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Single-step protein purification by back flush in ion exchange chromatography
Analytical Biochemistry 392 (2009) 174–176
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Single-step protein purification by back flush in ion exchange chromatography Ming-Kai Chern *, Wei-Jyh Shiah, Jyun-Jie Chen 1, Tzung-You Tsai 1, Hsin-Yin Lin, Chien-Wei Liu Graduate Institute of Life Sciences, Tamkang University, Tamsui 25137, Taiwan
a r t i c l e
i n f o
Article history: Received 2 February 2009 Available online 2 June 2009
a b s t r a c t Ion exchange chromatography, one of the major procedures for protein purification, seldom provides single-step purification due to a lack of specific affinity. In this work, a novel and simple method called ‘‘back flush” (i.e., reversing the flow direction of elution relative to that of sample loading) was developed to achieve single-step purification on an ion exchanger. Tips for the conditions and operation by back flush are presented. Our study demonstrates, for the first time, the feasibility and dramatic improvement for protein purification by the back-flush method. Ó 2009 Elsevier Inc. All rights reserved.
Ion exchange chromatography is one of the most common procedures for protein purification. It is based on the interactions between the charged groups on the proteins and the immobilized groups on the exchanger, which is usually described as an equilibrium model [1]. However, it seldom provides single-step purification due to a lack of specific affinity. In the analytical chemistry field, a novel and simple method called ‘‘back flush” has been developed to better separate inorganic ionic species on ion exchangers. By simply reversing the flow direction of elution relative to that of sample loading, this method is aimed to minimize peak spreading [2], avoid problems of irreversible ion exchange of sample ions [3], and reduce the dip peak [4]. Specifically, by loading the sample onto the column in the opposite direction to that in which it is eluted, the band broadening is minimized in the concentrator column through compression of the sample ions into a compact band during the initial stages of elution from the concentrator column [2]. Because the direction of process can affect the result of separation, not only equilibrium interactions but also nonequilibrium interactions may play a role in this method. In this study, we investigated the efficiency of the back-flush method in the purification of recombinant proteins. With the known sequences of recombinant proteins, we can calculate their theoretical isoelectric point (pI)2 values that serve as a reference for choosing the pH conditions for purification. Two aldehyde dehydrogenases (ALD4p and ALD6p) and one methyltransferase (Bud23p) from Saccharomyces cerevisiae were tested with this method for purification by back flush on an anion exchanger. * Corresponding author. Fax: +886 2 86311015. E-mail addresses: [email protected], [email protected] (M.K. Chern). 1 These authors contribute equally to this work. 2 Abbreviations used: pI, isoelectric point; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; 2D–PAGE, two-dimensional polyacrylamide gel electrophoresis. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.05.045
DNA sequence corresponding to the mature length of mitochondrial ALD4p or the full-length open reading frame of ALD6 was cloned and inserted at the restriction sites NdeI/SpeI of pET 43.1c (Novagen) for protein expression. For expression of Bud23p, fulllength open reading frame of Bud23 was cloned and inserted at the restriction sites NdeI/BamHI of pET 28c (Novagen). The theoretical pI values for recombinant ALD4p, ALD6p, and Bud23p are 6.07, 5.18, and 9.69, respectively. All three proteins were expressed in Escherichia coli strain BL21(DE3) (Novagen). Expression of ALD4p was induced at 30 °C, whereas that of ALD6p was induced at 24 °C to reduce degradation. Expression of Bud23p was induced at 18 °C in the presence of 0.5 M sorbitol and 2.5 mM betaine hydrochloride to reduce inclusion body [5]. For ALD4p and ALD6p, 200 ml of culture was used; for Bud23p, 50 ml was used. The cell pellet was then resuspended in 1/20 culture volume of lysis buffer (50 mM Tris–Cl [pH 7.4] containing 1 mM ethylenediaminetetraacetic acid [EDTA], 6 mM 2-mercaptoethanol, 10% glycerol, and 1 mM phenylmethylsulfonyl fluoride [PMSF]) prechilled at 4 °C. E. coli cells were broken on ice with a Branson sonicator Digital Sonifier 250 D equipped with a 1/8-inch microtip. Each 2s cycle was composed of alternating 1-s pulse of 28% amplitude and 1-s blank continued for 1 min. A total of 3 min of sonication was carried out with a 1-min interval on ice between each duty minute. The cell lysate was cleared at 5000g and 4 °C for 10 min. For purification of ALD4p and ALD6p, the supernatant was then applied onto a Q Sepharose Fast Flow column (1.6 cm diameter, 30 ml bed volume, Amersham Biosciences) preequilibrated with buffer A (50 mM Tris–HCl [pH 7.6] containing 1 mM EDTA, 6 mM 2mercaptoethanol, and 10% glycerol). The column used has a plunger filter to hold the ion exchanger in place so that the sample could be loaded either conventionally from the top or in a reverse manner from the bottom by connecting the sample tubing to either the top or bottom inlet of the column, respectively. The reverse loading mode has been called back flush [2,4]. For either loading mode, the column was then washed with buffer A and subse-
Notes & Tips / Anal. Biochem. 392 (2009) 174–176
175
quently eluted with a gradient from 0 to 100% of buffer B (with buffer A containing 1 M NaCl) in the top-down direction. For purification of Bud23p, buffer A was replaced with buffer C (50 mM boric acid [pH 8.8], 0.05 M NaCl, 6 mM 2-mercaptoethanol, 1 mM EDTA, and 10% glycerol) and buffer B replaced with buffer D (with buffer C containing 1 M NaCl). Both buffers C and D were adjusted to pH 8.8. Otherwise, the purification was carried out in the same way as for ALD4p and ADL6p. Throughout purification, the flow rate of 50 ml/h was used and the ambient temperature was 20 °C. Both the conventional and back-flush modes of sample loading for ALD4p were carried out and compared. The spread of proteins in the eluate was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). As shown in Fig. 1, with the conventional mode, ALD4p was centered in fractions corresponding to 55 to 70% of buffer B. Obviously, ALD4p in these fractions was mixed with a significant amount of impurities. In contrast, with the back-flush mode, ALD4p turned out to be homogeneous as eluted in 0 to 30% of buffer B (Fig. 2A). Thus, we were able to accomplish single-step purification of the recombinant ALD4p on an anion exchanger by inverting the flow direction of sample loading and elution. Similarly, ALD6p was also able to be
Fig. 2. Single-step purification of the recombinant ALD4p, ALD6p, and Bud23p by the back-flush method. (A) SDS–PAGE analysis of fractions of ALD4p eluted with buffer B. (B) SDS–PAGE analysis of fractions of ALD6p eluted with buffer B. (C) SDS– PAGE analysis of fractions of Bud23p eluted with buffer D. The range of gradient and the amount of protein (equivalent to 2.5 ll of the pooled fractions for ALD4p, 7.5 ll of the pooled fractions for ALD6p, and 10 ll of the pooled fractions for Bud23p) are indicated in the table below each gel. MW, molecular weight markers. The recombinant protein band is indicated by an arrow.
Fig. 1. SDS–PAGE analysis of fractions of ALD4p eluted with 0 to 45% (A) and 45 to 85% (B) of buffer B. The salt gradient was carried out in the same flow direction as that of sample loading. The range of gradient and the amount of protein (equivalent to 2.5 ll of the pooled fractions) are indicated in the table below each gel. CL, cleared lysate before purification; MW, molecular weight markers. The ALD4p band is indicated by an arrow.
separated to homogeneity by back flush in a single step. As shown in Fig. 2B, homogeneous ALD6p was eluted in 0 to 40% of buffer B. The purification tables for ALD4p and ALD6p are compiled in Table 1. The total amount of the overexpressed protein for ALD4p and ALD6p was quantified by subtracting the amount of total protein extract of the uninduced cells from that of the corresponding overexpressing cells, where both kinds of cells are of the same culture volume, 200 ml, and the same growing condition. The total protein
176
Notes & Tips / Anal. Biochem. 392 (2009) 174–176
Table 1 Purification of ALD4p and ALD6p by back flush. Total protein (mg)
Total activity (lmol/min)
Specific activity (lmol/min mg)
Yield (%)
Purification fold
ALD4p Cleared lysate Q Sepharose
156 5.4
3.3 1.2
0.021 0.22
100 36
1 10.5
ALD6p Cleared lysate Q Sepharose
216 3.5
1575 214
7.3 61
100 14
1 8.4
Note. The catalytic activity was determined at 22 °C with the following reaction mixtures: 0.6 mM propionaldehyde, 15 mM NADP+, 100 mM KCl, and 100 mM sodium phosphate (pH 7.4) for ALD4p; 1 mM propionaldehyde, 3 mM NADP+, 20 mM MgCl2, and 100 mM sodium phosphate (pH 7.4) for ALD6p. The progress of reaction was followed spectrophotometrically by absorbance at 340 nm using 6.22 mM 1 cm 1 for NADPH. Protein concentration was determined by the microassay method of Bradford [7].
in the cellular extract was precipitated and redissolved in urea [6] to eliminate the interference of nucleic acids in Bradford assay. The overexpressed protein obtained in this way was 13 mg for ALD4p and 18.5 mg for ALD6p. Originally, we managed to achieve one-step purification of recombinant Bud23p by engineering a His tag at the amino terminus. Unfortunately, the interaction between the tag and the chelating metal was too weak to recover pure Bud23p (data not shown), possibly due to a steric hindrance. We then needed to resort to the back-flush method. Initially, an attempt was made to purify Bud23p under the same conditions as for ALD4p and ALD6p; however, the separation was poor. Because the pH condition (7.6) used for ALD4p and ALD6p was higher than their pI values (6.07 and 5.18, respectively), whereas Bud23p has a theoretical pI of 9.69 (much higher than 7.6), we figured that by raising the pH of the buffer condition Bud23p may be better separated from the impurities. Therefore, buffers of pH 8.8 were employed for purification of Bud23p. As shown in Fig. 2C, Bud23p was indeed better separated from other impurities, particularly the fractions eluted in 40 to 44% of buffer D. The front fractions in buffer D lower than 40% almost did not contain any proteins according to SDS–PAGE analysis (not shown). The reason a buffer of pH higher than 9.69 was not used is that the Bud23p methyltransferase activity might not be tolerated at such a high pH. Interestingly, we noticed that all of the purified fractions of the three recombinant proteins were eluted earlier than other impurities that were left behind in the back fractions, that is, in the higher salt concentration range of the gradient. Thus, the empirical rule is that to achieve single-step purification by back flush on an anion exchanger with an increasing salt concentration gradient, the pH conditions used should be higher than the pI values of the target proteins; this is supposed to hold back the impurities on the column to be eluted behind the target proteins. Even the pH condition used for purification of Bud23p is still lower than the pI value of Bud23p; the relatively homogeneous protein could already be obtained in the front fractions. To further reveal the effects of the buffer pH values on the applicability of this method, purifications of ALD4p and ALD6p were also carried out in pH 6.0 (50 mM sodium phosphate, 0.05 M NaCl, 6 mM 2-mercaptoethanol, 1 mM EDTA, and 10% glycerol) and pH 8.8 (buffer C) buffers. With pH 6.0 buffer, both proteins failed to bind to the column (not shown). In contrast, with pH 8.8 buffer, both proteins retained so tenaciously to the column as to be eluted only along with contaminating proteins toward the high-salt range
(see Fig. S1 in supplementary material). Based on the pI values of ALD4p and ALD6p, a buffer 1.53 to 2.42 pH units higher than the pI value of the target protein may be recommended for back-flush purification. Considering that the distribution of the pI value and abundance of individual proteins in the mammalian cells are quite different from those of E. coli cells, as revealed in the two-dimensional polyacrylamide gel electrophoresis (2D–PAGE) profiles (see SWISS–2DPAGE, http://www.expasy.ch/ch2d, and references therein), and the overexpression levels of recombinant proteins in the mammalian cells are generally lower than those in the E. coli cells, the applicability of this method is likely limited when mammalian cells are used as the expression host. Finally, to investigate the effects of protein-to-resin ratios on the efficiency of purification, 100 and 50 ml of culture for purification of ALD6p were also tried on the fixed 30-ml bed volume of Q Sepharose using the pH 7.6 buffer. As shown in Fig. S2 and Table S1 in the supplementary material, an increase in the relative amount of resin tends to improve the yield of homogeneous ADL6p with the compromise of reduced concentration of purified ALD6p. Therefore, if the recovery of homogeneous ADL6p is to be optimized, 50 ml of culture to every 30 ml of resin is superior to the originally used 200 ml of culture. Taken together, the tips for single-step purification in anion exchange chromatography can be summarized as follows. First, calculate the pI value of the recombinant protein of interest or determine the pI value experimentally by isoelectric focusing analysis [6]. Second, choose a working pH higher than the pI value. Third, load the sample onto the column in the opposite direction to that in which it is eluted, and one can expect the homogeneous target protein to appear in the front fractions of elution. This study is, to the best of our knowledge, the first to demonstrate singlestep protein purification by the back-flush method. Acknowledgment This work was supported by grant NSC 97-2311-B032-001 from the National Science Council, Taiwan, Republic of China (to M-K. Chern).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ab.2009.05.045. References [1] J.C. Giddings, Unified Separation Science, Wiley-Interscience, New York, 1991. pp. 16–36. [2] P.R. Haddad, A.L. Heckenberg, Studies on sample preconcentration in ion chromatography, J. Chromatogr. 318 (1985) 279–288. [3] K.M. Roberts, D.T. Gjerde, J.S. Fritz, Single-column ion chromatography for the determination of chloride and sulfate in steam condensate and boiler feed water, Anal. Chem. 53 (1981) 1691–1695. [4] T. Okada, T. Kuwamoto, Trace analysis of anions by use of a back-flush method and large injection volumes in ion chromatography, J. Chrormatogr. 350 (1985) 317–323. [5] J.R. Blackwell, R. Horgan, A novel strategy for production of a highly expressed recombinant protein in an active form, FEBS Lett. 295 (1991) 10–12. [6] J.C. Anderson, S.C. Peck, A simple and rapid technique for detecting protein phosphorylation using one-dimensional isoelectric focusing gels and immunoblot analysis, Plant J. 55 (2008) 881–885. [7] G.L. Peterson, Determination of total protein, in: C.H.W. Hirs, S.N. Timasheff (Eds.), Methods in Enzymology, vol. 91: Enzyme Structure, Academic Press, New York, 1983, pp. 95–119.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Single-step protein purification by back flush in ion exchange chromatography Ming-Kai Chern *, Wei-Jyh Shiah, Jyun-Jie Chen 1, Tzung-You Tsai 1, Hsin-Yin Lin, Chien-Wei Liu Graduate Institute of Life Sciences, Tamkang University, Tamsui 25137, Taiwan
a r t i c l e
i n f o
Article history: Received 2 February 2009 Available online 2 June 2009
a b s t r a c t Ion exchange chromatography, one of the major procedures for protein purification, seldom provides single-step purification due to a lack of specific affinity. In this work, a novel and simple method called ‘‘back flush” (i.e., reversing the flow direction of elution relative to that of sample loading) was developed to achieve single-step purification on an ion exchanger. Tips for the conditions and operation by back flush are presented. Our study demonstrates, for the first time, the feasibility and dramatic improvement for protein purification by the back-flush method. Ó 2009 Elsevier Inc. All rights reserved.
Ion exchange chromatography is one of the most common procedures for protein purification. It is based on the interactions between the charged groups on the proteins and the immobilized groups on the exchanger, which is usually described as an equilibrium model [1]. However, it seldom provides single-step purification due to a lack of specific affinity. In the analytical chemistry field, a novel and simple method called ‘‘back flush” has been developed to better separate inorganic ionic species on ion exchangers. By simply reversing the flow direction of elution relative to that of sample loading, this method is aimed to minimize peak spreading [2], avoid problems of irreversible ion exchange of sample ions [3], and reduce the dip peak [4]. Specifically, by loading the sample onto the column in the opposite direction to that in which it is eluted, the band broadening is minimized in the concentrator column through compression of the sample ions into a compact band during the initial stages of elution from the concentrator column [2]. Because the direction of process can affect the result of separation, not only equilibrium interactions but also nonequilibrium interactions may play a role in this method. In this study, we investigated the efficiency of the back-flush method in the purification of recombinant proteins. With the known sequences of recombinant proteins, we can calculate their theoretical isoelectric point (pI)2 values that serve as a reference for choosing the pH conditions for purification. Two aldehyde dehydrogenases (ALD4p and ALD6p) and one methyltransferase (Bud23p) from Saccharomyces cerevisiae were tested with this method for purification by back flush on an anion exchanger. * Corresponding author. Fax: +886 2 86311015. E-mail addresses: [email protected], [email protected] (M.K. Chern). 1 These authors contribute equally to this work. 2 Abbreviations used: pI, isoelectric point; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; 2D–PAGE, two-dimensional polyacrylamide gel electrophoresis. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.05.045
DNA sequence corresponding to the mature length of mitochondrial ALD4p or the full-length open reading frame of ALD6 was cloned and inserted at the restriction sites NdeI/SpeI of pET 43.1c (Novagen) for protein expression. For expression of Bud23p, fulllength open reading frame of Bud23 was cloned and inserted at the restriction sites NdeI/BamHI of pET 28c (Novagen). The theoretical pI values for recombinant ALD4p, ALD6p, and Bud23p are 6.07, 5.18, and 9.69, respectively. All three proteins were expressed in Escherichia coli strain BL21(DE3) (Novagen). Expression of ALD4p was induced at 30 °C, whereas that of ALD6p was induced at 24 °C to reduce degradation. Expression of Bud23p was induced at 18 °C in the presence of 0.5 M sorbitol and 2.5 mM betaine hydrochloride to reduce inclusion body [5]. For ALD4p and ALD6p, 200 ml of culture was used; for Bud23p, 50 ml was used. The cell pellet was then resuspended in 1/20 culture volume of lysis buffer (50 mM Tris–Cl [pH 7.4] containing 1 mM ethylenediaminetetraacetic acid [EDTA], 6 mM 2-mercaptoethanol, 10% glycerol, and 1 mM phenylmethylsulfonyl fluoride [PMSF]) prechilled at 4 °C. E. coli cells were broken on ice with a Branson sonicator Digital Sonifier 250 D equipped with a 1/8-inch microtip. Each 2s cycle was composed of alternating 1-s pulse of 28% amplitude and 1-s blank continued for 1 min. A total of 3 min of sonication was carried out with a 1-min interval on ice between each duty minute. The cell lysate was cleared at 5000g and 4 °C for 10 min. For purification of ALD4p and ALD6p, the supernatant was then applied onto a Q Sepharose Fast Flow column (1.6 cm diameter, 30 ml bed volume, Amersham Biosciences) preequilibrated with buffer A (50 mM Tris–HCl [pH 7.6] containing 1 mM EDTA, 6 mM 2mercaptoethanol, and 10% glycerol). The column used has a plunger filter to hold the ion exchanger in place so that the sample could be loaded either conventionally from the top or in a reverse manner from the bottom by connecting the sample tubing to either the top or bottom inlet of the column, respectively. The reverse loading mode has been called back flush [2,4]. For either loading mode, the column was then washed with buffer A and subse-
Notes & Tips / Anal. Biochem. 392 (2009) 174–176
175
quently eluted with a gradient from 0 to 100% of buffer B (with buffer A containing 1 M NaCl) in the top-down direction. For purification of Bud23p, buffer A was replaced with buffer C (50 mM boric acid [pH 8.8], 0.05 M NaCl, 6 mM 2-mercaptoethanol, 1 mM EDTA, and 10% glycerol) and buffer B replaced with buffer D (with buffer C containing 1 M NaCl). Both buffers C and D were adjusted to pH 8.8. Otherwise, the purification was carried out in the same way as for ALD4p and ADL6p. Throughout purification, the flow rate of 50 ml/h was used and the ambient temperature was 20 °C. Both the conventional and back-flush modes of sample loading for ALD4p were carried out and compared. The spread of proteins in the eluate was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). As shown in Fig. 1, with the conventional mode, ALD4p was centered in fractions corresponding to 55 to 70% of buffer B. Obviously, ALD4p in these fractions was mixed with a significant amount of impurities. In contrast, with the back-flush mode, ALD4p turned out to be homogeneous as eluted in 0 to 30% of buffer B (Fig. 2A). Thus, we were able to accomplish single-step purification of the recombinant ALD4p on an anion exchanger by inverting the flow direction of sample loading and elution. Similarly, ALD6p was also able to be
Fig. 2. Single-step purification of the recombinant ALD4p, ALD6p, and Bud23p by the back-flush method. (A) SDS–PAGE analysis of fractions of ALD4p eluted with buffer B. (B) SDS–PAGE analysis of fractions of ALD6p eluted with buffer B. (C) SDS– PAGE analysis of fractions of Bud23p eluted with buffer D. The range of gradient and the amount of protein (equivalent to 2.5 ll of the pooled fractions for ALD4p, 7.5 ll of the pooled fractions for ALD6p, and 10 ll of the pooled fractions for Bud23p) are indicated in the table below each gel. MW, molecular weight markers. The recombinant protein band is indicated by an arrow.
Fig. 1. SDS–PAGE analysis of fractions of ALD4p eluted with 0 to 45% (A) and 45 to 85% (B) of buffer B. The salt gradient was carried out in the same flow direction as that of sample loading. The range of gradient and the amount of protein (equivalent to 2.5 ll of the pooled fractions) are indicated in the table below each gel. CL, cleared lysate before purification; MW, molecular weight markers. The ALD4p band is indicated by an arrow.
separated to homogeneity by back flush in a single step. As shown in Fig. 2B, homogeneous ALD6p was eluted in 0 to 40% of buffer B. The purification tables for ALD4p and ALD6p are compiled in Table 1. The total amount of the overexpressed protein for ALD4p and ALD6p was quantified by subtracting the amount of total protein extract of the uninduced cells from that of the corresponding overexpressing cells, where both kinds of cells are of the same culture volume, 200 ml, and the same growing condition. The total protein
176
Notes & Tips / Anal. Biochem. 392 (2009) 174–176
Table 1 Purification of ALD4p and ALD6p by back flush. Total protein (mg)
Total activity (lmol/min)
Specific activity (lmol/min mg)
Yield (%)
Purification fold
ALD4p Cleared lysate Q Sepharose
156 5.4
3.3 1.2
0.021 0.22
100 36
1 10.5
ALD6p Cleared lysate Q Sepharose
216 3.5
1575 214
7.3 61
100 14
1 8.4
Note. The catalytic activity was determined at 22 °C with the following reaction mixtures: 0.6 mM propionaldehyde, 15 mM NADP+, 100 mM KCl, and 100 mM sodium phosphate (pH 7.4) for ALD4p; 1 mM propionaldehyde, 3 mM NADP+, 20 mM MgCl2, and 100 mM sodium phosphate (pH 7.4) for ALD6p. The progress of reaction was followed spectrophotometrically by absorbance at 340 nm using 6.22 mM 1 cm 1 for NADPH. Protein concentration was determined by the microassay method of Bradford [7].
in the cellular extract was precipitated and redissolved in urea [6] to eliminate the interference of nucleic acids in Bradford assay. The overexpressed protein obtained in this way was 13 mg for ALD4p and 18.5 mg for ALD6p. Originally, we managed to achieve one-step purification of recombinant Bud23p by engineering a His tag at the amino terminus. Unfortunately, the interaction between the tag and the chelating metal was too weak to recover pure Bud23p (data not shown), possibly due to a steric hindrance. We then needed to resort to the back-flush method. Initially, an attempt was made to purify Bud23p under the same conditions as for ALD4p and ALD6p; however, the separation was poor. Because the pH condition (7.6) used for ALD4p and ALD6p was higher than their pI values (6.07 and 5.18, respectively), whereas Bud23p has a theoretical pI of 9.69 (much higher than 7.6), we figured that by raising the pH of the buffer condition Bud23p may be better separated from the impurities. Therefore, buffers of pH 8.8 were employed for purification of Bud23p. As shown in Fig. 2C, Bud23p was indeed better separated from other impurities, particularly the fractions eluted in 40 to 44% of buffer D. The front fractions in buffer D lower than 40% almost did not contain any proteins according to SDS–PAGE analysis (not shown). The reason a buffer of pH higher than 9.69 was not used is that the Bud23p methyltransferase activity might not be tolerated at such a high pH. Interestingly, we noticed that all of the purified fractions of the three recombinant proteins were eluted earlier than other impurities that were left behind in the back fractions, that is, in the higher salt concentration range of the gradient. Thus, the empirical rule is that to achieve single-step purification by back flush on an anion exchanger with an increasing salt concentration gradient, the pH conditions used should be higher than the pI values of the target proteins; this is supposed to hold back the impurities on the column to be eluted behind the target proteins. Even the pH condition used for purification of Bud23p is still lower than the pI value of Bud23p; the relatively homogeneous protein could already be obtained in the front fractions. To further reveal the effects of the buffer pH values on the applicability of this method, purifications of ALD4p and ALD6p were also carried out in pH 6.0 (50 mM sodium phosphate, 0.05 M NaCl, 6 mM 2-mercaptoethanol, 1 mM EDTA, and 10% glycerol) and pH 8.8 (buffer C) buffers. With pH 6.0 buffer, both proteins failed to bind to the column (not shown). In contrast, with pH 8.8 buffer, both proteins retained so tenaciously to the column as to be eluted only along with contaminating proteins toward the high-salt range
(see Fig. S1 in supplementary material). Based on the pI values of ALD4p and ALD6p, a buffer 1.53 to 2.42 pH units higher than the pI value of the target protein may be recommended for back-flush purification. Considering that the distribution of the pI value and abundance of individual proteins in the mammalian cells are quite different from those of E. coli cells, as revealed in the two-dimensional polyacrylamide gel electrophoresis (2D–PAGE) profiles (see SWISS–2DPAGE, http://www.expasy.ch/ch2d, and references therein), and the overexpression levels of recombinant proteins in the mammalian cells are generally lower than those in the E. coli cells, the applicability of this method is likely limited when mammalian cells are used as the expression host. Finally, to investigate the effects of protein-to-resin ratios on the efficiency of purification, 100 and 50 ml of culture for purification of ALD6p were also tried on the fixed 30-ml bed volume of Q Sepharose using the pH 7.6 buffer. As shown in Fig. S2 and Table S1 in the supplementary material, an increase in the relative amount of resin tends to improve the yield of homogeneous ADL6p with the compromise of reduced concentration of purified ALD6p. Therefore, if the recovery of homogeneous ADL6p is to be optimized, 50 ml of culture to every 30 ml of resin is superior to the originally used 200 ml of culture. Taken together, the tips for single-step purification in anion exchange chromatography can be summarized as follows. First, calculate the pI value of the recombinant protein of interest or determine the pI value experimentally by isoelectric focusing analysis [6]. Second, choose a working pH higher than the pI value. Third, load the sample onto the column in the opposite direction to that in which it is eluted, and one can expect the homogeneous target protein to appear in the front fractions of elution. This study is, to the best of our knowledge, the first to demonstrate singlestep protein purification by the back-flush method. Acknowledgment This work was supported by grant NSC 97-2311-B032-001 from the National Science Council, Taiwan, Republic of China (to M-K. Chern).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ab.2009.05.045. References [1] J.C. Giddings, Unified Separation Science, Wiley-Interscience, New York, 1991. pp. 16–36. [2] P.R. Haddad, A.L. Heckenberg, Studies on sample preconcentration in ion chromatography, J. Chromatogr. 318 (1985) 279–288. [3] K.M. Roberts, D.T. Gjerde, J.S. Fritz, Single-column ion chromatography for the determination of chloride and sulfate in steam condensate and boiler feed water, Anal. Chem. 53 (1981) 1691–1695. [4] T. Okada, T. Kuwamoto, Trace analysis of anions by use of a back-flush method and large injection volumes in ion chromatography, J. Chrormatogr. 350 (1985) 317–323. [5] J.R. Blackwell, R. Horgan, A novel strategy for production of a highly expressed recombinant protein in an active form, FEBS Lett. 295 (1991) 10–12. [6] J.C. Anderson, S.C. Peck, A simple and rapid technique for detecting protein phosphorylation using one-dimensional isoelectric focusing gels and immunoblot analysis, Plant J. 55 (2008) 881–885. [7] G.L. Peterson, Determination of total protein, in: C.H.W. Hirs, S.N. Timasheff (Eds.), Methods in Enzymology, vol. 91: Enzyme Structure, Academic Press, New York, 1983, pp. 95–119.