expression level
(% of total protein)
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.
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007
20
Table 2.5. Recombinant hosts used for protein production
Recombinant host
Saccharomyces cerevisiae
(yeast)
Insect cells
Mammalian cells
Some approved therapeutic
proteins in production
Novolog (engineered insulin)
Leukine (colony stimulating
factor)
Recombinvax, Comvax,
Infanrix, Twinrix, Primavex,
Hexavax (subunit vaccines)
Regranex (platelet-derived
growth factor)
Bayovac CSF E2 and Porcilis
Pesti (swine flu subunit
vaccines)
Insulins
Tissue plasminogen activator
Follicle-stimulating hormone
Interferon-
Erythropoietin
Glucocerebrosidease
Factor VIIa
Vaccines
Advantages of host
Well characterized genetics & fermentation
GRAS (“generally regarded as safe”) by
regulators
Rapid and inexpensive fermentations
Can carry out some post-translational
modifications of proteins
High-level recombinant protein expression
Performs post-translational modifications
Can be engineered to secrete recombinant
Proteins
Human pathogen-free
Cheaper to culture than mammalian cells
Ability to carry out necessary posttranslational modifications
protein glycosylation patterns most closely
mirrors that found in humans
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)
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007
21
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 90 oC or pH of <4.0 and >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.
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007
22
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.
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007
23
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
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007
24
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.
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007
25
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
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.
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007
26
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
0.50 M
Final
volume
20 mL
Comments
Sodium phosphate,
monobasic
1.0 M
20 mL
Store in the refrigerator
Sodium phosphate,
dibasic
PMSF in methanol
1.0 M
20 mL
Store in the refrigerator
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.
EDTA in purified H2O,
pH 8.0
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
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.
ACC Lab Manual, 2nd Edition
2007
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MEDIA PREP FORM
Control #
Name of Solution/Media:
Amount prepared:
Preparation Date:
Preparer(s):
Component
Brand/lot #
(Vendor)
Storage
conditions/
date received
FW or initial
concentration
Balance used
Calibration status
pH meter used
Calibration status
Initial pH
Final pH
Adjusted pH with
Prep temperature
Sterilization procedure/
sterility testing
Storage conditions
Amount
used
Final
concentration
Calculations/Comments:
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007
28
MEDIA PREP FORM
Control #
Name of Solution/Media:
Amount prepared:
Preparation Date:
Preparer(s):
Component
Brand/lot #
(Vendor)
Storage
conditions/
date received
FW or initial
concentration
Balance used
Calibration status
pH meter used
Calibration status
Initial pH
Final pH
Adjusted pH with
Prep temperature
Sterilization procedure/
sterility testing
Storage conditions
Amount
used
Final
concentration
Calculations/Comments:
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007
29
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 pK a: 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.
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007
30
Table 2.6. Some methods of cell disruption
Method
Gentle
Moderately harsh
Cell lysis
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
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
Abrasive grinding with glass beads or a mortar and
pestle, usually with frozen cells and sand or
alumina
Cells forced through small orifice at high pressure
and disrupted by rapid pressure drop and high
shear forces
Cells equilibrated with an inert gas (e.g. N2) at high
pressure, rapidly decompressed to 1 atm
Grinding
Vigorous
Underlying basis
Osmotic shock: rapid immersion in hypotonic
solution
French press cell
Explosive decompression
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.
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007
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Table 2.7. Some inhibitors used to control proteolysis
Type of tissue being disrupted
Inhibitors added (final
concentration)
PMSF (1 mM)
Benzamidine (1 mM)
Leupeptin (10g/mL)
Pepstatin (10 g/mL)
Aprotinin (1 g/mL)
Antipain (0.1 mM)
Stock solutions
Plant tissues
PMSF (1 mM)
Chmostatin (20 g/mL)
EDTA (1 mM)
0.2 M in methanol
1 mg/mL in DMSO
0.1 M in H2O
Yeasts, fungi
PMSF (1 mM)
Pepstatin (15 g/mL)
1,10-phenanthroline (5 mM)
0.2 M in methanol
5 mg/mL in methanol
1 M in ethanol
Bacteria
PMSF (1 mM)
EDTA (1 mM)
0.2 M in methanol
0.1 M in H2O
Animal tissues
0.2 M in methanol
0.1 M in H20
1 mg/mL in H2O
5 mg/mL in methanol
0.1 mg/mL in H2O
10 mM 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.
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007
32
Part I: Preparation of Extraction Buffer and Cell Suspension
Protocol
Comments, Observations, Calculations
1. Calculate the correct dilutions to prepare 15 mL
total volume for the entire class. Your final
concentrations should be:
Sodium phosphate, pH7.5
10 mM
EDTA pH 8.0
1 mM
PMSF
0.5 mM
The extraction buffer contains an unstable component
(PMSF), so should be made immediately before use.
2. 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.
3. 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.
4. 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.
5. 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
1. Measure approximately 0.20 mL of 0.45 m
diameter glass beads to each of 2
microcentrifuge tubes. (Select tubes that have
calibration marks.)
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007
33
2. Resuspend the cell suspension and transfer
300 L to each microcentrifuge tube.
Label each tube with its contents, your names, and the
date.
3. Vortex for 30 seconds, place on ice for one
minute. Repeat 5 times (six rounds of vortex,
cooling.).
4. Transfer the cell debris and buffer by
micropipeter to a labeled microcentrifuge tube
and store on ice.
5. 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.
6. 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.
7. Examine the pellet and supernatant formed
under a UV lamp and record your observations.
Where is the most GFP located?
8. Transfer the supernatant to clean
microcentrifuge tubes and store on ice.
9. Transfer 15 L of the supernatant to microfuge
tubes. Label and freeze for later analysis by
SDS-PAGE electrophoresis.
10. Continue with the analysis protocol, below.
Extraction Protocol 2: sonication
1. Transfer 6.0 mL of freeze-thawed cell
suspension to a 15 mL conical centrifuge tube.
Store on ice.
Label each tube with its contents, your names, and the
date.
2. Following instructions in the operator manual,
set up the sonicator with a 3 mm microtip.
3. 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
4. Repeat the sonication/cooling procedure for a
total of 8 times.
5. Transfer the sonicated cell suspension to
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007
34
microfuge tubes, balance the microfuge tubes,
and centrifuge in the microfuge at 10,000xg for
15 minutes at 4oC to pellet the cell debris.
6. Examine the pellet and supernatant formed
under a UV lamp and record your observations.
Where is the most GFP located?
7. 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.
8. Transfer a 15 L sample of the diluted
supernatant to a clean microcentrifuge tube.
Label and freeze for later analysis by SDSPAGE electrophoresis.
9. Continue with the analysis protocol, below.
Part III: Analysis Protocol, GFP compared to total protein
Protocol
Comments, Observations, Calculations
1. 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.
2. To get a quantitative estimate of your yields of
extracted GFP, transfer 1.0 mL to a UVtransparent 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.
3. 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.
4. Freeze your isolated protein at –20oC until the
next class.
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007
35
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?
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007
36
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 www.biomedcentral.com/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
BITC2411 Biotechnology Laboratory Instrumentation
Unit 2. Extraction of Proteins from Cells
ACC Lab Manual, 2nd Edition
2007