Overview of Protein Purification and
1
Characterization
AIMS AND OBJECTIVES
Protein purification has an over 200-year
history: the first attempts at isolating sub-
stances from plants having similar properties
to “egg albumen,” or egg white, were reported
in 1789 by Fourcroy. Many proteins from plants
were purified in the nineteenth century, though
most would not be considered pure by modern
standards. A century later, ovalbumin was the
first crystalline protein obtained (by Hofmeis-
ter in 1889). The year 1989 may not go down
in history as a milestone in protein chemistry,
but since then there has been a resurgence of
interest in proteins after more than a decade of
gene excitement.
The aims of protein purification, up until the
1940s, were simply academic. To then, even the
basic facts of protein structure were not fully
appreciated, and pure proteins were needed just
to study structure and test the rival theories of
the pre-DNA days. During the Second World
War, an acute need for blood proteins led to
development of the Cohn fractionation proce-
dure for purification of albumin and other pro-
teins from serum (Cohn et al., 1946). This was
the inception of large-scale protein purifica-
tions for commercial purposes; Cohn fractiona-
tion continues to be used to this day.
As more proteins, and particularly enzymes,
were purified and crystallized, they started to
be used increasingly in diagnostic assays and
enzymatic analyses, as well as in the large-
scale food, tanning, and detergent industries.
Many enzymes used in industry are not in fact
very pure, but as long as they do the job, that is
sufficient. “Process” enzymes such as α-amy-
lase, proteases, and lipases are pro-duced in ton
quantities, mainly as secretion products in bac-
terial cultures, and may undergo only limited
purification processes to mini-mize costs. At
the other extreme, enzyme products for re-
search and analysis require a high degree of
purification to ensure that contaminating activi-
ties do not interfere with the intended use.
Anyone familiar with molecular biology en-
zymes will appreciate how minute levels of
contamination of DNase or RNase can com-
pletely destroy carefully planned experiments.
The 1960s and 1970s could be described as
the peak years for protein and enzyme research,
and most of the methods used in protein puri-
Contributed by R.K. Scopes
Current Protocols in Protein Science (1995) 1.1.1-1.1.6
Copyright © 2000 by John Wiley & Sons, Inc.
UNIT 1.
fication were established by then, at least in
their principles. More recent developments
have been mainly in instrumentation designed
to optimize the application of each methodol-
ogy. Developments in instrumentation have
been stimulated by the rapid progress in mo-
lecular biology, because gene isolation has of-
ten been preceded by isolation of the gene
product. Because such products can now be
characterized sufficiently (i.e., partially se-
quenced) using minute amounts of protein, the
need for large-scale or even moderate-scale
procedures has decreased. Hence there has been
an explosive development of modern equip-
ment designed specifically for dealing with
amounts of protein in the milligram to micro-
gram range. On the other hand, structural stud-
ies using X-ray crystallography and nuclear
magnetic resonance (NMR) require hundreds
of milligrams of pure protein, so larger-scale
equipment and procedures are still needed in
the research laboratory.
The nature of the proteins studied has also
changed substantially. Whereas enzymes were
once the most favored subjects, they have now
been superceded by nonenzymatic proteins
such as growth factors, hormone receptors, vi-
ral antigens, and membrane transporters. Many
of these occur in minute amounts in the natural
source, and their purification can be a major
task. Heroic efforts in the past have used kilo-
gram quantities of rather unpleasant starting
materials, such as human organs, and ended up
with a few micrograms of pure product. It is
now more usual, however, to take the genetic
approach: clone the gene before the protein has
been isolated or even properly identified, then
express it in a suitable host cell culture or
organism. The expression level may be orders
of magnitude higher than in the original source,
which will make purification a relatively simple
task. It can be useful to know beforehand some
physical properties of the protein, to facilitate
the development of a suitable purification pro-
tocol from the recombinant source. On the other
hand, there are now several ways of preparing
fusion proteins, which can be purified by affin-
ity techniques without any knowledge of the
properties of the target protein. Moreover, there
are ways of modifying the expressed product Strategies of
to simplify purification further. Protein
Purification and
Characterization
1.1.1
CPPS
 Thus the approach to protein purification
must first take into account the reason it is being
done, as the methods will vary greatly with
different requirements. At one extreme is the
one-of-a-kind purification, in a well-financed
and equipped laboratory, that is carried out to
obtain a small amount of product for sequenc-
ing so that gene isolation can proceed. In this
case, expense of equipment and reagents may
be no problem, and a very low overall recovery
of product can be acceptable, provided it is pure
enough. At the other extreme are the require-
ments of commercial production of a protein in
large amounts on a continuing basis, where
high recovery and economy of processing are
the chief parameters to be considered. There are
many intermediate situations as well.
Many publications in the area of protein
research are entitled “Purification and charac-
terization of…,” and describe a purification
procedure in sufficient detail that it can be
reproduced in another laboratory. The charac-
terization section may include structural, func-
tional, and genetic information, and carrying
out such studies is likely to require at least
milligram quantities of pure protein. Ideally the
purification should involve a small number of
steps, with good recovery at each step. If the
recovery is poor (<50% at any step), however,
there should be some indication of what hap-
pened to the missing activity. Has it been dis-
carded in the other fractions for the sake of
purity, or does it represent a true loss of activity?
If the latter, then the end-product may be less
than fully active despite apparent homogeneity
indicated by standard analysis. The choice be-
tween recovery and purification at each step can
be problematical; taking a narrow cut of a
chromatographic peak may provide a very pure
fraction, at the expense of losing a good deal of
less pure active component on either side. In
making such decisions, the objective of the
exercise must be kept in mind: if yield is not
important, then the choice of poor yield for the
sake of purity may be logical.
By far the most important requirement of a
publication is reproducibility of the method
reported. It is not sufficient to have carried out
the process only once if it is expected that other
investigators will want to repeat it. There are
always factors that influence the process that
may be overlooked at first, and which if varied
slightly can have a major effect on the purifi-
cation procedure. The reported process should
Overview of always be repeated exactly as described before
Protein
Purification and submitting the manuscript for publication.
Characterization There is one exception, namely, the case where
1.1.2
purification was conducted simply to obtain
enough protein for sequencing and gene isola-
tion; if that was achieved, there should be no
need to provide instructions for repetition.
SOURCES OF MATERIAL FOR
PROTEIN PURIFICATION
For many people embarking on a protein
purification project, there is no choice of mate-
rial. They are studying a particular biological
tissue or organism, and the objective is to purify
a protein from that source. However, there may
be approaches that can make the project sim-
pler. If, for instance, the source is difficult to
obtain in large amounts, it may be best to carry
out at least preliminary trials on a source species
more readily obtained. The most obvious and
relevant example is when the species being
studied is Homo sapiens, and tissue samples are
not readily available for practical or ethical
reasons, or both. In this case, it is usual to go
to where mammalian tissue is readily available
(i.e., an abattoir) and work with bovine, ovine,
or porcine sources. Alternatively, if quantity of
tissue is not a problem, the humble laboratory
rat may suffice. Once a protocol for purifying
the protein from substitute sources has been
worked out, it will be much easier to develop
one using human material—the identical pro-
cedure may work satisfactorily. Proteins differ
to a fairly small extent between species that
have diverged within about 100 million years,
a time frame that groups together most higher
mammals. Thus the behavior of proteins de-
rived from different animals with respect to the
various fractionation procedures is likely to be
similar, and a protocol worked out for pig tis-
sues is likely to need only minor adjustments
for application to human tissues.
A second example is where the interest is
mainly on the function of a protein, especially
an enzyme, for which functions and actions
have generally been strongly conserved
through evolution. In that case, a preliminary
screening of potential sources, or, better still,
the literature, should come up with a raw ma-
terial that is best suited to the investigator’s
purposes. Considerations should include the
following: (1) What functions are required of
the end product? For instance, an enzyme hav-
ing a low Km may be needed, so selecting the
source with the highest activity may not suffice.
(2) How convenient is it to grow or obtain the
raw material, and are there problems concern-
ing pathogenicity or extractability? (3) Does
the quantity of the protein vary with growth
conditions or age, and does it deteriorate in situ
Current Protocols in Protein Science
if left too long? Obviously one requires a source
that reliably produces the highest amount of the
desired protein per unit volume to maximize
the chances of developing a good purification
procedure. (4) What storage conditions are re-
quired for the raw material? It is important to
consider that fresh raw material may not be
immediately available whenever a purification
is attempted.
The above considerations are relevant to the
traditional situation for commencing a protein
purification project. It is becoming increasingly
common, however, for proteins to be purified
as recombinant products using techniques in
which the gene is expressed in a host organism
or in cultured cells. This of course requires the
gene encoding the protein of interest to be
available. Until the mid-1980s, such material
was usually obtained by hybridization of an
oligonucleotide synthesized according to
amino acid sequence information. This re-
quired the protein to have been purified first, so
the initial task of protein purification still
needed to be done at least once. More recently,
genetic techniques have permitted the isolation
of many genes encoding known proteins, even
though the proteins may never have been stud-
ied directly. Moreover, with the expansion of
the Human Genome Project and related DNA
sequencing efforts, many genes for both known
and unknown proteins will become available
and will be able to be expressed in recombinant
form without ever being purified from the host
species. As a result some completely new con-
siderations for protein purification come into
play, including the possibility of modifying the
gene structure not only to increase expression
level and alter the protein product itself to
enhance a desired function, but equally impor-
tantly to aid in purification. Recombinant pro-
teins may be expressed in bacteria, yeasts, in-
sect cells, and animal tissue cultures. Further
details may be found in UNIT 1.2.
DETECTION AND ASSAY OF
PROTEINS
During a protein purification procedure
there are two measurements that need to be
made, preferably for each fraction. Measure-
ments both of the total protein and of the
amount (usually bioactivity) of the desired pro-
tein must be made. Details of the most com-
monly used assay methods are given in Chapter
3. It is not possible to isolate a protein without
a method of determining whether it is present;
an assay, either quantitative or at least
Current Protocols in Protein Science
semiquantitative, indicating which fraction
contains the most of the desired protein is
essential.
Assays may range from the quick-and-easy
type (e.g., instantaneous spectrophotometric
measurement of enzyme activity) to long and
tedious bioassays that may take days to produce
an answer. The latter situation is very difficult,
because by the time one knows where the pro-
tein is, it may be “was,” owing to degradation
or inactivation. Moreover, this may not become
clear until the next step has been completed and
its products assayed. Any assay that is quick is
therefore advantageous, even if it means a sac-
rifice of accuracy for speed.
Measurement of total protein is useful, as it
indicates the degree of purification at each step.
However, unless the next step critically de-
pends on how much protein is present, total
protein measurement is not extremely impor-
tant: a small sample can be put aside and meas-
ured later, when the purification is complete. It
is, however, very important to know how much
protein is present in the final, presumed pure
sample, as this will indicate the specific activity
(if the protein has an activity), which can be
compared with other preparations. The general
object is to obtain as high a specific activity as
possible (taking into account recovery consid-
erations), which means retaining as much of the
desired protein as possible while ending up
with as little total protein as possible.
METHODS FOR SEPARATION
AND PURIFICATION OF PROTEINS
The methods available for protein purifica-
tion range from simple precipitation pro-
cedures used since the nineteenth century to
sophisticated chromatographic and affinity
techniques that are constantly undergoing de-
velopment and improvement. Methods can be
classified in several alternative ways—perhaps
one of the best is based on the properties of the
proteins that are being exploited. Thus the
methods can be divided into four distinct but
interrelated groups depending on protein char-
acteristics: surface features, size and shape, net
charge, and bioproperties.
Methods Based on Surface Features of
Proteins
Surface features include charge distribution
and accessibility, surface distribution of hydro-
phobic amino acid side chains, and, to a lesser
extent, net charge at a given pH (see discussion Strategies of
Protein
of net charge). Methods exploiting surface fea- Purification and
Characterization
1.1.3
 tures mainly depend on solubility properties.
Differences in solubility result in precipitation
by various manipulations of the solvent in
which the proteins are solubilized. Methods for
obtaining an extract containing the desired pro-
tein in soluble form are given in Chapter 4. The
solvent, nearly always water containing a low
concentration of buffer salts, can be treated to
alter properties such as ionic strength, dielectric
constant, pH, temperature, and detergent con-
tent, any of which may selectively precipitate
some of the proteins present. Conversely, pro-
teins may be selectively solubilized from an
insoluble state by manipulation of the solvent
composition. The surface distribution of hydro-
phobic residues is an important determinant of
solubility properties; it is also exploited in hy-
drophobic chromatography, both in the re-
versed-phase mode (UNIT 11.6) and in aqueous-
phase hydrophobic-interaction chromatogra-
phy (UNIT 8.4).
Also included in this group is the highly
specific technique of immunoaffinity chroma-
tography, in which an antibody directed against
an epitope on the protein surface is used to pull
out the desired protein from a mixture.
Methods Based on Whole Structure:
Size and Shape
Although the size and shape of proteins can
have some influence on solubility properties,
the chief method of exploiting these properties
is gel-filtration chromatography (UNIT 8.3). In
addition, preparative gel electrophoresis makes
use of differences in molecular size. Proteins
range in size from the smallest classified as
proteins rather than polypeptides, around 5000
Da, up to macromolecular complexes of many
million daltons. Many proteins in the bioactive
state are oligomers of more than one polypep-
tide (see UNIT 1.2), and these can be dissociated,
though normally with loss of overall structure.
Thus many proteins have two “sizes”: that of
the native state, and that (or those) of the
polypeptides in the denatured and dissociated
state. Gel-filtration procedures normally deal
only with native proteins, whereas electro-
phoretic procedures commonly involve separa-
tion of dissociated and denatured polypeptides.
Methods Based on Net Charge
The two techniques that exploit the overall
charge of proteins are ion-exchange chroma-
tography (by far the most important) and elec-
Overview of trophoresis (Chapter 10). Ion exchangers bind
Protein
Purification and charged molecules, and there are essentially
Characterization only two types of ion exchangers, anion and
1.1.4
cation. The net charge of a protein depends on
the pH—positive at very low pH, negative at
high pH, and zero at some specific point in
between, termed the isoelectric point (pI). It
should be stressed that at the pI a protein has a
great many charges; it just happens that at this
pH the total negatives exactly equal the total
positives. The most charged state (disregarding
the charge sign) is in the pH range 6.0 to 9.0.
This is the most stable pH range for most
proteins, as it encompasses common physi-
ological pH values. Ion exchangers consist of
immobilized charged groups and attract oppo-
sitely charged proteins. They provide the mode
of separation that has the highest resolution for
native proteins. High-performance reversed-
phase chromatography has equivalent or even
better resolution, but it generally involves at
least partial denaturation during adsorption and
so is not recommended for sensitive proteins
such as enzymes. Protein purification using
ion-exchange chromatography has mainly em-
ployed positively charged anion exchangers,
for the simple reason that the majority of pro-
teins at neutral pH are negatively charged (i.e.,
have a low isoelectric point). Details of meth-
odology are found in UNIT 8.2.
Methods Based on Bioproperties
(Affinity)
A powerful method for separating the de-
sired protein from others is to use a biospecific
method in which the particular biological prop-
erty of the protein is exploited. The affinity
approach is limited to proteins that have a
specific binding property, except that proteins
are theoretically able to be purified by immu-
noaffinity chromatography (UNIT 9.1), which is
the most specific of all affinity techniques.
Most proteins of interest do have a specific
ligand: enzymes have substrates and cofactors,
and hormone-binding proteins and receptor
molecules are designed to bind specifically and
tightly to particular hormones and other factors.
Immobilization of the ligand to which the pro-
tein binds (or of antibody to the protein) enables
selective adsorption of the desired protein in
the technique known as affinity chromatogra-
phy (Chapter 9). There are also nonchroma-
tographic modes of exploiting biospecific in-
teractions.
CHARACTERIZATION OF THE
PROTEIN PRODUCT
Once a pure protein is obtained, it may be
employed for a specific purpose, such as enzy-
matic analysis (e.g., glucose oxidase and lac-
Current Protocols in Protein Science
tate dehydrogenase), or as a therapeutic agent
(e.g., insulin and growth hormone). However,
it is normal, when a protein has been isolated
for the first time, to characterize it in terms of
structure and function. Several features are gen-
erally expected in characterization of a new
protein. These include molecular weight, or at
least the size of the subunit(s), determined by
SDS-polyacrylamide gel electrophoresis
(Chapter 10) and/or gel filtration (UNIT 8.3).
Spectral properties such as the UV spectrum
(Trp and Tyr content), circular dichroism (CD)
spectrum (secondary structure), and special
characteristics of proteins with prosthetic
groups (e.g., quantitation and spectra) may be
presented. The quantity and nature of carbohy-
drates on glycoproteins should be determined
(Chapter 12). Also, if the gene has not already
been reported, some amino-terminal sequence
analysis should be given, if at all possible, along
with the results of a database search for similar
sequences (UNIT 2.1). Functional proteins should
be demonstrated to have the appropriate func-
tion, and for enzymes detailed kinetic charac-
terization is appropriate. Ultimately the full
three-dimensional structure of the protein may
be determined, which will require crystals: any
successful crystallization attempts should be
reported.
THE PROTEIN PURIFICATION
LABORATORY
The requirements for a protein purification
laboratory cannot be exactly formulated be-
cause they depend greatly on the types and
amounts of proteins being isolated. To cover all
eventualities, it would be necessary to have one
set of equipment to deal with submicrogram
quantities and another set to deal with multi-
gram quantities—a range of around 108! One
laboratory dedicated to protein purification
may not need small-scale equipment if, for
example, it works with plasma proteins that are
always available in large quantities. Another
may have all the latest in high-performance
equipment but not be able (nor need) to handle
quantities of protein in excess of a few milli-
grams.
If it is assumed that neither extreme in quan-
tity is to be attempted, and that the laboratory
is handling a variety of protein types and
sources, then certain basic pieces of equipment
are needed. Obtaining the starting material and
making an extract of it require homogenization
equipment and centrifuges to remove insoluble
residues. Preliminary fractionation, when start-
ing with a crude extract of tissue or cells,
Current Protocols in Protein Science
requires equipment and materials that will not
become clogged by particulates. Adsorbents
and similar materials used at the first step
should be relatively inexpensive so that when
performance falls off after a few uses, owing to
intransigent impurity buildup, they can be dis-
carded. It is also relevant that a larger amount
is handled at the initial step than later steps;
therefore, reagent expense can be an important
consideration. After the first one or two steps,
the sample should be sufficiently clean and
clear to enable use of high-performance equip-
ment.
High-performance liquid chromatography,
or HPLC, is a term with a variety of meanings.
To some it refers exclusively to reversed-phase
chromatography; to others it includes all sorts
of chromatography provided that the equip-
ment is fully automated and high-performance
adsorbents are used. A high-performance sys-
tem designed specifically for proteins—intro-
duced by Pharmacia Biotech (see SUPPLIERS AP-
PENDIX) called Fast Protein Liquid Chroma-
tography, or FPLC—uses standard protein
chromatographies such as ion exchange, hydro-
phobic interaction, and gel filtration. Scaleup
is possible with larger equipment based on the
FPLC design, so that laboratory development
can be quickly translated to large-scale produc-
tion. FPLC is designed to separate proteins in
their native active configuration, whereas re-
versed-phase HPLC often causes at least tran-
sient denaturation during adsorption and elu-
tion. Reversed-phase HPLC has a high resolv-
ing power, but it is best suited to peptides and
proteins smaller than ∼30 kDa. Chromatogra-
phy run with older-style low-pressure adsor-
bents is sometimes referred to as “low-perform-
ance” or “open-column” chromatography; nei-
ther of those descriptions is necessarily
accurate. Simple fraction collector and moni-
toring equipment is needed. This equipment
will be used for larger-scale operations (tens of
milligrams of protein and upward), probably at
an earlier stage in the protocol than with HPLC.
Various columns, both prepacked with pro-
prietary adsorbents and empty for self-packing,
will be needed, with the sizes and types depend-
ing on the scale of operations. Several anion-
exchange columns (different sizes), one or two
cation-exchange columns, and gel-filtration
media are essential, along with a range of alter-
native adsorbents such as hydrophobic interac-
tion materials, dyes, hydroxyapatite, and chro-
matofocusing and specialist affinity media. Strategies of
Protein
Fully equipped protein purification labora- Purification and
tories should also have preparative electropho- Characterization
1.1.5
 resis and isoelectric focusing apparatuses for
rare occasions when other techniques fail to
give sufficient separation.
In addition to equipment used in the actual
fractionation processes, a variety of other items
are needed. In particular it should be possible
to change buffers quickly and to concentrate
protein solutions with ease. These operations
require such things as dialysis membranes (AP-
PENDIX 3B), ultrafiltration cells, and gel-exclu-
sion columns of various sizes (UNIT 8.3).
Finally, equipment for assaying and analyz-
ing the preparations is needed. Most such
equipment is fairly standard in biochemical
laboratories and includes spectrophotometers,
scintillation counters, analytical gel and capil-
lary electrophoresis apparatuses, immunoblot-
ting materials, and immunochemical reagents.
A listing of standard equipment is found in
APPENDIX 2D.
Literature Cited
Cohn, E.J., Strong, L.E., Hughes, W.L., Mulford,
D.J., Ashworth, J.N., Melin, M., and Taylor, H.L.
1946. Preparation and properties of serum and
plasma proteins. IV. A system for the separation
into fractions of the proteins and lipoprotein
components of biological tissues and fluids. J.
Am. Chem. Soc. 68:459-475.
Overview of
Protein
Purification and
Characterization
1.1.6
Key References
Deutscher, M.P. (ed.) 1990. Guide to protein purifi-
cation. Methods Enzymol. 182:1-894.
Extensive collection of purification methods with
some general protocols and examples.
Janson, J.-C. and Ryden, L.G. 1989. Protein Purifi-
cation: Principles, High Resolution Methods,
and Applications. VCH Publishers, New York.
A useful collection of methods and examples.
Kennedy, J.F. and Cabral, J.M. (eds.) 1993. Recov-
ery Processes for Biological Materials. John
Wiley & Sons, New York.
A useful introduction to the problems of large-scale
methods.
Kenny, A. and Fowell, S. (eds.) 1992. Practical
protein chromatography. Methods Mol. Biol.
11:1-327.
Extensive descriptions of affinity chromatographic
techniques with protocols and recipes.
Scopes, R.K. 1993. Protein Purification, Principles
and Practice, 3rd ed. Springer-Verlag, New York
and Heidelberg.
General principles of all the main techniques used
in purifying proteins. A useful laboratory handbook;
does not include recipes or procedures for specific
proteins.
Contributed by R.K. Scopes
La Trobe University
Bundoora, Australia
Current Protocols in Protein Science