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Protein Purification by Affinity Chromatography

Chapter · January 2012

DOI: 10.5772/29947

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1 Chapter Number

2 Protein Purification by
3 Affinity Chromatography
4 Luana C. B. B. Coelho1, Andréa F. S. Santos2, Thiago H. Napoleão1,
5 Maria T. S. Correia1 and Patrícia M. G. Paiva1
6 1UniversidadeFederal de Pernambuco, Centro de Ciências Biológicas,
7 Departamento de Bioquímica, Av, Prof. Moraes Rego, Recife-PE,
8 2University of Minho, IBB-Institute for Biotechnology and Bioengineering,

9 Centre of Biological Engineering, Campus de Gualtar, Braga,

10 1Brazil

11 2Portugal

12 1. Introduction
13 Affinity chromatography is a method which depends essentially on the interaction between
14 the molecule to be purified and a solid phase that will allow the separation of contaminants.
15 Lectins are carbohydrate-binding proteins which can be purified by affinity
16 chromatography; also, the presence of multiple molecular forms of lectins in a preparation
17 can be separated. Immobilized lectins have been useful to affinity protein purification. In
18 immunoaffinity chromatography an antibody or an antigen is immobilized on a support so
19 as to purify the protein against which the antibody was developed. Monoclonal antibodies
20 are extremely useful as immunosorbents for purification of antigen. Immobilization of
21 monoclonal antibody on a suitable material to the column produces a support that will bind
22 with high selectivity to protein against which the antibody was developed. Affinity
23 chromatography containing DNA is a highly specific and important technique for the
24 purification of DNA-binding proteins involved in the transcription, replication and
25 recombination. The success of affinity chromatography depends on the conditions used in
26 each chromatographic step. So, the optimization of protocol is essential to achieve optimal
27 protein purification with maximum recovery.

28 2. Nomenclature and basic concepts

29 The term "affinity chromatography", first used by Cuatrecasas et al. (1968), refers to a
30 purification technique which depends essentially on the highly specific interaction between
31 the molecule to be purified and the solid phase that will allow the separation of
32 contaminants. This method has several other terms such as "bioselective adsorption", which
33 was appropriately used to denominate an adsorption chromatography that uses a very
34 special kind of affinity between the desired biological product and a biomolecule (Porath,
35 1973). For example, the biological affinity between an enzyme (protein with catalytic
36 activity) and its substrate and/or other small ligand – usually in the active or allosteric site
37 of the enzyme – results from a selective interaction.
 2 Protein Purification

1 The adsorption corresponds to the fixation of the molecules of a substance (the adsorbate)
2 on the surface of another substance (the adsorbent), which may be immobilized to an
3 insoluble support. The adsorbate and the adsorbent can be referred as bioligands. The
4 bioligands may be specific or may not have absolute specificity of interaction. Many
5 bioligands (e.g. NAD+, ATP, coenzyme A) may bind different enzymes, being then called
6 group-specific ligands. In the same manner, chitin (a polysaccharide composed by N-acetyl-
7 D-glucosamine units) may be an adsorbent for several different molecules if they possess a
8 group (e.g. a binding or catalytic site) able to interact with chitin.
9 Affinity chromatography is a powerful tool for the purification of substances in a complex
10 biological mixture. It can also serve to separate denatured and native forms of the same
11 substance. Thus, biomolecules which are difficult to purify have been obtained using
12 bioselective adsorbents, e.g. immobilized metal ions (Ni2+ and Zn2+) used to purify proteins
13 containing zinc finger domains with natural affinity to divalent ions (Voráčková et al., 2011).
14 The relative specificity degree of the affinity chromatography is due to the exploitation of
15 biochemical properties inherent in certain molecules, instead of using small differences in
16 physicochemical properties (such as size, form and ionic charge, which are employed by
17 other chromatographic methods).
18 Affinity chromatography may be used with different final objectives. If the aim is a rapid
19 purification of a macromolecule with high yield, many controls and careful attention are
20 necessary to establish the best conditions for a high bioselectivity of the system; the
21 researcher must be prepared to adjust the chromatographic conditions and to circumvent
22 possible absence of bioselectivity or low yields. If the objective is to first demonstrate a
23 bioselectivity for further purification, the choice of the bioselective adsorbent is dependent
24 on the physiological interaction between the bioselective component and the macromolecule
25 to be purified. In this case, the researcher must spend a lot of time establishing the
26 bioselectivity before starting the isolation experiments.
27 A good bioselectivity means that the affinity of the molecule by the ligand exceeds all factors
28 of non-specific adsorption that are present in the system. Also, the affinity should not be so
29 strong, since the biomolecule must be removed from the column. A well-designed affinity
30 method should consider the selection of the ligand molecule or the insoluble support to be
31 used; they must have specific and reversible binding affinity for the molecule being purified.
32 After defining the protocol, purification by affinity chromatography is a rapid method,
33 compared with others less specific. The technique also enables the concentration of the
34 molecule of interest resulting in a small volume of a concentrated product.
35 Standard procedures of protein purification result in obtainment of homogeneous protein.
36 However, a considerable cost of supplies and hours of work is often required and a low
37 yield is obtained after several steps. The power of affinity chromatography is often larger
38 than other chromatographic techniques, resulting in several hundred or thousand-fold
39 purification factors in a single step.

40 3. Supports for affinity chromatography

41 A good support for affinity chromatography should be chemically inert or have minimal
42 interaction with other molecules, having high porosity and large number of functional
 Protein Purification by Affinity Chromatography 3

1 groups capable of forming covalent bonds with the molecule to be immobilized. Many
2 materials are available (Table 1). A variety of supports with immobilized ligands, or stable
3 media for the immobilization of ligands through different functional groups are
4 commercially available. The ligand molecule to be used should contain a group capable of
5 being chemically modified, often an amino group, which will allow connection with the
6 matrix without destroying its capacity to bind to the molecule of interest.
7
Supports References
Affi-gel blue gel Wong et al., 2006
α-casein-Agarose Kocabiyik & Ozdemir, 2006
Chitin Sá et al., 2008; Coelho et al., 2009; Santana et
al., 2009; Napoleão et al., 2011a
Fetuin-fractogel Guzmán-Partida et al., 2004
Fetuin-Sepharose CL-4B Bhowal et al., 2005
Ferromagnetic levan composite Angeli et al., 2009
GalNac-Sepharose CL-4B Gade et al., 1981
Galactosyl-Sepharose Franco- Fraguas et al., 2003
Glutathione reduced (GSH)-Sepharose Hamed et al., 2011
Guar gel Coelho & Silva, 2000; Santos et al., 2009;
Nunes et al., 2011; Souza et al., 2011
IMAC (immobilized metal ion affinity Voráčková et al., 2011
chromatography)-Sepharose
Sephadex G25 Santana et al., 2009
Sephadex G50 Fenton-Navarro et al., 2003
Sephadex G75 Correia & Coelho, 1995
Sepharose-manose gel Latha et al., 2006
Lectin-Sepharose CL-4B Paiva et al., 2003; Silva et al., 2011
Trypsin-Agarose Leite et al., 2011

8 Table 1. Supports for affinity chromatography.

9 One example is the agarose, a polysaccharide obtained from agar, which provides numerous
10 free hydroxyl groups and is the most widely used (Chung et al., 2009). The ligand may be
11 covalently bound to it through a two step process. In the first step, the agarose reacts with
12 cyanogen bromide to form an "activated" intermediate which is stable and commercially
13 available. In the second step, the molecule to be immobilized reacts with agarose to form the
14 covalently bound product (Voet & Voet, 1995). A support containing trypsin immobilized
15 on agarose was used to purify trypsin inhibitor from liver of Oreochromis niloticus (Leite et
16 al., 2011). Chromatography on α-casein-Agarose was useful for purification of an
17 intracellular chymotrypsin-like serine protease from Thermoplasma volcanium (Kocabiyik
18 & Ozdemir, 2006).
19 Sepharose (a tradename of a registered product of GE Healthcare) is a beaded form of
20 agarose cross-linked through lysine side chains. It is a common support for
 4 Protein Purification

1 chromatographic separations of biomolecules and can also be activated with cyanogen

2 bromide. For example, glutathione S-transferases from Down syndrome and normal
3 children erythrocytes were purified by chromatography on matrix containing glutathione
4 reduced (GSH) immobilized on Sepharose (Hamed et al., 2011).
5 Insoluble polysaccharide matrices – such as chitin, guar gel and Sephadex – have been used
6 to purify lectins (carbohydrate-binding proteins) and will be discussed later.

7 4. Extraction and purification of proteins by affinity chromatography

8 To obtain a pure protein is essential for structural characterization and exploration of its
9 function in nature. These proteins should be free of contaminants if they will be used for
10 biotechnological purposes, such as the evaluation of their potentiality to purify and
11 characterize other molecules, as well as for studies on the ability to recognize receptors and
12 induce different cellular responses.
13 Proteins are dependent of environmental conditions to maintain their stability and for this
14 reason some parameters are crucial in all steps of the purification protocol: pH, ionic
15 strength, temperature and dielectric constant. The balance of these parameters, characteristic
16 for each protein, is essential for obtainment of the pure molecule in its native form. The
17 protein activity is due to the maintenance of protein structure that may be stabilized by
18 strong bonds, like disulfide bridges, and weak bonds, like hydrophobic interactions and
19 hydrogen, electrostatic or saline bonds.
20 In the purification processes of a protein, the following parameters should be considered:
21 the selection of the procedure for protein extraction from the biological source, the assays for
22 monitoring protein concentration in each step, the methods of solubilization, and the
23 environmental conditions for stabilization. The prior separation is based on differences in
24 solubility and usually corresponds to the preparation of a homogenate or extract. After
25 extraction and centrifugation, the separation can be based on molecular mass, electric charge
26 and protein affinity for other molecules.
27 Many proteins have the ability to bind strongly (but not covalently) to specific molecules
28 and thus can be purified by affinity chromatography. Figure 1 shows the steps of an affinity
29 chromatography for isolation of a protein. Initially, the affinity support must be equilibrated
30 with a binding buffer to achieve adequate conditions for affinity interaction between the
31 protein and the immobilized molecule (step 1). When an impure solution (crude extract or a
32 partially purified preparation) is passed through the affinity support, the protein of interest
33 interacts with the ligand (adsorption) and the other contaminants (other proteins or
34 molecules) are washed from the column with the binding buffer (step 2). The desired
35 molecule can be obtained highly purified by changing the elution conditions to release the
36 protein from the support (step 3). For example, the elution may be performed changing the
37 conditions of pH, ionic strength or temperature (non-bioselective desorption), or with a
38 solution containing a high concentration of free ligand that will compete for the binding-
39 sites of the protein (a bioselective desorption).
40 A crude extract can be directly applied in an affinity chromatography column. The
41 application of crude extract has the advantage of avoiding other steps that lengthen the
42 process. However, substances that may interfere in this process, like other proteins, nucleic
 Protein Purification by Affinity Chromatography 5

1 acids and lipids are present in higher concentrations in crude extracts. In general, before the
2 chromatography, one or more steps for partial separation of undesirable constituents are
3 incorporated into the purification protocol.

4
5 Fig. 1. Schematic representation of the equilibration (1), adsorption/washing (2) and
6 desorption (3) steps of an affinity chromatography for protein purification.
 6 Protein Purification

1 Among the parameters used to evaluate if a preparation is pure can be cited electrophoresis,
2 immunological and chromatographic methods. The homogeneity of a protein preparation
3 should not be judged by isolated parameters. The indication of protein purity is obtained by
4 analysis of various speculations.
5 Affinity chromatography is a useful tool in proteomics studies; this method plays an
6 essential role in the isolation of protein complexes and in the identification of protein–
7 protein interaction networks. In glycoproteomics, serial lectin affinity chromatography was
8 applied in the process for identification of over thirty proteins from the human blood with
9 O-glycosylation sites (Durham & Regnier, 2006). Affinity chromatography is also required
10 for quantification of protein expression by using isotope-coded affinity tags (Azarkan et al.,
11 2007).

12 5. Forces that stabilize proteins and affinity interactions

13 The protein structures are maintained by hydrophobic effects and interactions between
14 polar residues and other types of connections (Voet et al., 2008). For enzymes, the active
15 sites are constituted by amino acid residues in direct contact with the substrate and those
16 amino acid residues indirectly involved in substrate binding through a water molecule as
17 intermediate or by the side chain of an amino acid. Many of the mentioned residues may be
18 in contact with a single substrate; the connection can occur through various combinations of
19 hydrophobic interactions, ionic bonds, hydrogen bonds and charge transfer. The enzyme
20 specificity for a particular substrate depends mainly on the steric positioning of each amino
21 acid in the active site. Substrates or inhibitors can be accommodated in the active site; some
22 are adjusted better than others.
23 The ideal conditions for affinity chromatography correspond to those in which the
24 adsorbate-adsorbent interaction resembles an enzyme-substrate binding. However, in
25 general, the adsorbent support can interact with proteins applied to the column by ionic
26 interactions, hydrogen bonds, hydrophobic interactions, or other binding sites present on
27 the surface of the protein.
28 In affinity chromatography occur bioselective and non-bioselective interactions; the
29 contribution of these interactions is dependent of the medium used and the physico-
30 chemical characteristics of the preparation containing the protein to be purified. The
31 bioselective adsorption constitutes one of the most effective and complex methods of protein
32 separation.
33 In affinity chromatography the bioselective elution (desorption) should be attempted not
34 only to prove that a particular purification was possible due to a bioselective adsorption, but
35 also because the bioselective elution often provides high levels of purification. Large
36 numbers of reversible interactions (hydrophobic attraction and hydrogen or electrostatic
37 bonds) are involved in recognition of the free ligand (in elution solution) by the protein
38 which was adsorbed on the matrix (Scouten, 1981).

39 6. Lectins: Prototypes in protein purification by affinity chromatography

40 The term lectin (from Latin lectus, past participle of legere, which means “to select”) was
41 introduced by Boyd (1954) and describes a protein heterogeneous group of non-immune
 Protein Purification by Affinity Chromatography 7

1 origin, containing two or more binding sites for mono or oligosaccharides. These molecules
2 have the ability to agglutinate cells such as erythrocytes (hemagglutination), lymphocytes,
3 fibroblasts and bacteria, being also able to precipitate glycoconjugates (Goldstein et al., 1980;
4 Barondes, 1988; Kennedy et al., 1995; Correia et al., 2008; Sá et al., 2009a).
5 To be considered a lectin, the hemagglutinating activity should be inhibited by a
6 carbohydrate; when addition of mono or oligosaccharides neutralizes the agglutination
7 phenomenon, the protein is considered a potential lectin.
8 The lectin purification may be performed by conventional or high resolution techniques.
9 However, in most of the purification processes, the affinity chromatography is used. The
10 lectins are real models of protein purification exploring affinity interactions.
11 The lectin extracted from Canavalia ensiformis seeds (jack bean) named Concanavalin A (Con
12 A) was the first lectin to be crystallized. Since then, an increasing number of lectins with
13 similar or different specificities have been obtained.
14 Lectins have been purified from Cratylia mollis seeds using Sephadex (cross-linked dextran
15 gel) matrices allying the gel filtration property of this support and the ability of the lectins to
16 bind glucose (Paiva & Coelho, 1992; Correia & Coelho, 1995).
17 Guar gel beads produced by cross-linking of refined guar gum (a polysaccharide composed
18 of glucose and mannose) with epichlorohydrin in a mixture of water and 2-propanol (Gupta
19 et al., 1979) have been used to purify galactose-specific lectins (Santos et al., 2009; Nunes et
20 al., 2011).
21 Chitin-binding lectins can be isolated by affinity chromatography on columns containing
22 powder of chitin from crab shells hydrated with the equilibrating solution. This is a cheap,
23 efficient, and rapid technique to purify these lectins, which have great potential as
24 insecticidal and antimicrobial agents (Sá et al., 2009a; Sá et al; 2009b; Santana et al., 2009;
25 Coelho et al., 2009; Ferreira et al., 2011; Napoleão et al., 2011a; Napoleão et al., 2011b).
26 A ferromagnetic levan (a homopolysaccharide composed of D-fructofuranosyl) composite
27 was developed and efficiently used in purification of C. mollis lectin (Angeli et al., 2009). Egg
28 glycoproteins were immobilized and the affinity matrix was efficient to purify lectins from
29 extracts of Phaseolus vulgaris, Lens culinaris, and Triticum vulgaris (Zocatelli et al., 2003).
30 Lectins can be used for observation of the most diverse phenomena and the study of these
31 proteins allows the evaluation of different cell surfaces. It is known that all cells have a
32 membrane containing carbohydrates, consisting mainly of glycoproteins and glycolipids,
33 that are different for each cell and which may constitute the lectin receptors. In the same cell,
34 the surface structure can change characteristically due to normal development course or
35 cases of illness. The lectins have been used very successfully in histochemistry (Beltrão et al.,
36 1998, Lima et al., 2010) and electrochemistry (Souza et al., 2003, Oliveira et al., 2008, 2011b)
37 with diagnostic purposes.

38 6.1 Purification of lectins from autochthonous and introduced species at

39 Northeastern Brazil by affinity chromatography
40 The motivation to search lectins in autochthonous and introduced species from a particular
41 region of a country is primordially due to the perspectives to develop a biotechnological
 8 Protein Purification

1 leading edge. In the Laboratory of Glycoproteins from the Department of Biochemistry of

2 the Universidade Federal de Pernambuco (Brazil), the first plant tissues evaluated in order to
3 identify hemagglutinating activity (indicating the presence of lectins) were the seeds of C.
4 mollis (Paiva & Coelho, 1992; Correia & Coelho, 1995). This legume, also known as camaratu
5 bean, is important as human food and as native forage in the Semi-Arid Region from the
6 State of Pernambuco, northeastern Brazil. Since then, many other lectins have been purified.
7 Examples of lectins purified by affinity chromatography in the Laboratory of Glycoproteins
8 are shown in Table 2.
9
Lectin Plant (tissue) Affinity support References
used
BmoLL Bauhinia monandra (leaf) Guar gel Coelho & Silva (2000)
BmoRoL B. monandra (root) Guar gel Souza et al. (2011)
Cramoll Cratylia mollis (seeds) Sephadex Paiva & Coelho (1992);
Correia & Coelho (1995)
cMoL Moringa oleifera (seeds) Guar gel Santos et al. (2009)
WSMoL M. oleifera (seeds) Chitin Coelho et al. (2009)
MuBL Myracrodruon urundeuva (bark) Chitin Sá et al. (2009b)
MuHL M. urundeuva (heartwood) Chitin Sá et al. (2008)
MuLL M. urundeuva (leaf) Chitin Napoleão et al. (2011)
PpeL Parkia pendula (seed) Sephadex Lombardi et al. (1998)
PpyLL Phthirusa pyrifolia (leaf) Sephadex Costa et al. (2010)
OfiL Opuntia ficus indica (cladodes) Chitin Santana et al. (2009b)

10 Table 2. Lectins purified by affinity chromatography from different tissues of autochthonous

11 and introduced plants from northeastern Brazil.
12 Saline extract (0.15 M NaCl) from C. mollis seeds showed hemagglutinating activity on
13 erythrocytes from humans and other animals. The lectin activity was inhibited by glucose
14 and mannose. The extract was treated with ammonium sulfate (0-40% and 40-60%),
15 producing three fractions (F): 0-40F and 40-60F (precipitate fractions) and 40-60SF
16 (supernatant fraction) with hemagglutinating activity. The hemagglutinating activity was
17 concentrated (94%) in 40-60F, and a lectin (Cramoll 1) was purified by affinity
18 chromatography on Sephadex G-75 followed by ion exchange chromatography on CM-
19 cellulose (Correia & Coelho, 1995). Additionally, two other molecular forms were obtained
20 from 0-40F (Cramoll 3) and 40-60FS (Cramoll 2) through affinity chromatography on
21 Sephadex G-75, ion exchange using CM-Cellulose column, and molecular exclusion using
22 Bio-Gel P (Paiva & Coelho, 1992). The characterization of the isoforms was performed by
23 electrophoresis and immunological methods. Cramoll 1 was crystallized by Tavares et al.
24 (1996). C. mollis lectins showed several biological activities such as mitogenic effect on
25 human lymphocytes (Maciel et al., 2004), antitumor activity on Sarcoma 180 when
26 encapsulated into liposomes (Andrade et al., 2004), potential anti-helminthic against
27 Schistosoma mansoni (Melo et al., 2011a), healing activity on cutaneous wounds in healthy
28 and immunocompromised mices (Melo et al., 2011b), and induction of death on
29 epimastigotes of Trypanosoma cruzi (Fernandes et al., 2010).
 Protein Purification by Affinity Chromatography 9

1 Moringa oleifera is a multipurpose tree with great importance in industry and medicine.
2 Lectins have been found in extracts from distinct tissues of M. oleifera (Santos et al., 2009).
3 Seeds from moringa are used to treat water for human consumption and different lectins
4 were detected in this tissue (Santos et al., 2005; Katre et al., 2008; Santos et al., 2009; Coelho
5 et al., 2009). Santos et al. (2005) found a water-soluble M. oleifera lectin (WSMoL) that is the
6 unique M. oleifera lectin inhibited by fructose. WSMoL was isolated through affinity
7 chromatography on chitin column and showed larvicidal activity against fourth-stage larvae
8 of Aedes aegypti (Coelho et al., 2009). This lectin is also a potential natural biocoagulant for
9 water, reducing turbidity, suspended solids and bacteria (Ferreira et al., 2011). Genotoxicity
10 assessment of WSMoL showed that it was not mutagenic and was not able to promote
11 breaks in DNA structure (Rolim et al., 2011).
12 Santos et al. (2009) purified a lectin with coagulant properties from M. oleifera seeds (cMoL)
13 by affinity chromatography on guar gel. cMoL agglutinated erythrocytes from rabbit and
14 human, was insecticidal for Anagasta kuehniella and, when immobilized, served as an affinity
15 support able to interact with humic acids (Oliveira et al., 2011a; Santos et al., 2011).
16 Coelho & Silva (2000) purified a galactose-specific lectin (BmoLL) from the fresh leaves of
17 Bauhinia monandra. Also, other galactose-specific lectin was purified from B. monandra
18 secondary roots, BmoRoL (Souza et al., 2011). These lectins were purified in milligram
19 quantities by affinity chromatography on guar gel. BmoLL showed insecticidal activity on
20 Callosobruchus maculatus, Anagasta kuehniella and Zabrotes subfasciatus (Macedo et al., 2007)
21 while BmoRoL showed antifungal and termiticidal activities (Souza et al., 2011); thus, these
22 lectins have biotechnological potential for application in control of agricultural pests.
23 In our studies, the presence of lectin isoforms has been revealed. The exploration and
24 knowledgement of multiple molecular forms of lectins in extracts or in early stages of
25 fractionation is very important. A substantial proportion of proteins have been described
26 with multiple molecular forms having or not defined genetic origin.
27 The Parkia pendula (visgueiro) is a majestic tree from the Brazilian Atlantic Forest that stands
28 out by their generous production of vegetables. Extracts of its seeds showed
29 hemagglutinating activity with erythrocytes from humans and various animal species. The
30 best monosaccharide inhibitors of the hemagglutinating activity from P. pendula were α-
31 methyl D-mannoside, D (+)-mannose and D (+)-glucose, in descending order. To purify the
32 lectin, a seed extract in 0.15 M NaCl, was fractionated with ammonium sulfate (40%). The 0-
33 40F recovered 97% of total hemagglutinating activity. The dialyzed preparation was
34 chromatographed by affinity on Sephadex G-75, and eluted with 0.3 M glucose. The purity
35 of the obtained preparation allowed the crystallization of the lectin (Lombardi et al., 1998).
36 Other supports for purification of P. pendula lectin by affinity chromatography were also
37 exploited for its purification. Although the lectin was not inhibited by N-acetyl-D-
38 glucosamine, the support chitin was used to purify two molecular forms of lectin (Souza,
39 1989). The absence of inhibitory effect of carbohydrate on hemagglutinating activity does
40 not imply in an inability of lectin to adsorb on an affinity support containing this
41 carbohydrate (Lis & Sharon, 1981).
42 Myracrodruon urundeuva (aroeira-do-sertão) is a plant with importance in traditional
43 medicine and its heartwood is resistant to fungi and termite attack. Lectins were isolated
 10 Protein Purification

1 from M. urundeuva bark (MuBL), heartwood (MuHL) and leaf (MuLL) by affinity
2 chromatography on chitin columns. Similarly to P. pendula lectin, the hemagglutinating
3 activity of MuLL is not inhibited by N-acetyl-D-glucosamine but the lectin bind to chitin.
4 The affinity interaction between MuLL and this monosaccharide was demonstrated by
5 affinity chromatography on N-acetyl-D-glucosamine-Agarose column (Napoleão et al.,
6 2011a).
7 MuHL showed antimicrobial activity inhibiting the growth of bacteria and fungi (Sá et al.,
8 2009a). The three lectins showed termiticidal activity against Nasutitermes corniger and
9 insecticidal effect on fourth-stage larvae of A. aegypti (Sá et al., 2008; Sá et al., 2009b;
10 Napoleão et al., 2011a; Napoleão et al., 2011b).

11 6.2 Applications: Immobilized lectins as affinity supports for protein purification

12 Various applications of lectins have been developed from the binding of these versatile
13 molecules with free carbohydrates or glycoconjugates present in cell surfaces. The lectin
14 applications have emerged in parallel to their discovery in 1888, with the description of the
15 hemagglutination phenomenon, previously mentioned. Lectins have been applied for
16 different purposes.
17 An immobilized lectin, covalently attached to a support, can separate glycoproteins or
18 proteoglycans containing specific carbohydrate groups from a crude preparation. The
19 elution of adsorbed material can be performed by treatment of support with a solution
20 containing a competitive glycoside. The elution is usually performed near neutral pH, with
21 minimal deleterious effects to the glycoprotein.
22 The interaction of a glycoprotein with an immobilized lectin can be used as a suitable
23 technique to obtain preliminary information about the covalently linked carbohydrates to
24 the glycoconjugate in the study. Lectins with different carbohydrate specificities,
25 immobilized on Sepharose, have been applied as an analytical tool to assess and compare
26 the carbohydrate residues.
27 Coelho (1982), using columns containing lectins with different specificity, detected
28 microheterogeneities in human liver glycosidases. Con A revealed microheterogeneity in
29 type A and B isoenzymes of beta-N-acetylhexosaminidase purified from human placenta.
30 A preparation of lectin from C. mollis containing Cramoll 1,4 isoforms was immobilized on
31 inert support and used as an affinity matrix for purification of glycoproteins from human
32 plasma, including the lecithin cholesterol acyl transferase (Lima et al., 1997). C. mollis seed
33 lectins immobilized on cyanogen bromide-activated Sepharose 4B were used to purify a
34 trypsin inhibitor from Echinodorus paniculatus seeds (Paiva et al., 2003) and a soybean seed
35 protein with platelet antiaggregation and anticoagulant activities (Silva et al., 2011).
36 Immobilized Euonymus europaeus lectin was an efficient affinity ligand used in the capture
37 step for purification of human influenza A viruses derived from MDCK cells; the main
38 targets were two viral glycoproteins (Opitz et al., 2007).
39 Lectin affinity chromatography is a powerful fractionation technique in the identification of
40 glycobiomarkers. Immobilized Con A was successfully used in the glycoproteomic analysis
41 of pluripotent murine embryonic stem cells; differential patterns of binding to lectin allowed
42 the identification of stage-specific glycopeptides (Alvarez-Manilla et al., 2010).
 Protein Purification by Affinity Chromatography 11

1 7. Immunoaffinity chromatography
2 The immunoaffinity chromatography consists of an antibody (immunoglobulin)
3 immobilized on a support to purify the protein against which the antibody was
4 developed. Antibodies specific for the protein of interest are produced by the immune
5 system when the exogenous protein is inoculated in the animal; after, polyclonal antibodies
6 are extracted from the blood serum of the animal, isolated and immobilized to constitute a
7 matrix for purification of the protein of interest (Figure 2). Since the polyclonal antibodies
8 are products from many different cells of the immune system, they are heterogeneous,
9 differing in binding affinity for the protein inoculated on the animal.
10

11
12 Fig. 2. Immunoaffinity chromatography for protein purification. First, the antibody for a
13 specific protein is developed immunizing an animal. Next, the antibodies produced are
14 purified and immobilized for use as immunoaffinity support.
15 Polyclonal antibodies (IgY) were developed against Plasmodium falciparum proteins and
16 immobilized for use in immunoaffinity chromatography columns; the technique was more
17 efficient than conventional chromatography (using two anion exchange columns) in
18 purification of chimeric proteins expressed in Escherichia coli that are candidates for use as
19 vaccine to prevent malaria (Qu et al., 2011).
20 The native Cramoll 1 was used to develop a serum anti-Cramoll 1 produced by rabbits. The
21 anti-Cramoll 1 immunoglobulin (IgG anti-Cramoll 1) was obtained by affinity
22 chromatography on Protein A-Sepharose. The antibody was conjugated to peroxidase (IgG
23 anti-Cramoll 1-Per) and the conjugate was used to evaluate the structural assessment of the
24 lectin (Correia & Coelho, 1995).
 12 Protein Purification

1 Monoclonal antibodies can be developed by collecting lymphocytes B (producing the

2 desired antibody) from the spleen of the immunized animal and fusing them with a
3 myeloma (a tumor of lymphocytes B). The resulting cells (hybridoma) have an unlimited
4 capacity for division. When developed in culture they will produce large amounts of
5 monoclonal antibodies.
6 Monoclonal antibodies are extremely useful as immunosorbents for purification of antigens.
7 Immobilization of monoclonal antibody produces a support that can achieve a 10,000-fold
8 purification in a single step. Polyol-responsive monoclonal antibody that recognize a highly
9 conserved sequence in the β-subunit of bacterial RNA polymerase were used to purify RNA
10 polymerase from five species of bacteria in one immunoaffinity chromatography step
11 (Stalder et al., 2011). Nakamura et al. (2010) developed a sensitive and specific monoclonal
12 antibody against a soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1),
13 expressed prominently in atherosclerotic lesions as a specific biomarker to diagnostic acute
14 coronary syndrome at an early stage; this immunoassay was developed in order to establish
15 a more sensitive assay that may also be useful in predicting cardiovascular disease risk in
16 disease-free subjects.
17 The antigen-antibody complex often has a strong affinity and it is necessary that the release
18 of the protein is performed in drastic conditions of pH and/or ionic strength. The elution is
19 preferably performed in the reverse direction to sample application. To avoid denaturation
20 of the molecule, the conditions are adjusted immediately after the elution.

21 8. Purification of DNA-binding proteins by affinity chromatography

22 DNA-binding proteins can be purified using different techniques of affinity
23 chromatography. One of them is the affinity chromatography containing immobilized DNA,
24 which is a highly specific technique for the purification of DNA-binding proteins involved
25 in the transcription, replication and recombination. The affinity columns are usually
26 generated by immobilization of synthetic oligonucleotides consisting of tandem repeated
27 units or multiple copies of the same sequence (Gadgil et al., 2001). Purification factors can
28 reach 10,000-fold. Figure 3 shows a schematic representation of DNA-affinity
29 chromatography.
30 DNA-affinity chromatography is a powerful method with broad applicability; this
31 technology has been extended for purifying transcription factors, polymerases, and
32 nucleases (Chockalingam et al., 2001). Since affinity chromatography is based in the specific
33 interaction between molecules, it is highly selective and offers high yield and purity (Gadgil
34 et al., 2001). DNA affinity columns can be constructed depending on the DNA-binding
35 properties of a protein (non-specific, specific, double- or single-stranded DNA). Apart from
36 the purification of DNA-binding proteins, DNA affinity columns can also be used for the
37 purification of nucleic acids, such as RNA and DNA. Golovina et al. (2010) described a fast
38 and simple purification method for the 30S ribosomal subunits carrying lethal mutations
39 using DNA-affinity chromatography. Kerrigan & Kadonaga (2001) developed a DNA
40 affinity resin using agarose activated with cyanogen bromide.
41 Another technique used for isolation of DNA-binding proteins is ion metal affinity
42 chromatography. The Mvo10b is a DNA-binding protein member of the Sac10b family from
43 the mesophilic archaeon Methanococcus voltae, which may play an important role in the
 Protein Purification by Affinity Chromatography 13

1 organization and accessibility of genetic information in Archaea. This protein was purified
2 by polyethyleneimine precipitation followed by nickel affinity chromatography; this
3 protocol has potential application in the production of other thermophilic and mesophilic
4 proteins in the Sac10b family (Xuan et al., 2009). A telomeric DNA-binding protein (Stn1p)
5 from Saccharomyces cerevisae was purified by interaction with nickel-NTA resin followed by
6 chromatography on Superdex 200 column (Qian et al., 2010).

7
8 Fig. 3. Schematic representation of DNA-affinity chromatography. In the first step, the
9 support containing a synthetic oligonucleotide consisting of a tandem repeated unit is
10 washed equilibrated. Next, the mixture containing proteins is loaded and the DNA-binding
11 protein that recognizes the oligonucleotide adsorb on the support. The elution is performed
12 by solutions containing a very high concentration of salt.
 14 Protein Purification

1 9. Pitfalls in affinity chromatography

2 There are numerous problems in affinity chromatography, similar to any other techniques.
3 One of the most common ways to immobilize ligands with free amino groups is by a
4 reaction with Sepharose activated with cyanogen bromide. This method promotes the
5 formation of ionic groups that may cause non-selective electrostatic adsorption. The
6 immobilization of small ligands may create steric impediment problems that limit the
7 functional capacity of the columns.
8 To minimize the steric interference between the support and substances interacting with the
9 ligand, hydrocarbon spacer arms (such as in Sepharose CL-4B) are often interposed between
10 the substrate and the ligand. However, these spacer arms may cause hydrophobic effects.
11 In affinity chromatography, the adsorption step is often performed with buffers of low ionic
12 strength and the interference of non-selective electrostatic adsorption is inevitable. In
13 addition, non-selective desorption of the desired molecule, increasing the pH or the ionic
14 strength leads to at least a partial loss of activity, many times reversible but not always.
15 Special attention should be given to various affinity supports used under conditions of
16 saturation of the protein to be applied to matrix; possible contaminations can result from
17 non-selective interactions.
18 In immunoaffinity chromatography the disadvantages include the technical difficulty of
19 producing monoclonal antibodies and the drastic conditions that are often required to elute
20 the strongly adsorbed protein.

21 10. Acknowledgments
22 The authors express their gratitude to the Conselho Nacional de Desenvolvimento Científico e
23 Tecnológico (CNPq) for research grants and fellowships (LCBBC, MTSC and PMGP). We are
24 also grateful to the Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco
25 (FACEPE) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for
26 financial support. It is also acknowledged the Portuguese Fundação para Ciência e a Tecnologia
27 (FCT) through the Post-doctoral grant SFRH/BPD/37349/2007 (AFSS).

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