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Purification and very strong reversible immobilization of large proteins on anionic exchangers by controlling the support and the immobilization conditions
Enzyme and Microbial Technology 39 (2006) 909–915
Purification and very strong reversible immobilization of large proteins on anionic exchangers by controlling the support and the immobilization conditions Benevides C.C. Pessela a , Manuel Fuentes a , Cesar Mateo a , Roberto Munilla a , Alfonso V. Carrascosa b , Roberto Fernandez-Lafuente a,∗ , Jose M. Guisan a,∗ a
Departamento de Biocat´alisis, Instituto de Cat´alisis CSIC, Campus Universidad Aut´onoma, Cantoblanco, 28049 Madrid, Spain b Department of Microbiology, Instituto de Fermentaciones Industriales (CSIC), Spain Received 13 January 2006; accepted 18 January 2006
Abstract The interaction of two large beta-galactosidases (from Escherichia coli and from Thermus sp.) with tailor-made anion exchangers was studied. Using lowly activated supports (e.g., containing 2–3 mol of ionised groups per wet gram of support), large proteins selectively adsorbed and easily desorbed (e.g., using 200 mM of NaCl), giving highly purified proteins. However, these supports cannot be used to immobilize the enzymes for industrial use, because the weak adsorption. On the other hand, these large proteins strongly adsorb on very highly activated supports (e.g., containing 40 mol of ionic groups per wet gram of 4 BCL agarose). Thus, these supports may be not valid for large protein purification, but may be very suitable for immobilization of these proteins. Using high ionic strength (e.g., 300 mM NaCl), large proteins still may be adsorbed on these supports, while only around 20% of total proteins adsorb, permitting some purification of the large proteins but not a total one. Moreover, adsorption under these conditions increase the adsorption strength (now there are not desorption even using 800 mM NaCl). Thus, the purification and the strong reversible immobilization of both beta-galactosidases were performed in a very simple two-step process. The large proteins can be directly adsorbed on these supports after desorption (at 200 mM of NaCl) from poorly activated supports. Furthermore, adsorption on very highly activated supports promotes a significant thermal stabilization of both enzymes, mainly in dissociations conditions. © 2006 Elsevier Inc. All rights reserved. Keywords: Multimeric proteins purification; Multimeric proteins immobilization; Multimeric proteins stabilization; Selective adsorption; Multipoint adsorption
1. Introduction The understanding and control of the interaction between proteins and activated supports is in many instances a critical point in the optimization of immobilization and purification processes of proteins. Adsorption of proteins on ionic exchangers is one of the simplest and most used techniques for protein purification and immobilization [1–5]. Adsorption of proteins on these matrixes requires a multipoint adsorption, with several groups of the support interacting with several groups of the protein, the higher the
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Corresponding authors. Tel.: +34 91 585 48 09; fax: +34 91 585 47 60. E-mail addresses: [email protected] (R. Fernandez-Lafuente), [email protected] (J.M. Guisan). 0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.01.024
number of groups in the protein interacting with the support, the higher the adsorption strength [5,6]. These supports are able to adsorb a large percentage of proteins at pH 7 [7,8]. Standard protocols of purification using highly activated anionic exchangers involve the adsorption of most of the proteins in the sample and the selective desorption as a function of the adsorption strength. Lowly activated supports permit the selective adsorption of large proteins versus small proteins: only large proteins become adsorbed on these supports, permitting selective adsorption of multimeric proteins and high purification factors in just one-step [5,9]. Similar results could be achieved using immobilized metal chelate (IMAC) columns [10]. These lowly activated supports may be thus used to purify large proteins, but the adsorption of the proteins was too weak to use the adsorbed enzymes as immobilized biocatalysts.
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Ionic adsorption of proteins on ion exchangers is very rapid and simple, and permits the reuse of the support after the enzyme inactivation [11–13]. The main problem of this strategy is that standard proteins do not become strongly adsorbed on the supports and may be desorbed during the use of the biocatalysts by changes in pH or ionic strength. This leads to biocatalyst inactivation and product contamination [14–17]. Highly activated ionic exchangers may adsorb large and small proteins, and large proteins become strongly adsorbed on the supports. In fact, these supports have been discarded as supports to purify large protein because of the strong adsorption of these proteins [5,9]. However, this strong adsorption may become a very positive phenomenon if the supports are going to be used to immobilize large proteins. Thus, the ionic exchanger should be fully different if it is intended to selectively adsorb a large protein or if it is intended to produce an immobilized derivative. Moreover, we can expect that using highly activated anionic exchangers, but at high ionic strength, we can achieve also a selective adsorption: only proteins having the possibility of a very intense multi-interaction with the supports should become adsorbed. Moreover, these conditions force that the adsorption of the enzyme was in the way where the support–enzyme interaction was maximized, further increasing the adsorption strength of the immobilizing enzymes [18]. In this case, highly activated supports presenting large surfaces to interact with the proteins may be the best option to facilitate the multi-interaction. Sepabeads-EA, having ethylendiamine bond to the support via a secondary amino attachment, may fulfil these requirements. Sepabeads are prepared via a very intense polymerization in the presence of porogenic agents [19–21], therefore it offers large surfaces for the protein interaction (in fact, Sepabeads have been used to stabilize several enzymes via multipoint covalent attachment [21–23]). Thus, they can permit a very intense adsorption of large proteins. Moreover, the monoaminoethyl-N-aminoethyl (MANAE) (derived from the modification of aldehydes or epoxy groups with ethylendiamine) [24] groups have a double positive charge at neutral or acid pH value, and just one charge at moderate alkaline pH, permitting to alter the properties of the adsorbent by moderate changes in the pH value. 2. Materials and methods 2.1. Materials Cross-linked 4 agarose beads (BCL), (glyoxyl and MANAE) were generously donated by Hispanagar S.A. (Burgos, Spain) and prepared as previously described [24,25]. DEAE-6% cross-linked agarose beads was from Amershan Pharmacia Biotech, AB (Uppsala, Sweden). Toyopearl-DEAE 650 C and Toyopearl-Super Q 650 C (chromatographic resins) were obtained from TOSOH Bioscience, (GmbH, Germany). Sepabeads aminated supports (ECEA3) was kindly donated by Resindion S.R.L. (Milan Italy). o-Nitro-phenyl-d-galactopyranoside (o-NPG) was supplied by Sigma (St. Louis, MO, USA). Extracts containing beta-galactosidase from Escherichia coli (1%) was a kind gift from Dr. Jose L. Garcia (CIB-CSIC, Madrid, Spain), and prepared as previously described [4]. Extracts containing beta-galactosidase (5%) from Thermus sp. Strain T2 and the pure enzyme was prepared as previously described [26]. All other reagents were of analytical grade.
2.2. Methods All experiments were performed at least in triplicate and the results are presented as its mean value. Experimental error was never over 5%. 2.2.1. Assay of beta-galactosidase activity Beta-galactosidase activity of the enzyme and derivatives was usually determined spectrophotometrically using o-NPG as substrate. A sample of enzymatic solution or derivative suspension (25–100 L) was added to a cubette with 2 mL of a substrate solution (13 mM o-NPG in 100 mM sodium phosphate buffer, 2 mM MgCl2 pH 7.0 for beta-galactosidase from E. coli and 13 mM o-NPG in 50 mM sodium phosphate buffer pH 7 for beta-galactosidase from Thermus sp. strain T2) at 25 ◦ C. The activity was determined by recording the increment of absorbance at 405 nm (molar extinction coefficient of o-nitro-phenol (o-NP), 3100 M−1 cm−1 ). One beta-galactosidase unit was defined as the amount of enzyme which liberated 1 mol of o-NP per min under the described conditions. 2.2.2. Protein determination Protein concentration was determined by the method developed by Bradford [27]. Bovine serum albumin (BSA) was used as protein standard.
2.3. Preparation of monoaminoethyl-N-aminoethyl-agarose supports The protocol was similar to that previously described [24], 10 mL of glyoxyl agarose (4 and 10 BCL) were suspended in 90 mL (2 M ethylenediamine pH 10.05 and it was gently stirred for 2 h). Then, 1 g of solid NaBH4 was added and the suspension was reduced for 2 h. The reduced gels, monoaminoethylN-aminoethyl agarose, were filtered and washed with 100 mL of 0.1 M sodium acetate buffer, 1 M NaCl at pH 5.0, with 100 mL of 0.1 M sodium bicarbonate buffer, 1 M NaCl at pH 10.0 and finally with 500 mL of deionizer water. This permitted the full conversion of glyoxyl to MANAE groups. Therefore, by controlling the content in glyoxyl groups, it is very simple to control the concentration of amino groups [28].
2.4. Adsorption of proteins on anionic exchangers supports Five grams of different aminated supports (MANAE-agarose, DEAEagarose, Toyopearl-DEAE 650 C, Toyopearl-Super Q 650 C, and Sepabeadsamino (EC-EA3)) were suspended in 10 mL of enzyme solutions (5 mg of protein/per gram of support) at pH 7.0. In some instances, different NaCl concentrations were used. Previously, the supports were equilibrated at pH 7.0 and the indicated concentration of NaCl. The different suspensions were gently stirred at 25 ◦ C for during 1 h. Periodically, samples of supernatant and suspensions were taken and the enzymatic activity or protein concentration was measured. After, the preparations were washed five times with 4 volumes of the same adsorption buffer and finally, twice with 4 volumes of 5 mM sodium phosphate buffer pH 7.0 and then incubating in these conditions overnight at 4 ◦ C. A reference solution with soluble enzymes was submitted to the same treatment to detect any possible effect of salt concentration upon the enzyme activity.
2.5. Desorption of proteins from anionic exchangers supports The supports containing the immobilized proteins were suspended in a volume equivalent to that where the adsorption was carried out, in 5 mM sodium phosphate buffer pH 7.0 and 25 ◦ C. Then, NaCl was added to progressively increase its concentration. Samples were taken from the supernatant at each NaCl concentration after 30 min of the NaCl addition at room temperature. Longer incubation times (upper 3 h) did not result in a significant increment in desorbed proteins. Proteins desorbed from the matrices were measured by the Bradford’s method [27] and/or enzyme activity assays. Reference solutions with soluble samples were submitted to the same treatment to detect any possible effect of the NaCl on the activity. The percentage of desorbed enzymes/or proteins is referred to the adsorbed proteins and/or enzymes.
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Fig. 1. Adsorption of beta-galactosidases from Escherichia coli and Thermus sp. strain T2 on different activated MANAE-agarose (4BCL) supports. Process was carried out at 25 ◦ C and pH 7.0 during 1 h as described in Section 2.2. Thermus beta-galactosidase (䊉), E. coli beta-galactosidase (), crude proteins ().
2.6. Thermal stability of E. coli and Thermus sp. strain T2 beta-galactosidase preparations on different ionic exchanger supports To study the thermal stability of the enzymes, 1 U/mL of soluble enzyme or 10 U/g of immobilized enzymes (diluted 1/10) were incubated at different T and pH values. At different times aliquots of the suspensions were withdrawn and their residual activities were tested with o-NPG as described previously.
2.7. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis SDS-PAGE was performed as described by Laemmli [29]; in a SE 250Mighty small II electrophoretic unit (Hoefer Co.) using gels of 12% polyacrylamide in a separation zone of 9 cm × 6 cm and a concentration zone of 5% polyacrylamide. Gels were stained with Coomassie brilliant blue method. High and low molecular weight markers from Pharmacia were used (14,000–270,000 D).
3. Results 3.1. Adsorption of proteins on supports activated with different concentration of amino groups
Fig. 2. (A) Desorption of beta-galactosidase from Escherichia coli adsorbed on lowly activated MANAE-agarose. Adsorption was performed in supports activated with 3 mol of amino groups per g of support in 5 mM sodium phosphate buffer at pH 7.0. Desorption was performed after 30 min by incubating the adsorbed proteins at growing concentration of NaCl/5 mM sodium phosphate at pH 7.0 as described in methods. (B) SDS-PAGE (15%) of proteins adsorbed on and desorbed from MANAE-agarose activated with 3 mol of amino groups per g of support. Other specifications as described in Section 2.2. Lanes 1, high molecular marker; 2, crude extract of E. coli beta-galactosidase; 3, betagalactosidase from E. coli desorbed from 3 mol aminated agarose support with 200 mM of NaCl.
Crude protein preparations and pure beta-galactosidases from E. coli and Thermus were offered to MANAE supports progressively less activated (Fig. 1). Using crude extract of proteins, adsorption was very important (almost 80%) using fully activated supports, but it decreased progressively when the activation of the support diminished, with just a 5–7% protein adsorption using a support having 2 mol/wet g (Fig. 1). The adsorption of the beta-galactosidase from Thermus accounted by more than an 80% using supports having only 2 mol/wet g. However, only 20% of the beta-galactosidase from E. coli was adsorbed on this support. Using a support having 3 mol the adsorbed beta-galactosidase was more than 80%.
containing 3 mol MANAE/g. No enzyme was released using 50 mM of sodium chloride, but full desorption of the enzyme could be achieved using just 200 mM NaCl. The SDS-PAGE analysis of the proteins released from the support shows that the purity of the enzyme was quite high (over 50%) (Fig. 2). Thus, the purification factor by this simple adsorption/desorption step was around 30 with a yield higher than 85%. Similar results have been reported using the enzyme from Thermus [5]. The easy desorption of the protein has a double reading: this support is fine for large protein purification, but may be not used to immobilize these proteins.
3.2. Purification of beta-galactosidase from E. coli by selective adsorption on tailor-made activated supports
3.3. Effect of the ionic strength on the adsorption of proteins on highly activated supports
We offered a protein extract from E. coli where the betagalactosidase from E. coli had been super-expressed to a support
A more selective adsorption of the large enzymes using highly activated supports was intended. To achieve this goal,
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Fig. 3. Adsorption of beta-galactosidases from E. coli and Thermus on different ionic exchangers (A: MANAE-agarose, B: DEAE-agarose, C: DEAE-Toyopearl, D: MANAE-Sepabeads-(EC-EA3)) at different ionic strengths. The experiments were carried out at 25 ◦ C and pH 7.0 at different NaCl concentrations in 5 mM sodium phosphate during 1 h as described in methods. E. coli beta-galactosidase (), Thermus sp. Strain T2 beta-galactosidase (), crude protein extract (䊉).
experimental conditions where only proteins having the possibility of giving very intense adsorption was assayed: high ionic strength. Using the same support that in the previous study (MANAE-agarose) activated with 40 mol/g of support, it is possible to observe that the amount of proteins from the crude extract that become adsorbed on the support, decreased with the ionic strength, but around 20% of the proteins become adsorbed even using 500 mM NaCl. At this ionic strength, most of the beta-galactosidase from Thermus becomes adsorbed on the support, while only 30% of the beta-galactosidase from E. coli was adsorbed, suggesting that the thermophilic betagalactosidase was able to interact with the aminated support with a higher intensity than this mesophilic one. In order to improve the selectivity of the adsorption, some other supports were assayed: DEAE-agarose, DEAE-Toyopearl and ECEA3 Sepabeads. It was found that the influence of the ionic strength in the adsorption for the capability of each support to adsorb proteins was different (Fig. 3). Thus, DEAE-agarose only offered good results in the adsorption of beta-galatosidases until 300 mM, although DEAE groups are theoretically stronger adsorbents than MANAE groups. Toyopearl-DEAE, a support formed by very thin fibres (where multi-interaction is more difficult), is unable to adsorb proteins at a lower ionic strength than agarose. On the contrary, Sepabeads, a support formed by intense cross-linking in the presence of porogenic agents, giving large surface to interact with the proteins, fully adsorbed both beta-galactosidases even using 500 mM. However, all the supports absorb always around 20% of the proteins under con-
ditions where adsorption of the beta-galactosidases accounting for around 80%. It has been previously shown [5] that only large proteins become adsorbed on lowly activated supports. After desorption of the proteins from this lowly activated supports, it was found that 100% of the these proteins become adsorbed on the highly activated supports at 200 mM NaCl and pH 7. That is, all large proteins able to be adsorbed on lowly activated supports become adsorbed at high ionic strength on lowly activated supports. By offering the supernatant of the proteins that may be not adsorbed on lowly activated supports at highly activated supports, but this time at 300 mM NaCl, we could detect the adsorption of around 10% of the proteins. On the contrary, by offering the proteins not adsorbed at high ionic strength on highly activated supports, but at lowly activated supports (after reducing the ionic strength via dialysis), no immobilization of proteins was detected. This means that although selectivity may be increased at high ionic strength, there are a larger percentage of proteins able to become adsorbed on these highly activated supports at high ionic strength than on the lowly activated supports at low ionic strength. This could be due that, now, not only large proteins may become adsorbed, also small proteins having clusters with many negative charges may be adsorbed (even stronger than the large proteins). Therefore, this strategy, although permitted to get significant purification factors (around a 4fold), gave lower purification factors than using lowly activated supports.
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Fig. 4. Desorption of beta-galactosidase from Escherichia coli adsorbed on DEAE-agarose and MANAE-Sepabeads: adsorption was performed at 200 mM of NaCl/5 mM sodium phosphate buffer pH 7.0 and 25 ◦ C. The enzyme was released from the support by incubation on growing concentration of NaCl in 5 mM sodium phosphate buffer pH 7.0 and 25 ◦ C as described in methods. DEAE-agarose (), MANAE-Sepabeads ().
3.4. Effect of the support and adsorption conditions on the adsorption strength of the beta-galactosidase from E. coli The adsorption on highly activated supports in the presence of salt increased the adsorption strength of the enzyme on the support. Thus, now there are no significant desorption of the beta-galactosidase from Thermus sp. even at 800 mM NaCl (Fig. 4), while at low ionic strength a 30% of the protein could be desorbed at 400 mM.
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Fig. 6. Inactivation course of different preparations of beta-galactosidase from Thermus sp. T2. 10 U of soluble or immobilized beta-galactosidase were incubated in 5 mM sodium borate pH 8.5 at 70 ◦ C. Other specifications were described in methods. Soluble enzyme (䊉); MANAE-Sepabeads immobilized preparation ().
When the adsorption was performed at 200 mM NaCl, the desorption of the beta-galactosidase from E. coli from DEAE-agarose or MANAE-Sepabeads (Fig. 4) shows again that MANAE groups are able to adsorb the large protein much more strongly than DEAE. In fact, 60% of the protein is released from DEAE agarose only at 400 mM NaCl, while MANAESepabeads did not release the enzyme even at 800 mM NaCl. 3.5. Stability of beta-galactosidases reversibly immobilized on amino supports The enzymes were immobilized following the protocol described above and were submitted to inactivation on different conditions. Figs. 5 and 6 show that both enzymes become significantly stabilized by this immobilization protocol. This could be due to the simultaneous adsorption of several protein subunits on the support that reduce the risks of sub-unit dissociation [30–32]. 4. Conclusions
Fig. 5. Inactivation course of different preparations of E. coli beta-galactosidase. 10 U of soluble or immobilized beta-galactosidase were incubated in 5 mM sodium acetate pH 5.5 (A) or 5 mM sodium phosphate pH 7.0 (B) at 45 ◦ C. Other specifications were described in methods. Soluble enzyme (䊉), immobilized enzyme on MANAE-Sepabeads ().
Using tailor-made ionic exchangers we have been able to design supports useful for purification of large proteins (supports lowly activated) and supports able to adsorb these large proteins very strongly. Thus, the features of the support should be chosen having in mind the final use. Lowly activated supports may be very suitable for large protein purification via selective adsorption and mild desorption from the support. However, the adsorption is so weak that this strategy may no be used for reversible immobilization. Highly activated supports may be suitable for large proteins immobilization: adsorption of these enzymes become very strong. However, under standard conditions it is not a selective process. Moreover, the adsorption strength is too high to have an easy desorption from the supports. Thus, these supports may be useful for large protein immobilization but no for protein purification. To intend the one-step purification-reversible immobilization of large proteins using highly activated supports, ionic strength was increased during adsorption to favour the adsorption of proteins able to give many enzyme supports links. The use
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of high ionic strength decreased the number of small proteins able to become adsorbed on highly activated supports, permitting the adsorption of large proteins, but still 20% of the other proteins become adsorbed on the supports under similar conditions where large proteins are adsorbed. These enzymes could be medium–small ones having clusters with a high density of negatively charged groups, therefore also having a high potential to interact with the highly activated support. These enzymes cannot become adsorbed on poorly activated supports, because the distance between residues on the support is large enough to avoid multi-interaction with residues placed in a cluster, but may adsorb on very activated supports even under very drastic conditions of ionic strength because they may establish very intense multi-interaction with very activated supports, where several groups of the support may interact with the external clusters of the proteins. This feature of these proteins may be used to design future tailor-made ionic exchangers to get the selective adsorption of proteins. In any case, a 4-fold purification factor may be achieved using 200 mM NaCl during adsorption on highly activated supports. The nature of the ionic group and the internal nature of the support have also some influence in the adsorption of the proteins. Moreover, the presence of salt during adsorption makes that the adsorption was stronger, perhaps by forcing the enzyme to be adsorbed by the position where a more intense support–enzyme interaction may be obtained. Using SepabeadsMANAE and high ionic strength (200–300 mM NaCl) during adsorption, adsorption is very strong and permits some stabilization of the mulltimeric enzymes. As an example of how the supports nature may affect its interaction with proteins, and because the very simple step of purification using lowly activated supports, we can propose to use the selective adsorption on lowly activated support to purify large proteins, to desorbe the large proteins from these supports at moderately high ionic strength, and the further direct immobilization of these pure proteins on highly activated supports under these conditions, with a final yield higher than 80%. These derivatives with pure enzymes will be useful under very different conditions because the extremely strong adsorption of the large proteins on these very activated anionic exchangers. Acknowledgments This work was supported by grant O7G/002/2003 from the Comunidad Aut´onoma de Madrid and the EC Project (MATINOES G5RD-CT-2002-00752). The kind supply of Sepabeads from Resindion srl is gratefully recognized. The helpful com´ ments of M.Sc. Angel Berenguer (Departamento de Qu´ımica Inorg´anica, Universidad de Alicante) are gratefully recognized. Authors thank the MEC for a Ramon y Cajal contract for Dr. Mateo. References [1] Hutchens TW, Porath J. Thiophilic adsorption of immunoglobulinsanalysis of conditions optimal for selective immobilization and purification. Anal Biochem 1986:217–26.
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Purification and very strong reversible immobilization of large proteins on anionic exchangers by controlling the support and the immobilization conditions Benevides C.C. Pessela a , Manuel Fuentes a , Cesar Mateo a , Roberto Munilla a , Alfonso V. Carrascosa b , Roberto Fernandez-Lafuente a,∗ , Jose M. Guisan a,∗ a
Departamento de Biocat´alisis, Instituto de Cat´alisis CSIC, Campus Universidad Aut´onoma, Cantoblanco, 28049 Madrid, Spain b Department of Microbiology, Instituto de Fermentaciones Industriales (CSIC), Spain Received 13 January 2006; accepted 18 January 2006
Abstract The interaction of two large beta-galactosidases (from Escherichia coli and from Thermus sp.) with tailor-made anion exchangers was studied. Using lowly activated supports (e.g., containing 2–3 mol of ionised groups per wet gram of support), large proteins selectively adsorbed and easily desorbed (e.g., using 200 mM of NaCl), giving highly purified proteins. However, these supports cannot be used to immobilize the enzymes for industrial use, because the weak adsorption. On the other hand, these large proteins strongly adsorb on very highly activated supports (e.g., containing 40 mol of ionic groups per wet gram of 4 BCL agarose). Thus, these supports may be not valid for large protein purification, but may be very suitable for immobilization of these proteins. Using high ionic strength (e.g., 300 mM NaCl), large proteins still may be adsorbed on these supports, while only around 20% of total proteins adsorb, permitting some purification of the large proteins but not a total one. Moreover, adsorption under these conditions increase the adsorption strength (now there are not desorption even using 800 mM NaCl). Thus, the purification and the strong reversible immobilization of both beta-galactosidases were performed in a very simple two-step process. The large proteins can be directly adsorbed on these supports after desorption (at 200 mM of NaCl) from poorly activated supports. Furthermore, adsorption on very highly activated supports promotes a significant thermal stabilization of both enzymes, mainly in dissociations conditions. © 2006 Elsevier Inc. All rights reserved. Keywords: Multimeric proteins purification; Multimeric proteins immobilization; Multimeric proteins stabilization; Selective adsorption; Multipoint adsorption
1. Introduction The understanding and control of the interaction between proteins and activated supports is in many instances a critical point in the optimization of immobilization and purification processes of proteins. Adsorption of proteins on ionic exchangers is one of the simplest and most used techniques for protein purification and immobilization [1–5]. Adsorption of proteins on these matrixes requires a multipoint adsorption, with several groups of the support interacting with several groups of the protein, the higher the
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Corresponding authors. Tel.: +34 91 585 48 09; fax: +34 91 585 47 60. E-mail addresses: [email protected] (R. Fernandez-Lafuente), [email protected] (J.M. Guisan). 0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.01.024
number of groups in the protein interacting with the support, the higher the adsorption strength [5,6]. These supports are able to adsorb a large percentage of proteins at pH 7 [7,8]. Standard protocols of purification using highly activated anionic exchangers involve the adsorption of most of the proteins in the sample and the selective desorption as a function of the adsorption strength. Lowly activated supports permit the selective adsorption of large proteins versus small proteins: only large proteins become adsorbed on these supports, permitting selective adsorption of multimeric proteins and high purification factors in just one-step [5,9]. Similar results could be achieved using immobilized metal chelate (IMAC) columns [10]. These lowly activated supports may be thus used to purify large proteins, but the adsorption of the proteins was too weak to use the adsorbed enzymes as immobilized biocatalysts.
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Ionic adsorption of proteins on ion exchangers is very rapid and simple, and permits the reuse of the support after the enzyme inactivation [11–13]. The main problem of this strategy is that standard proteins do not become strongly adsorbed on the supports and may be desorbed during the use of the biocatalysts by changes in pH or ionic strength. This leads to biocatalyst inactivation and product contamination [14–17]. Highly activated ionic exchangers may adsorb large and small proteins, and large proteins become strongly adsorbed on the supports. In fact, these supports have been discarded as supports to purify large protein because of the strong adsorption of these proteins [5,9]. However, this strong adsorption may become a very positive phenomenon if the supports are going to be used to immobilize large proteins. Thus, the ionic exchanger should be fully different if it is intended to selectively adsorb a large protein or if it is intended to produce an immobilized derivative. Moreover, we can expect that using highly activated anionic exchangers, but at high ionic strength, we can achieve also a selective adsorption: only proteins having the possibility of a very intense multi-interaction with the supports should become adsorbed. Moreover, these conditions force that the adsorption of the enzyme was in the way where the support–enzyme interaction was maximized, further increasing the adsorption strength of the immobilizing enzymes [18]. In this case, highly activated supports presenting large surfaces to interact with the proteins may be the best option to facilitate the multi-interaction. Sepabeads-EA, having ethylendiamine bond to the support via a secondary amino attachment, may fulfil these requirements. Sepabeads are prepared via a very intense polymerization in the presence of porogenic agents [19–21], therefore it offers large surfaces for the protein interaction (in fact, Sepabeads have been used to stabilize several enzymes via multipoint covalent attachment [21–23]). Thus, they can permit a very intense adsorption of large proteins. Moreover, the monoaminoethyl-N-aminoethyl (MANAE) (derived from the modification of aldehydes or epoxy groups with ethylendiamine) [24] groups have a double positive charge at neutral or acid pH value, and just one charge at moderate alkaline pH, permitting to alter the properties of the adsorbent by moderate changes in the pH value. 2. Materials and methods 2.1. Materials Cross-linked 4 agarose beads (BCL), (glyoxyl and MANAE) were generously donated by Hispanagar S.A. (Burgos, Spain) and prepared as previously described [24,25]. DEAE-6% cross-linked agarose beads was from Amershan Pharmacia Biotech, AB (Uppsala, Sweden). Toyopearl-DEAE 650 C and Toyopearl-Super Q 650 C (chromatographic resins) were obtained from TOSOH Bioscience, (GmbH, Germany). Sepabeads aminated supports (ECEA3) was kindly donated by Resindion S.R.L. (Milan Italy). o-Nitro-phenyl-d-galactopyranoside (o-NPG) was supplied by Sigma (St. Louis, MO, USA). Extracts containing beta-galactosidase from Escherichia coli (1%) was a kind gift from Dr. Jose L. Garcia (CIB-CSIC, Madrid, Spain), and prepared as previously described [4]. Extracts containing beta-galactosidase (5%) from Thermus sp. Strain T2 and the pure enzyme was prepared as previously described [26]. All other reagents were of analytical grade.
2.2. Methods All experiments were performed at least in triplicate and the results are presented as its mean value. Experimental error was never over 5%. 2.2.1. Assay of beta-galactosidase activity Beta-galactosidase activity of the enzyme and derivatives was usually determined spectrophotometrically using o-NPG as substrate. A sample of enzymatic solution or derivative suspension (25–100 L) was added to a cubette with 2 mL of a substrate solution (13 mM o-NPG in 100 mM sodium phosphate buffer, 2 mM MgCl2 pH 7.0 for beta-galactosidase from E. coli and 13 mM o-NPG in 50 mM sodium phosphate buffer pH 7 for beta-galactosidase from Thermus sp. strain T2) at 25 ◦ C. The activity was determined by recording the increment of absorbance at 405 nm (molar extinction coefficient of o-nitro-phenol (o-NP), 3100 M−1 cm−1 ). One beta-galactosidase unit was defined as the amount of enzyme which liberated 1 mol of o-NP per min under the described conditions. 2.2.2. Protein determination Protein concentration was determined by the method developed by Bradford [27]. Bovine serum albumin (BSA) was used as protein standard.
2.3. Preparation of monoaminoethyl-N-aminoethyl-agarose supports The protocol was similar to that previously described [24], 10 mL of glyoxyl agarose (4 and 10 BCL) were suspended in 90 mL (2 M ethylenediamine pH 10.05 and it was gently stirred for 2 h). Then, 1 g of solid NaBH4 was added and the suspension was reduced for 2 h. The reduced gels, monoaminoethylN-aminoethyl agarose, were filtered and washed with 100 mL of 0.1 M sodium acetate buffer, 1 M NaCl at pH 5.0, with 100 mL of 0.1 M sodium bicarbonate buffer, 1 M NaCl at pH 10.0 and finally with 500 mL of deionizer water. This permitted the full conversion of glyoxyl to MANAE groups. Therefore, by controlling the content in glyoxyl groups, it is very simple to control the concentration of amino groups [28].
2.4. Adsorption of proteins on anionic exchangers supports Five grams of different aminated supports (MANAE-agarose, DEAEagarose, Toyopearl-DEAE 650 C, Toyopearl-Super Q 650 C, and Sepabeadsamino (EC-EA3)) were suspended in 10 mL of enzyme solutions (5 mg of protein/per gram of support) at pH 7.0. In some instances, different NaCl concentrations were used. Previously, the supports were equilibrated at pH 7.0 and the indicated concentration of NaCl. The different suspensions were gently stirred at 25 ◦ C for during 1 h. Periodically, samples of supernatant and suspensions were taken and the enzymatic activity or protein concentration was measured. After, the preparations were washed five times with 4 volumes of the same adsorption buffer and finally, twice with 4 volumes of 5 mM sodium phosphate buffer pH 7.0 and then incubating in these conditions overnight at 4 ◦ C. A reference solution with soluble enzymes was submitted to the same treatment to detect any possible effect of salt concentration upon the enzyme activity.
2.5. Desorption of proteins from anionic exchangers supports The supports containing the immobilized proteins were suspended in a volume equivalent to that where the adsorption was carried out, in 5 mM sodium phosphate buffer pH 7.0 and 25 ◦ C. Then, NaCl was added to progressively increase its concentration. Samples were taken from the supernatant at each NaCl concentration after 30 min of the NaCl addition at room temperature. Longer incubation times (upper 3 h) did not result in a significant increment in desorbed proteins. Proteins desorbed from the matrices were measured by the Bradford’s method [27] and/or enzyme activity assays. Reference solutions with soluble samples were submitted to the same treatment to detect any possible effect of the NaCl on the activity. The percentage of desorbed enzymes/or proteins is referred to the adsorbed proteins and/or enzymes.
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Fig. 1. Adsorption of beta-galactosidases from Escherichia coli and Thermus sp. strain T2 on different activated MANAE-agarose (4BCL) supports. Process was carried out at 25 ◦ C and pH 7.0 during 1 h as described in Section 2.2. Thermus beta-galactosidase (䊉), E. coli beta-galactosidase (), crude proteins ().
2.6. Thermal stability of E. coli and Thermus sp. strain T2 beta-galactosidase preparations on different ionic exchanger supports To study the thermal stability of the enzymes, 1 U/mL of soluble enzyme or 10 U/g of immobilized enzymes (diluted 1/10) were incubated at different T and pH values. At different times aliquots of the suspensions were withdrawn and their residual activities were tested with o-NPG as described previously.
2.7. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis SDS-PAGE was performed as described by Laemmli [29]; in a SE 250Mighty small II electrophoretic unit (Hoefer Co.) using gels of 12% polyacrylamide in a separation zone of 9 cm × 6 cm and a concentration zone of 5% polyacrylamide. Gels were stained with Coomassie brilliant blue method. High and low molecular weight markers from Pharmacia were used (14,000–270,000 D).
3. Results 3.1. Adsorption of proteins on supports activated with different concentration of amino groups
Fig. 2. (A) Desorption of beta-galactosidase from Escherichia coli adsorbed on lowly activated MANAE-agarose. Adsorption was performed in supports activated with 3 mol of amino groups per g of support in 5 mM sodium phosphate buffer at pH 7.0. Desorption was performed after 30 min by incubating the adsorbed proteins at growing concentration of NaCl/5 mM sodium phosphate at pH 7.0 as described in methods. (B) SDS-PAGE (15%) of proteins adsorbed on and desorbed from MANAE-agarose activated with 3 mol of amino groups per g of support. Other specifications as described in Section 2.2. Lanes 1, high molecular marker; 2, crude extract of E. coli beta-galactosidase; 3, betagalactosidase from E. coli desorbed from 3 mol aminated agarose support with 200 mM of NaCl.
Crude protein preparations and pure beta-galactosidases from E. coli and Thermus were offered to MANAE supports progressively less activated (Fig. 1). Using crude extract of proteins, adsorption was very important (almost 80%) using fully activated supports, but it decreased progressively when the activation of the support diminished, with just a 5–7% protein adsorption using a support having 2 mol/wet g (Fig. 1). The adsorption of the beta-galactosidase from Thermus accounted by more than an 80% using supports having only 2 mol/wet g. However, only 20% of the beta-galactosidase from E. coli was adsorbed on this support. Using a support having 3 mol the adsorbed beta-galactosidase was more than 80%.
containing 3 mol MANAE/g. No enzyme was released using 50 mM of sodium chloride, but full desorption of the enzyme could be achieved using just 200 mM NaCl. The SDS-PAGE analysis of the proteins released from the support shows that the purity of the enzyme was quite high (over 50%) (Fig. 2). Thus, the purification factor by this simple adsorption/desorption step was around 30 with a yield higher than 85%. Similar results have been reported using the enzyme from Thermus [5]. The easy desorption of the protein has a double reading: this support is fine for large protein purification, but may be not used to immobilize these proteins.
3.2. Purification of beta-galactosidase from E. coli by selective adsorption on tailor-made activated supports
3.3. Effect of the ionic strength on the adsorption of proteins on highly activated supports
We offered a protein extract from E. coli where the betagalactosidase from E. coli had been super-expressed to a support
A more selective adsorption of the large enzymes using highly activated supports was intended. To achieve this goal,
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Fig. 3. Adsorption of beta-galactosidases from E. coli and Thermus on different ionic exchangers (A: MANAE-agarose, B: DEAE-agarose, C: DEAE-Toyopearl, D: MANAE-Sepabeads-(EC-EA3)) at different ionic strengths. The experiments were carried out at 25 ◦ C and pH 7.0 at different NaCl concentrations in 5 mM sodium phosphate during 1 h as described in methods. E. coli beta-galactosidase (), Thermus sp. Strain T2 beta-galactosidase (), crude protein extract (䊉).
experimental conditions where only proteins having the possibility of giving very intense adsorption was assayed: high ionic strength. Using the same support that in the previous study (MANAE-agarose) activated with 40 mol/g of support, it is possible to observe that the amount of proteins from the crude extract that become adsorbed on the support, decreased with the ionic strength, but around 20% of the proteins become adsorbed even using 500 mM NaCl. At this ionic strength, most of the beta-galactosidase from Thermus becomes adsorbed on the support, while only 30% of the beta-galactosidase from E. coli was adsorbed, suggesting that the thermophilic betagalactosidase was able to interact with the aminated support with a higher intensity than this mesophilic one. In order to improve the selectivity of the adsorption, some other supports were assayed: DEAE-agarose, DEAE-Toyopearl and ECEA3 Sepabeads. It was found that the influence of the ionic strength in the adsorption for the capability of each support to adsorb proteins was different (Fig. 3). Thus, DEAE-agarose only offered good results in the adsorption of beta-galatosidases until 300 mM, although DEAE groups are theoretically stronger adsorbents than MANAE groups. Toyopearl-DEAE, a support formed by very thin fibres (where multi-interaction is more difficult), is unable to adsorb proteins at a lower ionic strength than agarose. On the contrary, Sepabeads, a support formed by intense cross-linking in the presence of porogenic agents, giving large surface to interact with the proteins, fully adsorbed both beta-galactosidases even using 500 mM. However, all the supports absorb always around 20% of the proteins under con-
ditions where adsorption of the beta-galactosidases accounting for around 80%. It has been previously shown [5] that only large proteins become adsorbed on lowly activated supports. After desorption of the proteins from this lowly activated supports, it was found that 100% of the these proteins become adsorbed on the highly activated supports at 200 mM NaCl and pH 7. That is, all large proteins able to be adsorbed on lowly activated supports become adsorbed at high ionic strength on lowly activated supports. By offering the supernatant of the proteins that may be not adsorbed on lowly activated supports at highly activated supports, but this time at 300 mM NaCl, we could detect the adsorption of around 10% of the proteins. On the contrary, by offering the proteins not adsorbed at high ionic strength on highly activated supports, but at lowly activated supports (after reducing the ionic strength via dialysis), no immobilization of proteins was detected. This means that although selectivity may be increased at high ionic strength, there are a larger percentage of proteins able to become adsorbed on these highly activated supports at high ionic strength than on the lowly activated supports at low ionic strength. This could be due that, now, not only large proteins may become adsorbed, also small proteins having clusters with many negative charges may be adsorbed (even stronger than the large proteins). Therefore, this strategy, although permitted to get significant purification factors (around a 4fold), gave lower purification factors than using lowly activated supports.
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Fig. 4. Desorption of beta-galactosidase from Escherichia coli adsorbed on DEAE-agarose and MANAE-Sepabeads: adsorption was performed at 200 mM of NaCl/5 mM sodium phosphate buffer pH 7.0 and 25 ◦ C. The enzyme was released from the support by incubation on growing concentration of NaCl in 5 mM sodium phosphate buffer pH 7.0 and 25 ◦ C as described in methods. DEAE-agarose (), MANAE-Sepabeads ().
3.4. Effect of the support and adsorption conditions on the adsorption strength of the beta-galactosidase from E. coli The adsorption on highly activated supports in the presence of salt increased the adsorption strength of the enzyme on the support. Thus, now there are no significant desorption of the beta-galactosidase from Thermus sp. even at 800 mM NaCl (Fig. 4), while at low ionic strength a 30% of the protein could be desorbed at 400 mM.
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Fig. 6. Inactivation course of different preparations of beta-galactosidase from Thermus sp. T2. 10 U of soluble or immobilized beta-galactosidase were incubated in 5 mM sodium borate pH 8.5 at 70 ◦ C. Other specifications were described in methods. Soluble enzyme (䊉); MANAE-Sepabeads immobilized preparation ().
When the adsorption was performed at 200 mM NaCl, the desorption of the beta-galactosidase from E. coli from DEAE-agarose or MANAE-Sepabeads (Fig. 4) shows again that MANAE groups are able to adsorb the large protein much more strongly than DEAE. In fact, 60% of the protein is released from DEAE agarose only at 400 mM NaCl, while MANAESepabeads did not release the enzyme even at 800 mM NaCl. 3.5. Stability of beta-galactosidases reversibly immobilized on amino supports The enzymes were immobilized following the protocol described above and were submitted to inactivation on different conditions. Figs. 5 and 6 show that both enzymes become significantly stabilized by this immobilization protocol. This could be due to the simultaneous adsorption of several protein subunits on the support that reduce the risks of sub-unit dissociation [30–32]. 4. Conclusions
Fig. 5. Inactivation course of different preparations of E. coli beta-galactosidase. 10 U of soluble or immobilized beta-galactosidase were incubated in 5 mM sodium acetate pH 5.5 (A) or 5 mM sodium phosphate pH 7.0 (B) at 45 ◦ C. Other specifications were described in methods. Soluble enzyme (䊉), immobilized enzyme on MANAE-Sepabeads ().
Using tailor-made ionic exchangers we have been able to design supports useful for purification of large proteins (supports lowly activated) and supports able to adsorb these large proteins very strongly. Thus, the features of the support should be chosen having in mind the final use. Lowly activated supports may be very suitable for large protein purification via selective adsorption and mild desorption from the support. However, the adsorption is so weak that this strategy may no be used for reversible immobilization. Highly activated supports may be suitable for large proteins immobilization: adsorption of these enzymes become very strong. However, under standard conditions it is not a selective process. Moreover, the adsorption strength is too high to have an easy desorption from the supports. Thus, these supports may be useful for large protein immobilization but no for protein purification. To intend the one-step purification-reversible immobilization of large proteins using highly activated supports, ionic strength was increased during adsorption to favour the adsorption of proteins able to give many enzyme supports links. The use
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of high ionic strength decreased the number of small proteins able to become adsorbed on highly activated supports, permitting the adsorption of large proteins, but still 20% of the other proteins become adsorbed on the supports under similar conditions where large proteins are adsorbed. These enzymes could be medium–small ones having clusters with a high density of negatively charged groups, therefore also having a high potential to interact with the highly activated support. These enzymes cannot become adsorbed on poorly activated supports, because the distance between residues on the support is large enough to avoid multi-interaction with residues placed in a cluster, but may adsorb on very activated supports even under very drastic conditions of ionic strength because they may establish very intense multi-interaction with very activated supports, where several groups of the support may interact with the external clusters of the proteins. This feature of these proteins may be used to design future tailor-made ionic exchangers to get the selective adsorption of proteins. In any case, a 4-fold purification factor may be achieved using 200 mM NaCl during adsorption on highly activated supports. The nature of the ionic group and the internal nature of the support have also some influence in the adsorption of the proteins. Moreover, the presence of salt during adsorption makes that the adsorption was stronger, perhaps by forcing the enzyme to be adsorbed by the position where a more intense support–enzyme interaction may be obtained. Using SepabeadsMANAE and high ionic strength (200–300 mM NaCl) during adsorption, adsorption is very strong and permits some stabilization of the mulltimeric enzymes. As an example of how the supports nature may affect its interaction with proteins, and because the very simple step of purification using lowly activated supports, we can propose to use the selective adsorption on lowly activated support to purify large proteins, to desorbe the large proteins from these supports at moderately high ionic strength, and the further direct immobilization of these pure proteins on highly activated supports under these conditions, with a final yield higher than 80%. These derivatives with pure enzymes will be useful under very different conditions because the extremely strong adsorption of the large proteins on these very activated anionic exchangers. Acknowledgments This work was supported by grant O7G/002/2003 from the Comunidad Aut´onoma de Madrid and the EC Project (MATINOES G5RD-CT-2002-00752). The kind supply of Sepabeads from Resindion srl is gratefully recognized. The helpful com´ ments of M.Sc. Angel Berenguer (Departamento de Qu´ımica Inorg´anica, Universidad de Alicante) are gratefully recognized. Authors thank the MEC for a Ramon y Cajal contract for Dr. Mateo. References [1] Hutchens TW, Porath J. Thiophilic adsorption of immunoglobulinsanalysis of conditions optimal for selective immobilization and purification. Anal Biochem 1986:217–26.
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