Bioconjugate Chem. 2005, 16, 1310−1315
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TECHNICAL NOTES Free Nonimmobilized Ligands as a Tool for Purification of Proteins Guy Patchornik†,* and Amnon Albeck§ Affisink Biotechnology Ltd., 11 Hamaccabee St. Kiryat-Ono 55572, Israel, and The Julius Spokojny Bioorganic Chemistry Laboratory, Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel. Received March 28, 2005; Revised Manuscript Received July 11, 2005
Purification of proteins on a large scale is a complex multistep process, and alternative economic strategies are required. This study presents a novel approach (Affinity Sinking, AS) for purification of native proteins utilizing nonimmobilized modified ligands. The nonimmobilized state of the ligand circumvents the need for immobilizing ligands to polymeric supports. Therefore, purification from large volumes can be accomplished without the use of industrial-scale affinity columns. The mechanism of product capture is formation and precipitation of a specific [target-protein/modified-ligand] complex by using a soluble interconnecting entity that generates an insoluble [target-protein/modified-ligand/ interconnecting entity] sediment containing the target protein. Rabbit IgG and two glycoproteins were purified accordingly, utilizing free avidin (as the interconnecting entity) and either desthiobiotinylatedprotein A (DB-ProA) or desthiobiotinylated-concanavalin A (DB-ConA) as the modified ligand. The recovery yields for the IgG and the two glycoproteins were 80-86% and 70-75%, respectively. Target proteins are eluted from the generated pellet nearly without disrupting the [modified-ligand/ interconnecting entity] macro-complex, thus enabling a practical procedure of recovering target proteins. Leaching of the DB-ProA ligand under eluting conditions (pH 3) was found to be lower than 1%. The two modified ligands, DB-ProA and DB-ConA, were regenerated without any chromatographic procedure in 80% and 85%-89% yield, respectively. The advantage of excluding the polymeric component from the purification process and obtaining highly purified proteins has been demonstrated, and it implies that other contaminants (e.g. endotoxins, prions, host DNA) could be excluded as well, thereby reducing the number of purification steps in a typical downstream process.
INTRODUCTION
There is a continuing search for alternative purification strategies capable of capturing minute amounts of target proteins from complex cell culture supernatants, plant extracts, or milk of transgenic animals containing excessive amounts of impurities (e.g. immunogenes, pyrogens, DNA, and perhaps endotoxins) (1, 2). The scope of purification techniques is limited by the inherent tendency of proteins to denature in the presence of organic solvents, high temperature, or extreme pH. Therefore, low concentration of the target proteins, their fragility, and the need to cope with large amounts of complex media may have led to the fact that the least well engineered part in bioproduction processes are the purification steps (3), typically resulting in 50-80% of the total production cost (4, 5). The largest purification factor within the multistep purification process is achieved by affinity chromatography (AC),1 exploiting the high specific binding interaction between a ligand immobilized to a polymeric support and a protein of interest (Figure 1, A) (6, 7). AC, perhaps the most widely used affinity technique at large-scale (8), has been used successfully in a number of biotech downstream processes, namely in the area of mammalian cell culture products (9). * Corresponding author. E-mail:
[email protected], Tel: 9728-9302575, Fax: 972-8-9302565. † Affisink Biotechnology Ltd. § Bar-Ilan University.
Like any purification approach, AC suffers from several limitations at industrial scale. These include (a) careful control over column loading which generally results in formation of large volumes of dilute eluent, requiring concentration before further processing; (b) high operation and maintenance costs due to the high pressure involved and the special requirement for affinity media; (c) column fouling which prevents the use of AC during early product isolation when process streams are too crude; (d) capacity and flow rate limitations with an upper limit of 2-10 kg/day productivity for each production line; (e) limited adsorbent lifetime due to the growth of microorganisms that frequently either lead to breakdown of the column matrix or contaminate the product with unknown bacteria and their byproducts, or may even result in possible digestion of the product by the bacterial extracellular proteases; (f) implementation of harsh sanitizing protocols raise the concern of ligand degradation that may contaminate the product and decrease the capacity of the column. Therefore, a wide array of alternative affinity purification approaches has 1Abbreviations: AC, affinity chromatography; AML, affinity macroligand; AP, Affinity precipitation; AS, affinity sinking; ConA, concanavalin A; DB, desthiobiotin; DB-ConA, desthiobiotinylated concanavalin A; DB-ProA, desthiobiotinylated protein A; EDTA, ethylenediaminetetraacetic acid; HPLC, high-pressure liquid chromatography; PAGE, poly acrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.
10.1021/bc050096s CCC: $30.25 © 2005 American Chemical Society Published on Web 08/27/2005
Technical Notes
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Figure 1. Comparison of the basic chemical architecture of AC, AP and AS. A. Ligands in AC are immobilized to insoluble polymeric matrixes. B. Ligands in AP are immobilized to water soluble polymers which would change reversibly to water insoluble upon a physiochemical change such as low pH. C. Ligands in AS are not immobilized but modified with a complexing entity enabling their precipitation upon addition of an appropriate mediator. Thus, no polymeric entity is present within the precipitation process and ligands are free in the medium.
been developed with the objective of meeting the unique difficulties addressed by large-scale purification (10-15). Affinity precipitation (AP) (secondary effect), first introduced by Schneider et al. (16), has been postulated to become the purification technique with the highest large-scale potential (4, 17-19). In AP, a water soluble polymer is conjugated with a specific ligand, generating an affinity macroligand (AML) (Figure 1, B). The AML forms a complex with the target molecule, which precipitates upon a critical physiochemical change in pH, temperature, or ionic strength. The target protein could then either be eluted directly from the [AML-target protein] complex or from the dissolved state of the complex under conditions which decrease the binding affinity between the AML and the target protein. The AML itself (without the target protein) is then precipitated again, leaving the target protein in the supernatant (20). The unique combination of specificity and precipitation features being utilized in all AP approaches has not yet led to an established downstream processing technique due to several difficulties: (a) steric hindrances induced by high molecular weight AML, limiting the access of the target molecule; (b) resolubilization of precipitated polymers was slow; (c) nonspecific adsorption and entrapment of impurities during precipitation was almost unavoidable, requiring up to three successive precipitation-resolubilization steps (1, 19, 21-23). This state of affairs prompted us to develop the present specific precipitation approach, affinity sinking (AS), lacking any polymeric component and with ligands that are free in solution. Specific precipitation is obtained within two successive steps: first, the nonimmobilized modified ligand and the target protein are incubated to allow specific binding. This generates a [modified ligandtarget protein] soluble complex. The latter complex is specifically precipitated by addition of an appropriate interconnecting entity (the mediator) such as free avidin that presumably forms macro networks with lower solubility properties (Figure 1, C). The approach was
performed with two distinct ligands: (a) Protein A: a 42 kDa factor produced by Staphylococcus aureus, which specifically binds the constant region of a wide range of antibodies from various classes and therefore is extensively utilized for purification of immunoglobulins (24). (b) Concanavalin A: a lectin that exhibits distinct sugar specificities toward R-D-mannopyranosides, R-D-glucopyranosides, or R-N-acetyl-D-glucosaminides (25) and specifically binds glycoproteins. Both ligands were modified with desthiobiotin N-hydroxysuccinimidyl ester (desthiobiotin-NHS) and used for purification of rabbit IgG, porcine thyroglobulin, and glucose oxidase. EXPERIMENTAL PROCEDURES
Materials. For SDS-PAGE, Precision plus protein standards and Gel Filtration Chromatography Standards were from Bio-Rad. All other reagents were of analytical grade. Tris (ultrapure) was from Bio-Lab, Jerusalem. Avidin, biotin, methyl R-D-glucopyranoside, concanavalin A Type IV, and EDTA were from Sigma. Porcine thyroglobulin and glucose oxidase were from GALAB Technologies GmbH, Germany. Desthiobiotin N-hydroxysuccinimidyl ester was from Berry & Associates, Dexter, MI. Recombinant protein A was from BioVision, Inc. (Palo Alto, CA). Rabbit IgG was from Sigma, Glucose oxidase kit was from Megazyme, Ireland. Protein Analysis. Protein content was estimated by the method of Bradford (26), using bovine serum albumin as a standard. Electrophoresis and Densitometry. Samples were loaded onto a 10% bis-Tris SDS-poly-acrylamide gel (1 mm thickness) according to Laemmli (27) and developed for 1 h at constant 120 V. All gels were stained with Coomassie Brilliant Blue R250, and the intensity of bands was measured by densitometry using the Scion Image program. These measurements were used for quantification of the recovery yields of the target proteins and their purity.
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Preparation of Desthiobiotinylated Protein A (DB-ProA) and Desthiobiotinylated Concanavalin A (DB-ConA). Recombinant protein A or concanavalin A (ConA) were modified with desthiobiotin N-hydroxysuccinimidyl ester according to Bayer et al. (28), with a molar ratio of 4.4:1 between the desthiobiotin reagent and ConA, while protein A was modified accordingly with a molar ratio of 20:1. Precipitation and Elution of Rabbit IgG. Precipitation was carried out at 4 °C in a medium containing: 50 mM sodium phosphate at pH 8, 0.23 mg/mL of DB-Pro A, 0.6 mg/mL rabbit IgG, and cell lysate in a total volume of 50 µL. A freshly prepared avidin solution (1.5 mg/mL final concentration) was added, and a precipitate was formed. This was followed by a short spin at 14000 rpm and removal of the supernatant. The pellet was resuspended once with 200 µL of 50 mM sodium phosphate buffer pH 8 and the supernatant discarded. To elute rabbit IgG, the pellet was further resuspended with 0.1 M sodium citrate pH 3, 0.9 M urea at 4°C for 10 min in a total volume of 50 µL with gentle agitation. After an additional spin, the supernatant was neutralized with 1 N NaOH and applied to the gel. Regeneration of DB-ProA. Recovery of DB-Pro A was achieved by incubating the pellets in 0.1 M sodium citrate pH 3 and 5 mM of biotin at 4 °C for 10 min. A spin at 14000 rpm was performed and the supernatant was neutralized with 1 N NaOH and applied to the gel. Precipitation of Porcine Thyroglobulin and Glucose Oxidase. Precipitations were carried out at 4 °C in a medium containing 20 mM Tris buffer pH 7.2, 3.3 mg/mL of DB-Con A, 2.2 mg/mL porcine thyroglobulin or 1.4 mg/mL glucose oxidase, and a mixture of native proteins (Biorad Gel Filtration Chromatography Standards), in a total volume of 30 µL. A freshly prepared avidin solution (3.3 mg/mL, final concentration) was then added, and a precipitate was formed. A short spin at 14 000 rpm allowed removal of the supernatant which contained the vast majority of the impurities, while the pellet was further washed by resuspending it once in 200 µL of 20 mM Tris buffer pH 7.2, and the supernatant discarded. Elution of Porcine Thyroglobulin and Glucose Oxidase from the Pellets. Washed pellets were incubated at 4 °C for 2-5 min in a medium containing 0.1 M NaHCO3 pH 7.8, 0.2 M methyl R-D-glucopyranoside (29), and 5 mM EDTA in a total volume of 30 µL with gentle agitation. After a short sedimentation at 14000 rpm, the supernatant of the pellet was applied to the gel. Regeneration of DB-Con A. Recovery of DB-ConA was identical to that described above for DB-ProA. Catalytic Activity of Glucose Oxidase. The catalytic activity of the purified glucose oxidase was studied with the glucose oxidase assay kit. Generation of E. coli Lysate. BL21 bacteria harboring a pACYCDuet vector (Novagen) were grown in 4.5 L of LB medium for 2 h at 37 °C. Protein expression was induced by the addition of 0.05 mM IPTG, and the bacteria continued to grow for an additional 4 h at 15 °C. The bacteria were lysed by freeze thawing in lysis buffer (Tris 50 mM pH 8, 0.1 M NaCl, 20 mM imidazole, protease inhibitor cocktail, lysozyme, and Dnase) followed by sonication. The bacterial lysate was then centrifuged by ultracentrifugation at 40000 rpm, and the supernatant was applied to a Ni column at 4 °C. (The lysate is the flow-through from this Ni capture).
Technical Notes
Figure 2. Purification of rabbit IgG from cell lysate, utilizing desthiobiotinylated protein A (DB-ProA) and free avidin. Lane 1 rabbit IgG; lane 2 DB-ProA; lane 3 bacterial cell lysate; lane 4 mixture of rabbit IgG, DB-ProA, and cell lysate; lane 5 Biorad prestained protein markers; lane 6 recovered IgG; lane 7 content of supernatant after specific precipitation of the IgG from the cell lysate. RESULTS
Specific Precipitation and Elution of Target Proteins. Rabbit IgG. To demonstrate the selectively of the approach, rabbit IgG was purified from bacterial cell lysate. Thus, a medium containing whole cell lysate, DB-ProA, and rabbit IgG was prepared (Figure 2, lane 4). Upon addition of avidin, a precipitate was generated and the resulting pellet was washed once with 200 µL of fresh buffer. The washed pellet was further incubated under eluting conditions (0.1 M sodium citrate at pH 3, 4 °C, for 15 min), and the supernatant of the resuspended pellet was applied to the gel after being neutralized to pH 7. The recovery yield of the IgG was 85% (Figure 2, lane 6). Comparing the total amount of impurities (Figure 2, lane 3) with the amount of impurities present in the supernatant after the addition of avidin (Figure 2, lane 7) indicates that 95% were excluded from the pellet, prior to the washing step. Since no DB-ProA was observed by Coomassie staining in the eluted IgG (lane 6), the degree of leached DB-ProA was assessed by silver staining to be below 1%. Porcine Thyroglobulin and Glucose Oxidase. Similar results, of target protein purification in high yield and high purity, were obtained for porcine thyroglobulin and glucose oxidase. In this case, the ligand was DB-ConA. The recovery yields of porcine thyroglobulin and glucose oxidase were 70-75% and the purity above 95% (by densitometry). Glucose oxidase and porcine thyroglobulin were not precipitated in the presence of native concanavalin A and avidin. The modified ligand (DB-ConA) was regenerated in 86%. The purification data of the three target proteins are summarized in Table 1. The Effect of Increased Background Contamination on the Purification Process. To study the effect of increased background contamination on the yield and purity of the purification process, identical amounts of rabbit IgG, avidin, and DB-ProA were added to increasing concentrations of either BSA (Figure 3, A) or cell lysate (Figure 3, B). All pellets were washed once with identical volumes of fresh buffer (200 µL) regardless of their contamination background and the IgG was eluted. The eluted IgG solutions exhibited similar purity and yield (Figure 3, A, lanes 2P-5P; B, lanes 3P-5P) whether BSA or the cell lysate served as the background contamination. Similar results were obtained with DB-ConA and the glycoproteins porcine thyroglobulin and glucose oxidase (data not shown).
Bioconjugate Chem., Vol. 16, No. 5, 2005 1313
Technical Notes
Figure 3. The effect of increased background contamination on the precipitation process. A. Lane 1 rabbit IgG; lanes 2-5 constant concentration of rabbit IgG and DB-ProA in the presence of increased BSA concentration; lane 6 Biorad prestained protein standards; lanes 2P-5P recovered IgG from pellets generated in lanes 2-5, respectively. B. Lane 1 rabbit IgG; lane 2 DB-ProA; lanes 3-5 constant concentration of rabbit IgG and DB-ProA in the presence of increased cell lysate concentration; lanes 3P-5P recovered IgG from pellets generated in lanes 3-5, respectively. Table 1. Recovery Yields and Purity of the Target Proteins and the Modified Ligands target protein
desthiobiotinylated ligand
recovery yield of target protein, %
purity of target protein, %
recovery yield of desthiobiotinylated ligand, %
rabbit IgG thyroglobulin glucose oxidase
protein A concanavalin A concanavalin A
80-86 70-75 70-75
97 95 95
80 85-89 85-89
Catalytic Activity of Recovered Glucose Oxidase. The purified glucose oxidase was assayed for its catalytic activity and was found to be 98 ( 3% active. This result emphasizes the mildness and lack of denaturation by the process. DISCUSSION
It has been argued that the ideal strategy for largescale purification of proteins would utilize a highly specific precipitation process rather than a chromatographic methodology (4, 19). Therefore, we aimed at developing a simple precipitation approach (which we call affinity sinking, AS) that would lead to highly purified proteins while eliminating the need for sophisticated instrumentation (e.g. HPLC). To the best of our knowledge, all known chromatographic and precipitation techniques require ligands covalently bound to various polymeric supports, while AS uses ligands at their free nonimmobilized state. The use of free ligands would circumvent the need for immobilizing ligands to polymers and would exclude polymers from the purification process. Figure 1 illustrates the differences in chemical architecture of well-established approaches (e.g. affinity chromatography, affinity precipitation) with that of AS. In AS, precipitation of the target protein requires two water soluble entities: a modified ligand and an interconnecting entity. The modified ligands used in this study were desthiobiotinylated protein A (DB-ProA) and desthiobiotinylated concanavalin A (DB-ConA). Incubation of the modified ligand with the target protein and addition of the interconnecting entity (free avidin) generated a precipitate, composed primarily of the [modified ligand-target protein-avidin] multicomplex (Figure 1, C). The target protein is then eluted from the generated sediment (i.e. pellet) under conditions that essentially do not dissociate the [modified ligand-avidin] multicomplex. Since antibody purification is a major scientific and industrial need, we tested the ability of the approach to specifically capture and purify rabbit IgG from cell lysate, utilizing DB-ProA as the ligand (Figures 2 and 3). The
high purity (97%) and yield (80-86%) of the recovered IgG demonstrates the feasibility of the approach. The majority of impurities are excluded from the pellet already in the precipitation step (Figure 2, lane 7), prior to the washing step. This emphasizes the advantage of the present purification approach, which lack any polymeric matrix onto which impurities would probably have been adsorbed nonspecifically. Similar precipitation and recovery behavior was observed with a desthiobiotinylated concanavalin A derivative (DB-ConA), used for the capture of glucose oxidase and porcine thyroglobulin (Table 1). These consistent results with two distinct ligands indicate that other ligands may be similarly utilized and lead to highly purified proteins with good recovery yields. Native protein A or concanavalin A lacking bound desthiobiotin (DB) did not lead to precipitation of the target proteins (data not shown). The use of nonimmobilized ligands may raise the concern of ligand leaching. Nevertheless, leaching was not observed by Coomassie staining (Figure 2 lane 6; Figure 3, A, lanes 2P-5P, B, lanes 3P-5P). Therefore, gels were visualized by silver staining and the degree of leached DB-ProA was found to be less than 1% (data not shown). Since these values were obtained under highly acidic conditions (pH 3), one would expect lower levels of leaching under milder eluting conditions. These observations suggest that target proteins can be eluted directly from the generated precipitates, while keeping the [modified ligand-avidin] multicomplex intact in the precipitate. This feature may be advantageous for largescale protein purification, where obtaining a relatively pure protein in high concentration by direct elution of the target protein from the pellet is a major advantage (2). Furthermore, since all ligands according to AS are modified with a complexing entity (e.g. desthiobiotin, metal chelator), removal of minute amounts (
