Improved isolation of proteins tagged with glutathione S-transferase

Protein Expression and Purification 75 (2011) 161–164

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Improved isolation of proteins tagged with glutathione S-transferase Nicholas K. Vinckier, Arkadiusz Chworos, Stanley M. Parsons ⇑ Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA

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Article history: Received 14 July 2010 and in revised form 3 September 2010 Available online 16 September 2010 Keywords: Glutathione S-transferase S-butylglutathione Glutathione Fusion protein Gamma-hydroxybutyrate dehydrogenase GHB

a b s t r a c t A common affinity tag used to express and purify fusion proteins is glutathione S-transferase. However, many researchers have reported difficulty eluting GST-tagged proteins from the affinity matrix. This report demonstrates that the problem likely is due to the propensity of glutathione S-transferase to dimerize combined with a propensity of the tagged protein to oligomerize, which results in formation of large oligomers of fusion protein that are chelated by the affinity matrix. The solution to the problem is to use S-butylglutathione instead of glutathione to elute, as S-butylglutathione binds more tightly to glutathione S-transferase and overcomes the chelate effect. Moreover, in contrast to glutathione, S-butylglutathione has no reducing capability that might inactivate a tagged protein. Ó 2010 Elsevier Inc. All rights reserved.

Introduction An improved protocol for isolation of proteins tagged with glutathione S-transferase (GST)1 is reported using the example of gammahydroxybutyrate dehydrogenase (GHB-DH) from Ralstonia eutropha. GST is a desirable affinity tag, as it can inhibit incorporation of fusion protein into insoluble inclusion bodies when heterologous proteins are over expressed in host bacteria [1,2]. GST-tagged fusion proteins adsorb well to affinity matrices containing covalently bound GSH. After washing away contaminating proteins, the fusion protein sometimes can be eluted in essentially pure state using glutathione (GSH) [3]. The method has been used in both column and batch approaches. This laboratory developed an expression and purification procedure for GST/GHB-DH fusion protein that utilizes GSH for elution [4]. However, GHB-DH activity is inactivated by 15 mM GSH with a half-life of 8 h at 4 °C. Moreover, yields using the procedure have been erratic due to variable efficiency of elution, and the eluted protein is dilute. These circumstances require (a) immediate precipitation of eluted fusion protein with ammonium sulfate in order to concentrate it and remove most of the GSH, and (b) dialysis of the solubilized pellet of GST/GHB-DH in order to remove residual GSH before long-term storage. GST forms homodimers, which suggests that tagged proteins might at minimum form homodimers [5–9]. If the native tagged ⇑ Corresponding author. Fax: +1 805 893 4120. E-mail address: [email protected] (S.M. Parsons). Abbreviations used: GSC4, S-butylglutathione; GHB-DH, gamma-hydroxybutyrate dehydrogenase; GSH, glutathione; GST, glutathione S-transferase from Schistosoma japonicum; GST/GHB-DH fusion protein, GST fused at the C-terminus via a short peptide linker to the N-terminus of gamma-hydroxybutyrate dehydrogenase. 1

1046-5928/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2010.09.006

protein also forms oligomers, very large oligomers of the fusion protein might form. An example of linear polymerization of indefinite extent that could occur is shown in Fig. 1. However, depending on the relative positions of the interfaces mediating dimerization of GST and self-association of the tagged protein, highly oligomerized fusion protein might form non-linear shapes. An online search of scientific forums revealed that a number of researchers have had difficulty eluting GST-tagged proteins from affinity matrix [10,11]. The problem has not been investigated in detail. The only online suggestions were to use higher GSH concentrations, different salt concentrations, or different pH values. All of these changes could be deleterious to the tagged protein. Several researchers have suggested in peer-reviewed publications that GST is unsuitable for tagging oligomeric proteins, but no further comment was made as to why [9,12]. GHB-DH is an Fe(II)-dependent alcohol dehydrogenase [13]. It has sequence homology to other Fe(II)-dependent alcohol dehydrogenases that form dimers, tetramers, hexamers, and decamers [14–19]. Thus, GST/GHB-DH is a candidate for formation of large oligomers. Because they generally are very large at the molecular scale, the pores in affinity matrices likely can accommodate even large oligomers and present multiple immobilized GSH ligands to them. Such a circumstance could create a strong chelate effect when the oligomer is a GST-tagged protein. As the dissociation constant for GSH binding to GST is only about 100 lM [20], it is not surprising that accessible concentrations of GSH cannot compete against affinity matrix well enough to release highly oligomerized fusion proteins. S-alkylated derivatives of GSH bind to GST tighter than GSH does [6,21,22]. They have been used to elute GST itself from affinity columns

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GHB

GSH

GHB

GSH

GHB

GSH

GHB

GSH

Fig. 1. Hypothetical polymerization scheme for the GST/GHB-DH fusion protein. A rectangle is GST, a rectangle with rounded corners is GHB-DH, and the thick horizontal line between them is the covalent linker region in the fusion protein. Each type of monomer forms a non-covalent, face-to-face homodimer having a C2 symmetry axis through the center of each dimer and perpendicular to the plane of the drawing. A filled monomer is the backside of a shaded monomer in a homodimer. The type of ligand binding site in each monomer is indicated.

[6]. Observations presented here demonstrate that oligomerization of the tagged protein is a likely source of difficulty in elution of GST-tagged proteins. A simple solution is use of S-butylglutathione (GSC4) instead of GSH. Materials and methods Materials Bradford reagent was obtained from Bio-Rad Laboratories, Inc (Hercules, CA). Tween 20Ò and tris(hydroxymethyl)aminomethane (tris base) (Molecular Biology Grade) were obtained from Thermo Fisher Scientific Inc (Pittsburg, PA). Aprotinin was obtained from MP Biomedicals LLC (Solon, OH). Gamma-hydroxybutyric acid (GHB), benzamidine, phenylmethylsulfonyl fluoride (PMSF), isopropyl b-D-1-thiogalactopyranoside (IPTG), bovine pancreatic deoxyribonuclease I (DNase I, 569 Kunitz units/mg protein), chicken egg white lysozyme, phenylmethanesulfonyl fluoride (PMSF), 3-(N-morpholino)propanesulfonic acid (MOPS), sodium 3,3-[(phenylamino) carbonyl]-3,4-tetrazolium-bis(4-methoxy-6nitro) benzenesulfonic acid hydrate (XTT), nicotinamide adenine dinucleotide (NAD+), phenazine methosulfate (PMS), GSH-agarose affinity matrix (lyophilized powder stabilized with lactose), and GSC4 were obtained from Sigma–Aldrich Co (St. Louis, MO). All other chemicals were obtained in the highest standard purity from common suppliers. Assay for GHB-DH activity GHB-DH activity was measured by adding a 5 lL sample to a plastic cuvette (1 cm path length) containing 1 mL of assay medium consisting of 0.16 mM XTT, 1.0 mM NAD+, 16.0 mM GHB, and 0.05 mM PMS in 100 mM Tris/HCl buffer at pH 8.5 and 23 °C. Absorbance increase at 450 nm was measured for 60 s (standard assay). Background activity was determined without addition of enzyme and subtracted from reported activity. It was very small in all cases. Improved isolation of GST/GHB-DH fusion protein E. coli harboring recombinant pGEX-2T vector was grown at 37 °C in 1 L of LB medium containing 50 lg ampicillin/mL [4]. The vector coded for GST from Schistosoma japonicum fused to a short linker that in turn was fused to the N-terminus of GHB-DH (R. eutropha). When the culture reached an apparent absorbance of 1 at 650 nm, the temperature was lowered to 19 °C, and 0.1 mM IPTG was added to the vigorously shaking cells to induce expression of fusion protein. After shaking for 20 h, 1.0 mM benzamidine and 1.0 mM PMSF were added to inhibit proteolysis. Cells

were harvested by centrifugal pelleting at 2740g for 30 min. Many buffers used in the rest of the procedure are based on Buffer A (30 mM MOPS adjusted to pH 7.4 with NaOH). Pelleted cells were resuspended in 20 mL of ice-cold Buffer A that also contained 50 mM NaCl, 0.6 lM aprotinin, 1 mM PMSF and 1.5 mM benzamidine. Cells were loaded into a 50 mL plastic Falcon™ tube with about 20 mg of lysozyme. The tube was capped and submerged in liquid nitrogen until the suspension froze, after which it was placed in room-temperature water until the suspension thawed. The tube was opened and about 20 mg of DNase I was added, after which the tube was capped, and the suspension was frozen and thawed. Lysed cells were centrifuged (17,600 g for 30 min) to remove debris. The supernatant was filtered through a 1.2 lm MF-Millipore™ membrane filter. Subsequent steps were conducted at 4 °C. Fusion protein was isolated by affinity chromatography on a column packed with 30 mL bed volume of GSH-agarose matrix equilibrated with Buffer A (also containing 50 mM NaCl). Clarified supernatant was flowed into the column and allowed to adsorb for about 20 min. Buffer A (20 mL) containing 0.5 M NaCl and 1% Tween 20Ò was flowed through the column followed by Buffer A (20 mL). Then, 20 mL of 30 mM MOPS adjusted to pH 7.0 with NaOH (Buffer B) containing 10 mM GSC4 was flowed into the column and allowed to incubate for 1 h, after which more of the same elution buffer was flowed slowly into the column. Eluate fractions (5 mL) were collected and immediately assayed for GHB-DH activity until little was found. Finally, Buffer A (30 mL) containing 0.2% NaN3 was flowed into the column for long-term storage of the affinity matrix. Fractions containing most of the GHB-DH activity (typically about 50 mL) were pooled, after which GHB-DH activity and protein content in the pool were measured. The pool was divided into 2 mL aliquots in sealed cryotubes, quick-frozen in liquid N2, and stored at 80 °C. Size exclusion chromatography-multiple angle light scattering (SEC-MALS) Buffer B was filtered using a 0.1 lm Millipore StericupÒ (1 L capacity) before being placed at 0.9 atm of vacuum overnight to remove dissolved air. Purified GST/GHB-DH fusion protein (200 lL) was centrifuged at 16,000g for 10 min to remove particulates. After flushing the SEC-MALS system thoroughly with Buffer B, 100 lL of purified fusion protein was injected and chromatographed at 0.5 mL/min for 45 min on a size exclusion column of 0 300 Å A pore size (Wyatt WTC-030S5). The eluate was passed through sequential monitors measuring absorbance at 280 nm, light scattering at 658 nm (Wyatt miniDAWN™ TREOS MALS instrument), and change in refractive index at 658 nm (Wyatt OptilabÒ T-rEX refractometer). The intensity of scattered light collected by the LS detector is directly proportional to the weight-average molecular weight (Mw) and the solute concentration (c). The RI signal detected is also directly proportional to the solute concentration and together these measurements were used to estimate shape-independent molecular weight [23,24]. Statistics Quoted errors are one standard deviation. Results and discussion Typical elution profiles from the affinity column for GHB-DH activity in the GST/GHB-DH fusion protein isolated by the old and new protocols are shown in Fig. 2. In both protocols, some GHB-DH activity does not adsorb to the column and flows directly through. In the old protocol, the column was extensively washed,

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1.1 ΔRI

Old Isolation New Isolation

1.5

(67.5 kDa)

(66 kDa)

1.0 GSH

GSC4

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Relative Intensity .

A450/min/5μL

2.0

0.7

0.3

UV LS

-0.1

0.0 0

20

40

60

80

100

120

10

Fig. 2. Elution profiles for GHB-DH isolations using both old and new elution protocols. In the old protocol (- - - -), 15 mM GSH was used to elute, and in the new protocol (—), 10 mM GSC4was was used to elute. Inset is SDS–PAGE of pooled GST/GHB-DH fusion protein (left lane) isolated by the new protocol and bovine serum albumin (right lane).

and fusion protein was eluted with 15 mM GSH. The amounts of purified activity obtained in different preparations were quite variable, meaning that some preparations were very disappointing. A large amount of GHB-DH activity remained bound to the affinity matrix in those cases, as shown by (a) opening the top of the column, (b) manually removing a few microliters of the matrix, and (c) assaying the matrix by suspending it in the standard assay solution. In the improved protocol, after brief washing of the loaded affinity column, fusion protein was eluted with 10 mM GSC4, which binds GST 25-fold tighter than GSH does [20,22,25]. From 1-L cultures of transformed and induced bacteria, 161 ± 32 mg of purified fusion protein at 3.2 ± 0.9 mg/mL in a total of 52 ± 6.5 mL having a specific activity of 44 ± 17 mA450/lg/min is obtained (n = 6). This is 3-fold more protein and higher specific enzymatic activity than the averages obtained by the original protocol. There is no need to concentrate the purified fusion protein by precipitation or remove harmful GSH by dialysis. Overall the new protocol is far superior. However, in addition to the buffer components, the new preparation contains 10 mM of GSC4. In most applications this does not matter. An isolation using S-octylglutathione, which binds to GST tighter than GSC4 does, also was attempted, but this ligand is a surfactant that causes troublesome foaming. Fig. 2 inset shows an SDS–PAGE of fusion protein obtained with the improved protocol. The predicted molecular weight of the fusion protein based on its amino acid sequence is 67.5 kDa, which agrees with its slightly slower mobility relative to BSA (66 kDa). Trace amounts of two contaminating polypeptides were present. These might be DNase I (30.1 kDa) and lysozyme (14.3 kDa) added to assist freeze–thaw lysis of the bacteria and viscosity lowering. Thrombin does not cleave in the linker between the GST and GHB-DH domains under these conditions (not shown). Because native GST from S. japonicum forms homodimers, the minimal molecular weight of native GST/GHB-DH fusion protein likely is 135 kDa [5,7,8]. The actual molecular weight distribution was determined by several methods. Fig. 3 shows superimposed profiles for light scattering (LS), absorbance at 280 nm (UV), and change in refractive index (DRI) of purified fusion protein chromatographed on a size exclusion column. Four main peaks of protein eluted at 13, 14, 15, and 16.6 min. The weight-average molecular weights (Mw) for the peaks are 2000, 650, 400, and 330 kDa, respectively. The values are computed rather than interpolated from the elution volumes of standards and are independent of particle shape. They correspond to oligomers of fusion protein containing about 30, 10, 6, and 5 monomers.

12

14

16

18

20

Time (min)

Volume Eluted (mL)

Fig. 3. Size exclusion chromatography of purified GST/GHB-DH. Light scattering (LS), absorbance at 280 nm (UV), and change in refractive index (DRI) are shown. A BSA control exhibited a major monomer peak of 66 kDa and a minor dimer peak, as expected.

Dynamic-light-scattering measurements indicated the presence of particles spanning a similar Mw range (not shown). Atomic force microscopy of 100-fold diluted fusion protein also indicated the presence of particle sizes ranging from dimers to hexamers (not shown). The evidence demonstrates that native GST/GHB-DH fusion protein exists as discrete oligomers of various sizes. The prominent presence of larger oligomers indicates that GHB-DH interacts with itself. Taken together, the observations indicate that difficulty eluting GST-tagged proteins from affinity matrix arises when they form large oligomers due to a propensity of the tagged protein to form oligomers. Such large oligomers probably allow the affinity matrix to chelate the tagged protein. Nevertheless, the greater affinity of GSC4 compared to GSH outcompetes even the chelate effect. Any tag might destabilize a protein, and whether this event happens must be determined empirically. For example, a large tag might block formation of stabilizing oligomers by the tagged protein. That possibility apparently is not a problem in GHB-DH from R. eutropha, as properly stored GST/GHB-DH fusion protein is stable indefinitely. However, even small tags can present problems. In our original cloning effort, N-terminal tagging with hexa-histidine yielded no GHB-DH activity (although protein was expressed), possibly because the tag blocked loading of Fe(II) into the nascent dehydrogenase. Furthermore, some proteins contain critical cysteines, disulfides or even mixtures of the two that must be maintained in a proper redox state during purification to obtain active protein [26]. Elution with GSC4 can be carried out under nonreducing or reducing conditions by inclusion of simple thiolcontaining compounds like beta-mercaptoethanol, whereas elution with GSH is only reducing. Often a researcher does not know whether a novel protein exhibits any of the sensitivities just described when first tagging and isolating it. Availability of multiple options for tagging can be important to finding one that works. GSC4 broadens the range of applications for which GST tagging is appropriate, and it can shorten the purification procedure with concomitant improvements in yield and specific activity. We recommend routine consideration of GSC4 instead of GSH for elution of GST-tagged proteins. Acknowledgments We thank Armand Vartanian, Javin Oza, and Krystyna Brzezinska for assistance in size exclusion chromatography and light-scattering measurements. We thank Ethan McSpadden and Joey Ostrand for assistance in isolating protein and determining the half-life for inactivation of GHB-DH at 4 °C by 15 mM GSH.

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