Purification of recombinant C-reactive protein mutants

Abstract

C-reactive protein (CRP) is an evolutionarily conserved protein, a component of the innate immune system, and an acute phase protein in humans. In addition to its raised level in blood in inflammatory states, CRP is also localized at sites of inflammation including atherosclerotic lesions, arthritic joints and amyloid plaque deposits. Results of in vivo experiments in animal models of inflammatory diseases indicate that CRP is an anti-pneumococcal, anti-atherosclerotic, anti-arthritic and an anti-amyloidogenic molecule. The mechanisms through which CRP functions in inflammatory diseases are not fully defined; however, the ligand recognition function of CRP in its native and non-native pentameric structural conformations and the complement-activating ability of ligand-complexed CRP have been suggested to play a role. One tool to understand the structure-function relationships of CRP and determine the contributions of the recognition and effector functions of CRP in host defense is to employ site-directed mutagenesis to create mutants for experimentation. For example, CRP mutants incapable of binding to phosphocholine are generated to investigate the importance of the phosphocholine-binding property of CRP in mediating host defense. Recombinant CRP mutants can be expressed in mammalian cells and, if expressed, can be purified from the cell culture media. While the methods to purify wild-type CRP are well established, different purification strategies are needed to purify various mutant forms of CRP if the mutant does not bind to either calcium or phosphocholine. In this article, we report the methods used to purify pentameric recombinant wild-type and mutant CRP expressed in and secreted by mammalian cells.

1. Introduction

C-reactive protein (CRP), which is a component of the acute phase response in humans, participates in innate immunity against pneumococcal infection (Agrawal et al., 2008; Kushner, 1982; Simons et al., 2014; Volanakis, 2001). In humans, the concentration of CRP in circulation increases rapidly within hours of an inflammatory stimulus due to its increased hepatic synthesis under transcriptional and post-transcriptional control (Bode et al., 2012; Kim et al., 2015; Singh et al., 2007; Voleti and Agrawal, 2005; Voleti et al., 2012; Zhang et al., 1995). CRP has also been shown to be deposited at localized sites of inflammation such as at atherosclerotic lesions, in the joints of arthritic patients, and surrounding the amyloid plaque deposits in patients with Alzheimer’s disease (Gitlin et al., 1977; Hatanaka et al., 1995; Iwamoto et al., 1994). However, the functions of circulating and locally deposited CRP in chronic and acute inflammatory diseases are not known. CRP has not been found to be pro-inflammatory in healthy human volunteers (Lane et al., 2014). Also, there are no reports of either deficiency of CRP or mutations in the CRP gene in any individual, suggesting that CRP plays a critical role in host defense and that its functions are not confined to any particular inflammatory disease.

Human CRP, a cyclic pentamer of five identical monomers, has two recognition functions in vitro (Agrawal et al., 2014). In its native pentameric structural conformation, CRP binds to phosphocholine (PCh) groups present on the surface of damaged cells and microbes in a Ca²⁺-dependent interaction (Volanakis and Kaplan, 1971). CRP, in a Ca²⁺-dependent interaction, also binds to phosphoethanolamine (PEt) (Agrawal et al., 2002; Mikolajek et al., 2011). In its alternate pentameric structural conformation, CRP binds to deposited, aggregated and immobilized proteins including amyloid β peptide, oxidized low-density lipoprotein and complement regulator factor H, in a Ca²⁺-independent manner (Agrawal, 2013; Agrawal et al., 2014; Hakobyan et al., 2008; Hammond et al., 2010; Singh et al., 2012; Suresh et al., 2004). The significance of the recognition function of CRP in its alternate structural pentameric conformation in host defense is unknown. However, ex vivo and in vivo experiments in animal models of inflammatory diseases indicate that CRP exerts, although limited, anti-pneumococcal, anti-atherosclerotic, anti-arthritic and anti-amyloidogenic functions (Agrawal, 2005; Agrawal et al., 2008, 2010; Chang et al., 2012; Gang et al., 2012, 2015; Jiang et al., 2006; Jones et al., 2011; Kovacs et al., 2007; Nayeri et al., 2010; Ngwa et al., 2016; Ozawa et al., 2016; Simons et al., 2014; Singh et al., 2008; Suresh et al., 2006, 2007; Szalai et al., 1995; Yother et al., 1982). CRP has also been shown to alleviate experimental autoimmune encephalomyelitis (Zhang et al., 2015). The ligand recognition function of CRP in its native and non-native pentameric structural conformations and the complement-activating ability of ligand-complexed CRP have been suggested to play a role in its host defense functions (Agrawal, 2005; Agrawal et al., 2008, 2014; Jarva et al., 1999).

One tool to understand the structure-function relationships of CRP and determine the contributions of the recognition and effector functions of CRP in host defense is to use site-directed mutagenesis to create mutants for use in in vitro and in vivo experiments. For example, CRP mutants incapable of binding to PCh are generated to investigate the importance of the PCh-binding property of CRP in pneumococcal infection (Agrawal et al., 2002; Gang et al., 2012, 2015; Suresh et al., 2006, 2007). Recombinant CRP mutants can be expressed in mammalian cells and, if expressed, can be purified from the cell culture media. However, while the methods to purify wild-type (WT) CRP are well established (Agrawal et al., 2001, 2002; Gang et al., 2015; Macintyre, 1988; Nunomura et al., 1990; Pepys et al., 1977, 2012; Riley and Coleman, 1970; Volanakis et al., 1978), different purification strategies are needed to purify various mutant forms of CRP if the mutant does not bind to either Ca²⁺ or PCh. Although several expression systems have been employed to express recombinant CRP, including Leishmania tarentolae, Escherichia coli, Pichia pastoris and baculovirus-infected cell lines and Trichoplusia ni larvae (Dortay et al., 2011; Kilpatrick et al., 2012; Marnell et al., 1995; Potempa et al., 2015; Tanaka et al., 2002), in this article, we report the methods used to purify pentameric recombinant WT and mutant CRP expressed in and secreted by mammalian cells.

2. Purification Methods

Native and recombinant WT CRP can be purified from body fluids and cell culture media of transfected mammalian cells, respectively. Recombinant CRP mutants can be expressed in either COS or CHO cells and are purified from the culture supernatants. The methods to purify recombinant WT CRP are the same that are used to purify native WT CRP (Agrawal et al., 2001; Macintyre, 1988; Nunomura et al., 1990; Pepys et al., 1977, 2012; Riley and Coleman, 1970; Volanakis et al., 1978). Similar to the methods used to purify WT CRP, three different chromatographic steps are used to purify recombinant CRP mutants: affinity chromatography, anion exchange chromatography and gel filtration chromatography, as described below.

3. Affinity Chromatography

Depending upon the nature of the mutation introduced in CRP, either ligand-affinity chromatography or immunoaffinity chromatography is used to purify mutant CRP. Again, depending upon the nature of the mutation introduced in CRP, one of the two ligand-affinity chromatography methods, PCh-affinity chromatography and PEt-affinity chromatography, is used to purify mutant CRP. Ligand-affinity chromatography involves Ca²⁺-dependent binding of CRP to PCh or PEt and EDTA-mediated elution. A fixed molarity of EDTA is used to elute bound proteins from the affinity column and generate the chromatogram. CRP is eluted from the affinity column as a single peak in the elution profile. Immunoaffinity chromatography involves acidic pH-mediated dissociation of CRP from anti-CRP antibody column. A fixed acidic pH is used to elute bound CRP from the affinity column and CRP is eluted as a single peak in the elution profile; the chromatogram shows only one peak. The three affinity chromatography methods are described below.

3.1. PCh-affinity Chromatography

PCh-affinity chromatography is used to purify WT CRP and those CRP mutants that retain their PCh-binding specificity. Commercially available PCh-conjugated Sepharose beads (Pierce) are used to prepare the column for chromatography according to manufacturer’s instructions. The capacity of these PCh-Sepharose beads is to bind more than 5 mg of CRP per ml of packed beads. The beads are packed into a column, and before using the column for chromatography, the column is equilibrated with 10 ml of 0.1 M borate-buffered saline (BBS, pH 8.3) containing 3 mM CaCl₂ (BBS-Ca) per ml packed beads.

The cell culture medium containing CRP is first centrifuged at 10,000 rpm for 30 min to remove floating dead cells if present. The supernatant is then diluted with an equal volume of BBS-Ca and passed through the column preferably at a slow flow rate (20 drops/min), and at 4°C if a large volume has to be passed. The flow-through from the column is collected and saved. The flow-through is tested by ELISA to ensure that there is no CRP left in the flow-through, and that the amount of packed beads in the column was sufficient for capturing all CRP present in the supernatant. The column is then washed with BBS-Ca with monitoring of absorbance at 280 nm (A₂₈₀), till there is no protein coming out in the wash buffer (A₂₈₀<0.02). Bound CRP is eluted from the column by using BBS containing 5 mM EDTA (BBS-EDTA). Depending upon the amount of CRP in the supernatant and the amount of beads in the column, the fraction volume of the eluate is decided. If the packed column volume is 1.0 ml, then bound CRP is eluted in the first 10 ml or less. The A₂₈₀ for each fraction is measured. If it is a small-scale purification such that CRP cannot be detected by measuring A₂₈₀ of the fractions, then ELISA is used to locate fractions containing CRP. The elution process is continued till there is no CRP being eluted (A₂₈₀<0.02). After all bound CRP is eluted, fractions containing CRP are pooled and dialyzed against the binding buffer (Mono Q buffer A; See anion exchange chromatography below) for 24 h at 4°C with 2 changes of buffer, to prepare for the next chromatographic step which is anion exchange chromatography on a Mono Q column. The affinity column can be washed with another 10 column volumes of BBS-EDTA followed by equilibration with 10 column volumes of BBS-Ca and stored at 4°C.

3.2. PEt-affinity Chromatography

PEt-affinity chromatography is used to purify those CRP mutants that have lost their PCh-binding activity but not their PEt-binding activity (Agrawal et al., 2002; Gang et al., 2012). For example, CRP mutants F66A/E81A and F66A/T76Y/E81A do not bind PCh, but retain their PEt-binding activity. The mutations may even result in increased binding of CRP to PEt compared to that of WT CRP. For example, CRP mutant F66A/T76Y/E81A has been shown to possess PEt-binding activity several fold higher than that of WT CRP (Gang et al., 2015). These CRP mutants can be purified using PEt-conjugated Sepharose beads.

PEt-conjugated Sepharose beads are prepared as follows (Gang et al., 2012). Twenty five ml of packed ECH-Sepharose 4B beads (GE Healthcare) are first washed with deionized water (pH 4.5) and then with 0.5 M NaCl. Then, 180 mg of PEt (catalog number P0503, Sigma-Aldrich) is dissolved in 25 ml of deionized water (pH 4.5) and added to the washed beads. Next, 500 mg of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (catalog number E6383, Sigma-Aldrich) is added to the slurry and the mixture is stirred for 1 h at room temperature, all the while ensuring that the pH is 4.5. After monitoring the pH for 1 h to ensure that the pH stayed at 4.5, the mixture is left overnight at 4°C with slow stirring. The beads are first washed with 0.1 M acetate buffer, pH 4.0, followed by washing with 100 mM Tris-HCl, pH 8.0, containing 0.5 M NaCl. The beads are then washed with the two buffers three additional times (4 x 25 ml washes for each buffer), by alternating between the acetate and Tris buffers. After the last wash with Tris buffer, the beads are washed two times, 25 ml for each wash, using de-ionized water. Finally, the beads are washed two times with 25 ml of 20 mM Tris-HCl buffer, pH 8.0, containing 150 mM NaCl and 2 mM CaCl₂. The beads are then packed into a column for chromatography.

Before use in chromatography, the column is equilibrated with 10 column volumes of BBS-Ca. Preparation of cell culture supernatant containing CRP for applying to the column and purification by using PEt-Sepharose column involves steps exactly as described above for chromatography using PCh-Sepharose column. After chromatography, CRP-containing fractions are processed for anion exchange chromatography exactly as described above for CRP eluted from the PCh-Sepharose column.

3.3. Immunoaffinity Chromatography

Immunoaffinity chromatography is used to purify those CRP mutants that bind to neither PCh nor PEt. For example, CRP mutant F66A/E81A, which does not bind PCh and binds poorly to PEt, can be purified by immunoaffinity chromatography employing affinity-purified polyclonal anti-CRP antibody-conjugated Sepharose beads. Polyclonal anti-CRP antibody is purified from rabbit anti-human CRP antiserum (Sigma-Aldrich) by a separate affinity chromatography using a CRP-conjugated Sepharose column. The conjugation of CRP or anti-CRP antibody to Sepharose beads can be performed using the AminoLink Immobilization kit (Pierce) according to manufacturer’s instructions (Agrawal et al., 2002; Suresh et al., 2007). The capacity of the anti-CRP-Sepharose beads to bind CRP per ml of packed beads is determined by using a known amount of purified WT CRP.

Anti-CRP-conjugated Sepharose beads are packed into a column. The cell culture supernatant is diluted 1:1 in 10 mM Tris-HCl buffer, pH 7.2, containing 150 mM NaCl (TBS) and is passed, once or twice, through the immunoaffinity column, as described above for ligand-affinity chromatography. The flow-through from the column is collected and saved. The flow-through is tested by ELISA to ensure that there is no CRP left in the flow-through, and that the amount of packed beads in the column was sufficient to capture all CRP present in the supernatant. The column is then washed with TBS till there is no protein coming out in the wash buffer (A₂₈₀<0.02). Bound CRP is eluted from the column by using 50 mM Glycine-HCl buffer, pH 3.0, and the pH of the fractions is immediately neutralized with 1 M Tris, pH 9.0. The volume of 1 M Tris, pH 9.0 needed to neutralize the pH 3.0 eluate can be predetermined by titration. The fractions can be collected in tubes containing the neutralizing volume of pH 9.0 buffer. The A₂₈₀ for each fraction is measured. If it is a small-scale purification such that CRP cannot be detected by measuring A₂₈₀ of the fractions, then ELISA is used to locate CRP-containing fractions. The elution process is continued till there is no CRP being eluted (A₂₈₀<0.02). After all bound CRP is eluted, CRP-fractions containing CRP are pooled and dialyzed against TBS, and then further processed for anion exchange chromatography exactly as described above for CRP eluted from the ligand-affinity columns. The affinity column can be washed with another 10 column volumes of TBS and stored at 4°C.

4. Anion Exchange Chromatography

Two buffers are required: 1. Binding buffer (buffer A) which is 20 mM Tris-HCl, pH 7.8, containing 150 mM NaCl and either 2 mM CaCl₂ or 0.1 mM EDTA, and 2. Elution buffer (buffer B) which is 20 mM Tris-HCl, pH 7.8, containing 1 M NaCl and either 2 mM CaCl₂ or 0.1 mM EDTA (Figure 1). The buffers are filtered and degassed. The MonoQ anion exchange column 5/50 GL (GE healthcare) is used for chromatography. The column is connected to a protein purification system, such as the Biologic Duo Flow Protein Purification System (Biorad).

Fig. 1.

Anion exchange chromatography of CRP. CRP-containing eluate from the affinity chromatography columns was applied to the MonoQ column. Bound CRP was eluted by using a linear 150–1000 mM NaCl gradient. Elution profiles were generated by the BioRad’s Biologic DuoFlow protein purification system. A typical chromatogram is shown. (A) Chromatography of WT CRP eluted from the PCh-affinity column, performed in buffers containing CaCl₂. (B) Chromatography of F66A/T76Y/E81A CRP mutant eluted from the PEt-affinity column, performed in buffers containing EDTA. (C) Chromatography of F66A/E81A CRP mutant eluted from the immunoaffinity column, performed in buffers containing EDTA.

The column is equilibrated with 10 ml of buffer A. CRP eluate from the affinity chromatography column is dialyzed against buffer A, filtered, degassed, and then applied to the MonoQ column. Alternatively, CRP eluate from the affinity chromatography column can be diluted 1:4 in buffer A and applied to the MonoQ column. The flow rate should be 0.5 ml/min. The flow through is collected and saved. The column is washed till A₂₈₀<0.02 (baseline absorbance). When washing is complete, an ionic strength gradient (NaCl gradient) using buffer A and B is applied to the column to elute bound CRP. As shown in Fig. 1A, WT CRP is eluted at 30% buffer B (300 mM NaCl, conductivity 160 mS). As shown in Figs. 1B and 1C, CRP mutants F66A/T76Y/E81A and F66A/E81A are eluted at 20% buffer B (200 mM NaCl, conductivity 120 mS). Fractions (1 ml) are collected and A₂₈₀ measured. A single peak appears in the chromatograms. CRP-containing fractions are pooled. After measuring the volume and A₂₈₀ of the pool, CRP eluate is dialyzed against TBS containing 2 mM CaCl₂ (TBS-Ca). A₂₈₀ is measured again and, assuming that there is no other protein present in the eluate except CRP, the concentration of CRP is determined by using the extinction coefficient of CRP which is 19.5 at A₂₈₀. CRP is concentrated to 5 mg/ml and either stored frozen or processed for the last step of purification using gel filtration chromatography.

5. Gel Filtration Chromatography

As shown in Fig. 1, a single peak is seen in the chromatogram after MonoQ anion exchange chromatography. If CRP is stored frozen after anion exchange chromatography, then, upon thawing, some CRP may dissociate into monomers. Even if CRP is not stored frozen and processed directly for further purification, the gel filtration chromatography ensures the purity of the CRP preparation. Before using CRP in experiments, it should be purified by gel filtration to remove any monomeric CRP or aggregates of monomeric CRP. Superose 12 (10/300 GL Pharmacia) column is used for gel filtration which is connected to the BioRad’s Biologic Duo Flow Protein Purification System. The column is equilibrated with 25 ml of filter-sterilized and degassed TBS-Ca or TBS containing 5 mM EDTA (TBS-EDTA), at a flow rate of 0.3 ml/min.

Concentrated CRP (5 mg/ml) obtained after MonoQ anion exchange chromatography is injected into the Superose12 column using a 400 μl loop; 250 μl of which enters into the column. CRP is eluted with 25 ml of either TBS-Ca (for WT CRP) or TBS-EDTA (for CRP mutants). Fractions are collected and A₂₈₀ measured to locate the elution volume of CRP from the column. A single peak corresponding to pentameric CRP is eluted at 13.0–13.5 ml (Fig. 2) of the elution buffer. CRP-containing fractions are pooled, and dialyzed against TBS-Ca if CRP (mutants) was eluted by using TBS-EDTA, and stored at 4°C. This final CRP preparation containing pentameric CRP only is now ready for use in in vitro and in vivo experiments. The purity of CRP is confirmed by using denaturing SDS-PAGE (Fig. 3).

Fig. 2.

Gel filtration chromatography of CRP. CRP-containing eluate from the MonoQ column was applied to the Superose12 column. Chromatography of WT CRP was performed in TBS-Ca. Chromatography of CRP mutants was performed in TBS-EDTA. Elution profiles were generated by the BioRad’s Biologic DuoFlow protein purification system. A typical chromatogram is shown. (A) Chromatography of WT CRP eluted from the MonoQ column. (B) Chromatography of F66A/T76Y/E81A CRP mutant eluted from the MonoQ column. (C) Chromatography of F66A/E81A CRP mutant eluted from the MonoQ column.

Fig. 3.

Gel electrophoresis of CRP. CRP (10 μg) preparations were analyzed by SDS-PAGE, under reducing and denaturing conditions. A representative gel (4–20%) stained with Coomassie brilliant blue is shown. Lane 1, molecular weight markers; Lane 2, WT CRP after PCh-affinity chromatography; Lane 3, WT CRP after MonoQ anion exchange chromatography; Lane 4, WT CRP after gel filtration; Lane 5, F66Y/T76Y/E81A CRP mutant after PEt-affinity chromatography; Lane 6, F66Y/T76Y/E81A CRP mutant after MonoQ anion exchange chromatography; Lane 7, F66Y/T76Y/E81A CRP mutant after gel filtration; Lane 8, F66Y/E81A CRP mutant after immunoaffinity chromatography; Lane 9, F66Y/E81A CRP mutant after MonoQ anion exchange chromatography; Lane 10, F66Y/E81A CRP mutant after gel filtration.

6. Discussion

Storage of purified CRP in the absence of Ca²⁺ causes spontaneous dissociation of pentameric CRP into monomers (Potempa et al., 2015; Singh et al., 2009; Zouki et al., 2001). We have reported monomerization of pentameric CRP upon prolonged storage even in the presence of Ca²⁺ (Singh et al., 2009). The functions of monomeric CRP are different than that of pentameric CRP; monomeric CRP is pro-inflammatory while pentameric CRP is not (Agrawal et al., 2014; Eisenhardt et al., 2009; Lane et al., 2014; Mihlan et al., 2011; Potempa et al., 1987; Thiele et al., 2014; Zouki et al., 2001). Therefore, on the day of the experiments, CRP is re-purified by gel filtration to remove any form of modified CRP generated due to storage of CRP. Freshly purified CRP is stored in TBS-Ca at 4°C and can be used for a week. For these reasons, it is advisable that CRP purified after anion exchange chromatography is stored frozen without the gel filtration step. Gel filtration can be performed on the day when CRP is needed for use in experiments. Use of freshly purified CRP eliminates the possibility of artefactual results generated due to low concentration of CRP monomers in preparations of pentameric CRP. If in vivo experiments are to be performed, endotoxin removal and testing can be performed before freezing CRP after anion exchange chromatography, so that on the day of the experiment, CRP can quickly be further purified by gel filtration, the level of endotoxin checked again, and used in experiments. To remove endotoxin, CRP is treated with the Detoxi-Gel Endotoxin Removing Gel (Thermo Scientific) according to manufacturer’s instructions. The concentration of endotoxin in CRP is determined by using the Limulus Amebocyte Lysate kit QCL-1000 (Lonza) according to manufacturer’s instructions.

PEt-affinity chromatography and immunoaffinity chromatography techniques can also be used to purify WT CRP (Agrawal et al., 2002; Nunomura et al., 1990; Pepys et al., 1977). It is critical that the acidic pH of CRP eluate from immunoaffinity column be neutralized quickly using a basic pH buffer. As shown in Fig. 3 (lane 8), in which CRP eluted from the immunoaffinity column was analyzed after neutralizing the pH, the CRP band was not as strong as in other lanes, suggesting that the pH of CRP was not neutral; it was basic after addition of the basic pH, and therefore CRP did not enter the gel. However, as shown in Fig. 3 (lanes 9, 10), if the pH of CRP is basic after attempting to neutralize the acidic pH, the basic pH is preferred over acidic pH because CRP was not found denatured at basic pH and CRP could be recovered after anion exchange chromatography when the pH was close to neutral.

As shown in Figs. 1B and 1C, the elution conditions for CRP mutants from the anion exchange column were different from that of WT CRP, suggesting that the mutations had affected the overall charge on the protein. However, as shown in Fig. 2, CRP mutants were pentameric and the elution volumes of WT and mutant CRP from the gel filtration column were similar. For some CRP mutants it may be necessary to perform anion exchange chromatography and gel filtration in TBS-EDTA, instead of in TBS-Ca, because mutant CRP may react with the beads in the column. CRP should immediately be subjected to dialysis against TBS-Ca if the chromatography was performed in TBS-EDTA.

As shown in Fig. 3 (lanes 2, 5, 8), recombinant CRP was found to be pure after the first affinity chromatography step and could be used in the experiments as is. Subjecting CRP to two additional chromatographic steps provides data on the effects of the mutations on the overall charge on CRP and ensures that pentameric CRP is devoid of any monomers of CRP. However, if the goal is to obtain pure pentameric CRP devoid of monomeric CRP, the anion exchange chromatography step can be skipped. There are no differences in any function of CRP purified either by using three chromatography or two chromatography steps. If WT CRP is purified from body fluids, then a contaminant is always seen after PCh-affinity chromatography, and in this case, all three chromatographic steps are required to obtain pure CRP (Agrawal et al., 2002; Suresh et al., 2004).

CRP is an evolutionarily conserved protein. The methods reported in this paper to purify CRP apply only to human pentameric CRP. The methods to purify monomeric CRP and CRP from animals, such as fish and Limulus, may require different or additional steps for their purification (Armstrong, 2015; Elvitigala et al., 2015; Huong Giang et al., 2010; Li et al., 2013; Mattecka et al., 2013; Mihlan et al., 2011; Pathak et al., 2016; Pepys et al., 1982; Potempa et al., 2015; Singh et al., 2009; Suresh et al., 2004).

Highlights.

• CRP is an anti-pneumococcal, anti-atherosclerotic, anti-arthritic and anti-amyloidogenic molecule.

• The ligand recognition functions of CRP play a role in host defense.

• CRP mutants are used to understand the structure-function relationships of CRP.

• Different purification strategies are needed to purify CRP mutants.

Abbreviations

CRP

C-reactive protein

PCh

phosphocholine

PnC

pneumococcal C-polysaccharide

BBS

0.1 M borate buffer, pH 8.3, containing 150 mM NaCl

BBS-Ca

BBS containing 3 mM CaCl₂

BBS-EDTA

BBS containing 5 mM EDTA

TBS

10 mM Tris-HCl buffer, pH 7.2, containing 150 mM NaCl

TBS-Ca

TBS containing 2 mM CaCl₂

TBS-EDTA

TBS containing 5 mM EDTA

WT

wild-type