Protein A vs. thiophilic resin

Historically, Protein A has been the preferred method of immunoglobulin purification. However, there are certain types of antibodies, such as the single-chain antibodies IgE, IgY, and IgM, that cannot be purified using Protein A. Thiophilic adsorption chromatography is ideal for these types of applications, as well as immunoglobulin purification in general. In addition to providing broader selectivity and higher stability, thiophilic adsorption chromatography offers a faster protocol than Protein A under neutral buffer conditions (Figure 1). Purification with thiophilic Resin allows elution in any buffer with a pH of 7.0. In contrast, Protein A requires elution in a buffer with a pH lower than 7.0. Protein A also requires a dialysis step, which can be omitted with thiophilic resin.

Figure 1. Thiophilic resin purifies antibodies at neutral pH and in less time than Protein A.

Thiophilic ligand allows salt-dependent purification of a wide range of antibody types

Thiophilic adsorption chromatography (TAC) was developed by Porath, et al., in 1984. TAC is a group-specific, salt-dependent purification technique with distinct adsorption affinity towards immunoglobulins and alpha-2-macroglobulins. The term "thiophilic" refers to the affinity that proteins have for sulfone groups that lie in close proximity to thioether groups (Figure 2; Porath 1985). With this technique, protein adsorbs to a sulfone thioether ligand.

The adsorption of different proteins can be promoted by adding different salts to the mixture. Varying the concentration of the loading salt can affect the adsorption affinities of IgG, IgM, IgA, Fab and Fc fragments, and C3 and C4 complement factors. Several sulfate salts can be used to promote the adsorption of target proteins. The most commonly used salts are potassium sulfate, sodium sulfate, and ammonium sulfate. In addition, salt concentration can differentially affect the adsorption kinetics of IgG, IgM, IgA, Fab and Fc fragments, and complement factors C3 and C4 (Lutomski et al. 1995; Oscarsson et al. 1992; Schulze et al. 1994; Yurov et al. 1994).

TAC is an economical technique for purifying immunoglobulins from whole serum and tissue cultures (Porath & Belew 1987; Scoble & Scopes 1997). In comparison to Protein A-based immunoadsorbents, thiophilic adsorbents have broader affinity towards immunoglobulins (Hutchens & Porath 1986). Furthermore, >99% of total proteins are recovered using a thiophilic adsorbent in comparison to less than 92% for phenyl and 75% for octyl agarose adsorbents (Oscarsson et al. 1995).

Purification of single-chain antibodies

Recombinant, single-chain antibodies are becoming increasingly common in research use because they can be genetically manipulated to bind different proteins and to perform specific, desired functions. However, standard antibody purification methods such as Protein A and Protein G do not work well for single-chain antibodies because these antibodies lack the Fc domain that natural antibodies possess. Protein A usually binds to this Fc domain. Because thiophilic resin binds to a region other than the Fc domain on single chain antibodies, it is able to purify them (Figure 3).

Figure 2. Structure of thiophilic resin.

 

Figure 3. Single-chain antibody purification with thiophilic resin. SDS-PAGE analysis of the following samples: bacterial lysate expressing scFv215 (Lane 2), periplasmic fraction (Lane 3), peak fraction from Ni-NTA (Lane 4) and peak fraction from thiophilic resin (Lane 5). (Shultze, et al. 1994; permission to reprint obtained).

Purification of IgY

Generating antibodies in chickens rather than rabbits is becoming a common method of immunoglobulin production. Antibodies produced in immunized chickens, which are transferred to the egg yolk, can be collected daily from eggs rather than animal serum. Also, chicken egg yolk provides higher amounts of a specific immunoglobulin than rabbit serum (Hansen et al. 1998). Standard immunoglobulin purification methods do not work well for IgY because it does not adsorb to Protein A. In contrast, IgY does adsorb to thiophilic resin (Figure 4), which is ideal for this type of purification because it provides a fast, simple, and inexpensive way to obtain large amounts of purified IgY.

Figure 4. Purification of IgY from chicken egg using thiophilic resin. Panel A. SDS-PAGE analysis of fractions from purification of chicken egg immunoglobulins. Lanes 7 & 10 each contain 10 mg of protein, and all other lanes, 25 mg each. Lane 1: Egg yolk extract supernatant. Lane 2: Supernatant after 60% SAS (Saturated concentration of Ammonium Sulfate). Lane 3: Wash with 60% SAS. Lane 4: Pellet after 60% SAS. Lane 5: Column load. Lane 6: Unbound material. Lane 7: Eluted material. Lanes 8–10: Purification with another thiophilic resin. Panel B. Immunoblot using 1/10 the amount of material in Panel A. IgY was detected with polyclonal rabbit anti-chicken HRP-conjugate. M = molecular weight. (Hansen et al. 1998; permission to reprint obtained).

Flexibility: suitable for batch, gravity-flow, or FPLC applications

Thiophilic-Superflow Resin (Table I) can be used for batch and gravity-flow purification, as well as FPLC. This resin is prepared using Superflow 6 agarose crosslinked beads, which permit linear flow rates as high as 5 cm/min. The agarose beads were activated with divinylsulfone, and mercaptoethanol was coupled to the activated resin. This resin can be regenerated and reused without detrimental effects on specificity and capacity.

Figure 5. Thiophilic-Superflow Resin purifies IgG at a high flow rate and neutral pH. Whole rabbit serum in 0.5 M sodium sulfate was purified on a Thiophilic-Superflow Resin column and eluted with 0.05 M sodium sulfate (Peak II).

When a Thiophilic-Superflow Resin column was used to purify whole rabbit serum, albumin was effectively removed in the nonadsorbed fraction, allowing the elution of highly purified intact IgG (Figures 5 & 6). Thiophilic adsorbents can also purify other types of proteins such as horseradish peroxidase (Chaga et al. 1992), allergens (Goubran-Botros et al. 1998), glutathione peroxidase (Huang et al. 1994), procollagen (Pedersen & Bonde 1994), acetolactate synthase (Poulsen & Stougaard 1989), insect hemolymph proteins (Samaraweera et al. 1992), serpins (Rosenkrands et al. 1994), lactate dehydrogenase (Kminkova & Kucera 1998), and tuberculosis antigen proteins (Rosenkrands et al. 1998).

Figure 6. Analysis of purified IgG fractions. Analytical size exclusion chromatography was performed on the purified fractions from Figure 5. Results indicate that the albumin, which constitutes 60–70% of the whole serum, is removed in the nonadsorbed fraction from whole rabbit serum (Panel A) and wash (Panel B). Then, the intact IgG from Peak II is eluted (Panel C).

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