Protein Purification Flow Charts
Protein purification flow charts are pre-
sented to give a broad outline of the methods
used for different types of proteins. They cannot
give any detail, as the process appropriate for
each protein will have its own variations at each
stage. In most cases, the first stage is to obtain
a solution containing the desired protein, after
which it can be dealt with by the many separa-
tion techniques described in the following
chapters. In some cases the insolubility of the
desired protein can be exploited by removing
soluble fractions. Purification procedures are
commonly divided into three stages: (a) the
primary steps, which deal with crude mixtures
of proteins and other molecules present in the
raw material; (b) the secondary processing,
which generates a product near to homogene-
ity; and (c) the polishing steps, which remove
minor contaminants, a process that is especially
important for therapeutic proteins.
SOLUBLE RECOMBINANT
PROTEINS
Proteins expressed in a recombinant manner
may be (1) soluble in the cytoplasm, (2) insol-
uble as inclusion bodies (see section on Insol-
uble Recombinant Proteins), (3) excreted from
the cells into the culture medium, (4) excreted
into the periplasmic space (e.g., in gram-nega-
tive bacteria), or (5) associated with organelles
or membrane fractions. In addition they may
be expressed (6) as the normal, mature, natu-
rally occurring protein, (7) containing a natural
leader peptide that would normally be proc-
essed, (8) as a fusion protein with a peptide that
is not natural to the protein, or (9) lacking
glycosylation or other post-translational modi-
fication, or incorrectly modified. Possibilities
(1) to (5) affect the method of extraction used
to obtain the starting material for purification.
Cases (6) to (9) can affect the methods used for
purification.
The scheme for purifying soluble recombi-
nant proteins is outlined in Figure 1.3.1. The
first stage is to obtain a clarified solution con-
taining the desired protein, with as little in the
way of unwanted proteins as possible. For sol-
uble cytoplasmic proteins, case (1), it is not
normally possible to exclude any significant
amount of unwanted soluble proteins, but in
cases (2) to (5) the compartmentalization away
from the cytoplasm allows such separation in
the initial stage.
It may be necessary to carry out a concen-
Contributed by R.K. Scopes
Current Protocols in Protein Science (1995) 1.3.1-1.3.7
Copyright © 2000 by John Wiley & Sons, Inc.
UNIT 1.
tration step before proceeding, especially if the
protein has been excreted into the culture me-
dium. Normally ultrafiltration is used, although
other techniques are possible, especially if the
extract contains particulates that block ultrafil-
tration membranes.
Recombinant expression in the cytoplasm
of bacteria, followed by extraction via total cell
disruption, results in large amounts of nucleic
acids being solubilized with the protein. A num-
ber of treatments to remove nucleic acids are
possible. Streptomycin is used to precipitate
ribosomal material, and cationic polymers such
as protamine (a basic protein) and polyethyl-
enimine will form insoluble complexes (at low
ionic strength) with nucleic acids. In addition,
viscosity caused by DNA can be reduced by
adding small amounts of DNase.
INSOLUBLE RECOMBINANT
PROTEINS
It has been found that many proteins ex-
pressed in bacteria (mainly in Escherichia coli)
do not fold correctly, and as a result aggregation
occurs, leading to large insoluble inclusion
bodies within the cytoplasm of the cells (see
Chapter 6). Although this creates major diffi-
culties in obtaining satisfactory amounts of
active native product, it greatly simplifies the
initial stage of purification.
The purification scheme for recombinant
insoluble proteins is outlined in Figure 1.3.2.
After cell disruption, inclusion bodies can be
obtained in a fairly pure state by differential
centrifugation. They must now be solubilized,
however, and the active protein generated by
encouraging correct folding. Solubilization is
usually accomplished with guanidine hydro-
chloride and/or urea, and thiols such as 2-mer-
captoethanol or glutathione are included to dis-
rupt any disulfides that have formed and pre-
vent more from forming. Folding the protein
correctly may require a variety of additions to
the solution, as well as slow removal of the
denaturant. The latter can be carried out by
simple dilution or by dialysis. Folding occurs
best at low protein concentrations, so dilution
may be adequate. If the native protein does
contain disulfides, then it is important to create
redox conditions such that some (but not exces-
sive) oxidation of thiols can occur. A combina-
tion of oxidized and reduced glutathione is Strategies of
commonly used. In addition, the action of the Protein
Purification and
enzyme protein disulfide isomerase, which can Characterization
1.3.1
CPPS
 cells with cells with cells with protein cells with
protein soluble protein excreted in periplasmic protein organelle-
in cytoplasm to medium space associated

break up cells; centrifuge or filter gently treat separate organelles;

centrifuge or to remove with lysozyme to perform differential
ultrafilter to cells minimize cell centrifugation
remove lysis
insolubles

treat to remove remove protoplasts extract to

nucleic acids and cell debris solubilize proteins,
remove insolubles

perform
concentration
step

starting material:
soluble fraction containing
the protein

perform purification steps

use salt fractionation, for tagged fusion protein,

ion-exchange chromatography, use affinity column
hydrophobic chromatography, chromatography and
affinity and pseudoaffinity polishing steps
chromatography, gel filtration,
or other less conventional steps
(i.e., preparative electrophoresis)

purified protein

Figure 1.3.1 Purification scheme for soluble recombinant proteins, which may be excreted or
located in the periplasm, in the membrane fraction, or most commonly the cytoplasm. The first step
is to obtain an extract containing the desired protein in soluble form. After this, conventional
purification steps may be carried out, or affinity purification of tagged fused proteins can be
performed.

make and unmake disulfides by exchange reac-
tions, has been found to be beneficial in many
cases. If the native protein is of intracellular
origin, it probably will not contain disulfides;
it will, however, contain cysteines, so a full
reducing potential should be maintained. Spe-
cific methodology is discussed in UNIT 6.5.
Not all proteins can fold unassisted by other
cellular components. Chaperonins are proteins
Protein whose role is to refold denatured proteins such
Purification as those that form during heat shock (Zeilstra-
Flow Charts
Ryalls et al., 1991). The most studied, and just
becoming commercially available as of 1995,
are the E. coli chaperonins GroEL and GroES,
both of which are needed, together with ATP,
to renature many proteins. Proline residues
can adopt two isomeric conformations in pro-
teins, and the wrong conformation is switched
to the correct one by the enzyme prolyl
isomerase, aiding the process of protein fold-
ing. At present these are not large-scale pros-
pects, both because of the cost of the chap-

1.3.2
Current Protocols in Protein Science
 cells containing protein

disrupt cells

perform differential centrifugation

wash inclusion bodies

dissolve in denaturing agents

dilute with buffer or dialyze

to dilute denaturing agents

add appropriate reducing agents

and/or folding factors

concentrate

perform purification procedure:

remove unwanted proteins and
incorrectly folded species
(e.g., by ion-exchange chromatography,
immunoaffinity methods, gel filtration)

purified protein

Figure 1.3.2 Purification scheme for insoluble recombinant proteins that are produced as inclu-
sion bodies in the cytoplasm of host cells. The cells must be broken open, and then the insoluble
inclusion bodies are separated by differential centrifugation. Solubilization is achieved by the use
of denaturing solvents, and renaturation of the dissolved protein occurs on removal of the
denaturant. Further polishing steps will be needed to remove small amounts of contaminating
proteins as well as incorrectly folded species. Additional information can be found in UNIT 6.3-6.5.

eronins and because the agents operate best in SOLUBLE NONRECOMBINANT

vitro at very low protein concentrations.
Once the proteins are folded, the purification
process consists of removing small amounts of
still incorrectly folded protein plus any other
host proteins that were trapped with the original
inclusion bodies. The former may be difficult,
since incorrectly folded species have a size and
charge similar to those of the correct product.
However, subtle differences arising from the
folded conformation can be exploited by chro-
matographic techniques. In ideal cases immu-
noaffinity techniques using antibodies specific
for either the incorrectly folded form or the
correct one can be used to resolve the mixture.
PROTEINS
There are so many sources of soluble pro-
teins that it is not possible to give a complete
overview of methods used to obtain starting
extracts from which a desired protein can be
isolated. The sources can be classified as either
microorganisms, plants, or animals, as shown
in Figure 1.3.3, but these in turn should be
subdivided according to how the starting ex-
tract is obtained. In particular there is a distinc-
tion between extracellular and intracellular
proteins. With the latter it is necessary to disrupt
the cells and release the proteins, whereas with
Strategies of
the former, if the extracellular fluid can be Protein
obtained directly, there need be no contamina- Purification and
tion with intracellular proteins. Extracellular Characterization

1.3.3
Current Protocols in Protein Science
 microorganisms plants animals

disrupt cells, disrupt cells, homogenize with

e.g., using French press, e.g., using French press 2-5 vol buffer or
bead mill, or sonication or by grinding with sand mince and stir
with 5-10 vol buffer using 1-2 vol buffer with buffer

treat to remove treat to remove

nucleic acids phenols

centrifuge

extracellular
starting extract proteins
(5-20 mg/ml protein)

perform initial fractionation:

use salt fractionation, precipitation
with organic solvents,
affinity methods, and/or
two-phase partitioning

perform secondary fractionation:

use ion-exchange chromatography,
hydrophobic chromatography,
affinity methods, other adsorbents,
and/or gel filtration

perform polishing step:

use HPLC– reversed phase,
HPLC– ion exchange,
or isoelectric focusing

purified protein

Figure 1.3.3 Purification scheme for soluble proteins present in their natural host cells. Cells must
be disrupted to release the proteins, usually in the presence of 2 to 10 ml of a suitable buffer per
gram weight. After removal of insoluble material, the process will generally require several steps,
using various standard fractionation procedures in a suitable order. For production of highly pure
protein, a final polishing step may be required to remove final trace contaminants. Additional
information can be found in UNIT 6.2.

sources include microorganism culture me-
dium, plant and animal tissue culture medium,
venoms, milk, blood, and cerebrospinal fluids.
Soluble proteins may also occur within organ-
elles such as mitochondria; these may be best
obtained by first isolating the organelle, then
Protein
Purification disrupting it to release the contents.
Flow Charts The starting extract normally contains be-
tween 5 and 20 mg protein per milliliter, though
lesser concentrations can be dealt with, espe-
cially if working on a small scale. It may be
necessary to include a concentration step before
starting the purification process in order to
approach that level. There are exceptions to
every rule, however, and very high protein
concentrations can be handled, for example,

1.3.4
Current Protocols in Protein Science
 whole tissue

homogenize disrupt cells; gently

without detergent; isolate organelles
centrifuge

supernatant residue

homogenize
with detergent;
centrifuge

supernatant containing residue containing

membrane-associated insoluble proteins
proteins

suspend and extract

perform primary fractionation: with other solvents
use hydrophobic chromatography, (e.g., ethanol, urea, or SDS)
precipitation with organic solvents,
ion-exchange chromatography,
and/or phase partitioning

purified insoluble
perform secondary fractionation: protein residue
use ion-exchange chromatography,
affinity methods, and/or
gel filtration

perform polishing step:

use HPLC or isoelectric focusing

purified protein

Figure 1.3.4 Purification scheme for membrane-associated and poorly soluble proteins (nonre-
combinant). An initial purification can be achieved by isolation of organelles containing the desired
protein. Membrane proteins are normally solubilized with a nonionic detergent, although loosely
associated proteins may be extracted without detergent at high pH, with EDTA, or with small
amounts of an organic solvent such as N-butanol. Normal fractionation procedures may need some
modification if the detergent is required throughout to maintain the integrity of the protein.

with two-phase partitioning (Walter and Jo-
hansson, 1994). When isolating proteins on a
large scale, the volumes being manipulated
become of increasing concern, so maximizing
protein concentration can be an important aim.
The starting extract should be clarified, usually
by centrifugation; on a large scale, ultrafiltra-
tion methods are becoming more widely used.
Pretreatment of certain extracts to remove ex-
cessive amounts of nucleic acids, phenolics,
and lipids may be necessary in order to obtain
an extract that is amenable to standard frac-
tionation procedures.
Fractionation procedures can somewhat ar-
bitrarily be divided into three steps: initial frac-
tionation, secondary fractionation, and polish- Strategies of
Protein
ing. Initial processing, which deals with a large Purification and
amount of extract that is not all protein, may Characterization

1.3.5
Current Protocols in Protein Science
 involve materials that become soiled and un-
able to be used many times. Consequently,
preferred methods do not require expensive
reagents or adsorbents such as are used in high-
performance liquid chromatography (HPLC).
Classic salt fractionation and the less-used or-
ganic solvent fractionation can achieve, if not
a high degree of purification, a useful level of
concentration and removal of much unwanted
nonproteinaceous material. Alternatively, a
highly selective affinity procedure may be used
as the first step, but only if the affinity material
is inexpensive to make and/or the extract is a
simple, clear solution, as opposed to a turbid
whole-cell homogenate.
Secondary processing achieves the main pu-
rification, and may involve two or more steps
in difficult situations. Ion-exchange and hydro-
phobic-interaction chromatography, gel filtra-
tion, and affinity techniques (see Chapter 8 and
Chapter 9) are the main procedures. Finally, it
may be necessary to remove traces of contami-
nants by “polishing,” using high-resolution
procedures such as reversed-phase HPLC (UNIT
11.6) and isoelectric focusing (IEF; UNIT 10.2 &
UNIT 10.4). Because every protein has unique
characteristics, it is impossible to make general
statements about procedures to be followed.
MEMBRANE-ASSOCIATED AND
INSOLUBLE NONRECOMBINANT
PROTEINS
Proteins that are not physiologically soluble
can be purified after extracting and removing
soluble proteins, thereby achieving a substan-
tial degree of purification at the extraction step
(Fig. 1.3.4; also see UNITS 6.1 & 6.2). To carry out
a purification it is nearly always necessary to
obtain the desired protein in a soluble form,
which will often require the addition of solubi-
lizing agents such as detergents. Some proteins
remain insoluble even with detergent treatment,
and so can be substantially purified by remov-
ing the soluble fractions. Some membrane-as-
sociated proteins become partly solubilized
during breaking up of the tissue, and recovery
in the particulate fraction may be poor. In such
cases it may be best to solubilize the whole
tissue by including detergent in the homogeniz-
ing buffer. Extraction of insoluble residues us-
ing detergents can be done differentially; some
proteins are released at low detergent concen-
tration, whereas others require complete solu-
bilization of the membrane fraction. Suitable
detergents include nonionic (e.g., Triton) and
Protein
Purification weakly acidic types (e.g., cholic acid deriva-
Flow Charts
1.3.6
tives). Strongly acidic detergents such as sulfate
esters (e.g., sodium dodecyl sulfate) usually
result in denaturation.
Detergents can be removed either by adsorp-
tion of the protein on a column, and subsequent
elution without detergent, by use of special
detergent-adsorbing beads, or even by extrac-
tion with nonmiscible organic solvents in
which the detergent partitions. On the other
hand, many membrane proteins require the
presence of detergent at all times in order to
remain in solution and in a native conformation.
These include most integral membrane pro-
teins, for example, cytochrome: P450, trans-
membrane receptors, and transporters. The
most sensitive proteins require a particular
combination of natural lipids (in addition to the
detergent) to maintain structural integrity. Pu-
rification methods include most of those used
for soluble proteins, but some techniques are
not recommended if detergent is needed at all
times. For instance, ammonium sulfate precipi-
tation will often cause a detergent-protein com-
plex to come out of solution and float rather
than sink on centrifugation—this can be useful,
but the “floatate,” when redissolved, may have
a high detergent content. Hydrophobic chroma-
tography can be very useful, as membrane pro-
teins are naturally hydrophobic.
Integral membrane proteins that are com-
pletely insoluble in normal detergents may be
solubilized by denaturation using compounds
such as sodium dodecyl sulfate and guanidine
hydrochloride. Some cross-linked proteins
such as elastin are not soluble without disrup-
tion of the covalent linkages.
Literature Cited
Walter, H. and Johansson, G. (eds.) 1994. Aqueous
two-phase systems. Methods Enzymol. 228:1-
725.
Zeilstra-Ryalls, J., Fayet, O., and Georgopoulos, C.
1991. The universally-conserved GroE (Hsp60)
chaperonins. Annu. Rev. Microbiol. 45:301-325.
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.
Current Protocols in Protein Science
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

Strategies of
Protein
Purification and
Characterization

1.3.7
Current Protocols in Protein Science