Expression in Escherichia coli and Purification of Hexahistidine ...

Protein Expression and Purification 24, 366–373 (2002) doi:10.1006/prep.2001.1587, available online at http://www.idealibrary.com on

Expression in Escherichia coli and Purification of Hexahistidine-Tagged Human Tissue Transglutaminase Qingli Shi,*,†,1 Soo-Youl Kim,*,† John P. Blass,*,†,‡ and Arthur J. L. Cooper*,†,§ *Department of Neurology and Neuroscience, ‡Department of Medicine and §Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021; and †Burke Medical Research Institute, White Plains, New York 10605

Received August 10, 2001 and in revised form November 18, 2001

Recent evidence suggests that aberrant transglutaminase activity is associated with a wide variety of diseases. Tissue transglutaminase is the most widely distributed of the six well-characterized transglutaminases in humans. We describe a method for expressing hexahistidine-tagged human tissue transglutaminase in Escherichia coli BL21(DE3) using the pET-30 Ek/LIC expression vector. Purification of the expressed enzyme from suspensions of E. coli cells treated with CelLytic B Bacterial Cell Lysis/Extraction Reagent was accomplished by immobilized metal (Ni2+) affinity column chromatography. The procedure typically yields highly purified and highly active recombinant human tissue transglutaminase in about 1 day (about 0.6 mg/ from a 1-liter culture). 䉷 2002 Elsevier Science (USA) Key Words: hexahistidine tag; human tissue transglutaminase; immobilized metal affinity chromatography.

Transglutaminases (TGases)2 are a group of enzymes that catalyze the Ca2+-dependent covalent attachment of the carboxamide moiety of a glutaminyl (Q) residue 1 To whom correspondence and reprint requests should be addressed at Burke Medical Research Institute, 785 Mamaroneck Avenue, White Plains, NY 10605. Fax: (914) 597 2757. E-mail: qshi@ burke.org. 2 Abbreviations used: bp, basepairs; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; his6, exahistidine; HRP, horseradish peroxidase; htTGase, human tissue transglutaminase; IMAC, immobilized metal affinity chromatography; CGG, N-␣ -carbobenzoxyglutaminylglycine; IMAC, immobilized metal affinity chromatography; IPTG, isopropyl ␤ -D-thiogalactopyranoside; LB, Luria–ßertani; LIC, ligation-independent cloning; NTA, nitrolotriacetic acid; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PVDF, polyvinylidene difluoride; rhtTGase, recombinant human tissue transglutaminase; SDS, sodium dodecyl sulfate; TBS, Tris-

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of a protein/polypeptide (acyl donor, amine acceptor) to the ␧ -amino group of a lysyl (K) residue of a protein/ polypeptide (acyl acceptor, amine donor) (1–3). The acyl donor may also be an amine, diamine, or polyamine (1–3). Tissue transglutaminase (tTGase; TGase 2) is the most ubiquitous of the six human TGases that have been well characterized (3). Increased or defective tTGase activity may contribute to, or be a factor in, many pathological processes. They include cataracts, atherosclerosis, inflammation, fibrosis, diabetes, cancer metastases, celiac disease, autoimmune diseases, lamellar ichthyosis, and psoriasis (reviewed in ref. 4). Recent evidence suggests that TGases may also play a role in neurodegenerative diseases, such as Huntington disease, Alzheimer disease, Parkinson disease, and supranuclear palsy. (For recent reviews see refs. 4–10.) Because of the apparent role of TGases in neurodegenerative diseases, several authors have suggested that inhibitors of these enzymes (especially tTGase) may have clinical benefit (e.g., 5–13). One general method for obtaining inhibitors of target enzymes is to rapidly screen large chemical libraries. In order to carry out such screening for human tTGase (htTGase) it is important to obtain the enzyme in relatively high yield and purity. Several procedures for purifying tTGases with high specific activity have been published. For example, highly purified tTGase has been obtained from guinea pig liver by classical purification procedures (14–17).

buffered saline; TCA, trichloroacetic acid; TGase, transglutaminase; TTBS, Tris-buffered saline containing 0.05% v/v Tween 20; tTGase, tissue transglutaminase. 1046-5928/02 $35.00 䉷 2002 Elsevier Science (USA) All rights reserved.

PURIFICATION OF HIS6-RECOMBINANT HUMAN TISSUE TRANSGLUTAMINASE

As much as 25 mg can be obtained from 140 g of guinea pig liver (17). Guinea pig liver tissue transglutaminase has also been expressed in Escherichia coli and purified (18). The expressed protein lacks the N-terminal acetyl group found in the natural liver enzyme but is kinetically indistinct from the natural enzyme (18). A tissuetype TGase from red sea bream (Pagrus major) has been cloned, purified, and sequenced (19, 20). tTGase has been obtained from human erythrocytes (21, 22) and cultured human A431 tumor cells (23). Particularly relevant to the present work is the study of Lee et al. (24), who showed that it is possible to express human recombinant TGase (hrtTGase) in E. coli and purify the active enzyme. Building on the previous work of Lee et al., it should be possible to obtain recombinant htTGase (rhtTGase) in high yields by using the approach of attaching a terminal hexahistidine (His6) tag followed by immobilized metal affinity chromatography (IMAC). Here we report a method for bacterial expression of rhtTGase tagged at the N terminus with His6, and for rapid purification of the expressed enzyme. MATERIALS AND METHODS Materials A fibroblast cDNA library was obtained from Clontech (Palo Alto, CA). The bacterial (E. coli) host and the cloning vector pET-30 Ek/LIC (which contains the nucleotide sequence coding for six histidines) were obtained from Novagen (Madison, WI). Taq polymerase and nucleotides were purchased from Roche (Indianapolis, IN). Primers were synthesized at Invitrogen (Carlsbad, CA). The kit for cycle sequencing was purchased from Perkin–Elmer (Foster City, CA). The polymerase chain reaction (PCR) purification kit, gel extraction kit, and Ni-NTA Superflow column matrix were obtained from QIAGEN (Valencia, CA). The HisTrap kit and [1,4-14C]putrescine dihydrochloride (111 ␮Ci/ ␮mol) were purchased from Amersham Pharmacia Biotech Inc. (Piscataway, NJ). Tris–HCl Ready gels (10% polyacrylamide) and prestained SDS–PAGE standards (broad range) were from Bio–Rad (Hercules, CA). GelCode Blue Stain Reagent and IgG (H ⫹ L) antibody labeled with rabbit horseradish peroxidase (HRP) were from Pierce (Rockford, IL). Anti-tTGase polyclonal antibody was from Abcam Ltd. (Cambridge, UK). CelLyticB Bacterial Cell Lysis/Extraction Reagent, Luria– Bertani (LB) broth, isopropyl ␤ -D-thiogalactopyranoside (IPTG), kanamycin, Sigma-Fluor High Performance liquid scintillation cocktail, sodium dodecyl sulfate (SDS), dithiothreitol (DTT), Tris, glycine, N-␣ carbobenzoxyglutaminylglycine (CGG), hydroxylamine HCl, N,N-dimethylcasein, ethylenediaminetetraacetic acid (EDTA), imidazole, semipurified guinea pig liver tissue transglutaminase (lyophilized powder, 1.5–3.0 units/mg of protein in the hydroxamate reaction; see

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below), and bromophenol blue were obtained from Sigma (St. Louis, MO). Construction of Expression Plasmid The tTGase cDNA sequence was obtained via nested PCR amplification using a human fibroblast cDNA library. (See Table 1 for primers.) The first round of PCR amplification was performed using PCR primers (P1 and P2, Table 1), the design of which was based on sequences located between ⫺72 and 2388 bp of the htTGase gene. The ⫹2388 bp is located very near the poly(A) tail. The PCR product was purified by means of a QIAquick PCR purification kit (QIAGEN) and used as a template for the second round of PCR amplification. The TGase cDNA construct was designed for insertion into the pET-30 Ek/LIC expression vector (Novagen). This vector contains an initiation codon under the direction of an IPTG-inducible T7 lac promoter. The primers used in the second round of PCR amplification (P3 and P4, Table 1) were designed to contain nucleotide sequences complementary to the sequences within the extensions of the pET-30 Ek/LIC vector. The conditions used for both PCR amplifications were 95⬚C for 4 min followed by 30 cycles at 95⬚C for 30 s, 55⬚C for 30 s, 72⬚C for 1 min. After amplification, the PCR product was purified using a QIAquick PCR purification kit and ligated into the expression vector. The vector was then transformed into the host cell [E. coli BL21(DE3)]. For verification purposes, the rhtTGase insert was sequenced. Expression of His6-rhtTGase The bacteria containing the desired clone inserted into the pET-30 Ek/LIC expression vector were grown overnight at 37⬚C in 50 ml of LB medium supplemented with 50 ␮g/ml of kanamycin. A portion (10 ml) of the bacterial suspension was then subinoculated into 1 liter of fresh LB medium and the bacteria were allowed to grow at 37⬚C until the optical density of the medium at 600 nm reached 1.0. Thereafter, the expression of TABLE 1 DNA Oligonucleotide Primers Primer P1 P2 P3 P4

Sequence (5⬘ → 3⬘) CCC CCT TAA AGC ATA AAT CTC TTA GGC GGG GCC AAT GAT GAC GAC GAC GAC AAG ATG GCC GAG CTG GTC TTA GAG AGG TGT GAT CTG GAG GAG AAG CCC GGT TAT CAA ATG ATG ACA TTC CGG AAG CCC TT

Note. P1 and P2 were used in the first round of PCR amplification and P3 and P4 were used for nested PCR cloning. The underlined letters in P3 and P4 are complementary extensions compatible to sequences in the pET-30 Ek/LIC cloning vector.

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the rhtTGase was induced by addition of IPTG to a final concentration of 1 mM. After a further 3-h incubation at 37⬚C, the bacterial cells were collected by centrifugation for 10 min at 5000g (see Results). The rhtTGase appeared to be resistant to whatever endogenous proteases were present, and addition of proteases inhibitors was found not to increase yield.

Cell Lysate Preparation Using Different Disruption Methods After induction of his6-rhtTGase by IPTG for 3 h at 37⬚C, the 1-liter bacterial suspension was divided into five portions. Each 200-ml portion was centrifuged at 5000g for 10 min, and each pellet was resuspended in ⬃10 ml of 50 mM Tris-acetate buffer (pH 8.0) containing 0.5 mM EDTA and 10 mM DTT. The cells were then repelleted by centrifugation and the pellets were stored at ⫺80⬚C. To compare the efficiency of different disruption methods, individual pellets were suspended with 10 ml of 20 mM Tris-acetate buffer (pH 8.0) containing about 20 mg of lysozyme (preparation A), or 10 ml of 20 mM Tris-acetate buffer (pH 8.0) (preparation B), or 10 ml of CelLyticB Bacterial Cell Lysis/Extraction Reagent (preparation C). The cells in preparations A–C were disrupted by incubation on ice for 30 min, sonication at 0⬚C, and incubation at room temperature for 15–20 min, respectively. The cell debris was removed by centrifugation at 10,000g for 10 min. The tTGase activities of the supernatants from the three different disruption methods were compared using a fluorometric assay (see Enzyme Assays below).

Purification of His6-rhtTGase Unless otherwise stated, all procedures were carried out at 0–4⬚C. The packed cells from each 200-ml portion (⬃1g) were suspended in 10 ml of the CelLyticB Bacterial Cell Lysis/Extraction Reagent and incubated at room temperature for 15–20 min. After centrifugation, the supernatant was then applied to a 2-ml column of Ni-NTA equilibrated with 20 mM Tris-acetate, 0.5 M NaCl, pH 8.0 (buffer A), containing 10 mM imidazole, and the column was washed with 20 ml of buffer A containing 10 mM imidazole followed by elution with 10 ml of buffer A containing 100 mM imidazole. The 100 mM imidazole fraction was dialyzed against 1 liter of buffer A containing 10 mM imidazole for 2 h. The dialyzed sample was then applied to a 1-ml HisTrap column equilibrated with buffer A containing 10 mM imidazole. The column was eluted successively with buffer A containing 10 mM imidazole (20 ml), 20 mM imidazole (5 ml), 80 mM (5 ml), and 100 mM (5 ml). Fractions of 1 ml were collected.

Stability of His6-Tagged rhtTGase In order to study the stability of His6-tagged rhtTGase, the purified enzyme was dialyzed against 50 mM Tris-acetate buffer (pH 8.0) for about 2–3 h at 4⬚C and the dialyzed enzyme was then divided into three portions. The buffer in each portion was adjusted as follows: Buffer I contained 25 mM Tris-acetate buffer (pH 8.0), 0.5 mM EDTA, and 10 mM DTT; buffer II contained 25 mM Tris-acetate buffer (pH 8.0), 1% (w/ v) bovine serum albumin, and 30% (v/v) glycerol; buffer III contained 25 mM Tris-acetate buffer (pH 8.0) and 30% (v/v) glycerol. Aliquots were stored at 4⬚C and ⫺80⬚C, respectively. At days 0, 3, 5, and 10, the tTGase activities of the aliquots were determined using the fluorometric assay (see Enzyme Assays below). SDS–PAGE The protein sample (typically 10 ␮l) was mixed with 10 ␮l of loading buffer [100 mM Tris–HCl (pH 6.8), 200 mM DTT, 4% w/v SDS, 0.2% w/v bromophenol blue, and 20% v/v glycerol] and boiled for 5 min prior to loading onto a Tris–HCl ready gel (10% polyacrylamide) for electrophoresis. Both the upper and lower reservoirs contained 25 mM Tris, 192 mM glycine, and 0.1% w/v SDS. The conditions were voltage, 150 V; run time, 1 h; temperature, ambient. Proteins in the gel were visualized by staining with GelCode Blue reagent (Coomassie G-250) according to the instructions of the manufacturer (Pierce). Western Blotting For Western blotting, following SDS–PAGE the gel was soaked in transfer buffer (48 mM Tris, 39 mM glycine, 0.037% w/v SDS in 20% v/v methanol) and a polyvinylidene difluoride (PVDF) membrane (Pierce) was soaked in 100% methanol. Protein transfer from the gel to the PVDF membrane was accomplished electrophoretically (0.65 mA/cm2 for 2 h at 4⬚C). The PVDF membrane was then blotted with Superblot buffer (Pierce) at 4⬚C overnight, followed by washing three times with TTBS [Tris-buffered saline (TBS; 137 mM NaCl, 2.7 mM KCl, 25 mM Tris, pH 7.4) containing 0.05% v/v Tween 20] for 5 min each wash. Next, the membrane was incubated with rabbit anti-tTGase polyclonal antibody (Abcam Ltd., Cambridge, UK) (1:1000) in the Superblot buffer for 1 h at room temperature. The membrane was then washed extensively three times with TTBS for 5 min each time, followed by incubation with rabbit IgG (H ⫹ L) antibody conjugated with horseradish peroxidase (HRP) (1:5000) for 1 h at room temperature. The unbound HRP-conjugated secondary antibody was removed by thorough washing (⫻6) with TTBS for 10 min each wash. Finally, the membrane was incubated with SuperSignal West Pico


Substrate working solution (Pierce) for 10 min and then pressed against an autoradiographic film (Kodak) for 10 s to 1 min in a cassette. The film was removed and developed in a Kodak X-Omat M20 Processor. Enzyme Assays rhtTGase was routinely assayed by a fluorometric method (9, 25). The assay mixture (final volume 200 ␮l) contained 0.5 mM dansylcadaverine (acyl acceptor), 0.2 mg of N,N-dimethylcasein (acyl donor), 10 mM CaCl2, 10 mM DTT, and 100 mM Tris–HCl (pH 7.5). The reaction was started by the addition of enzyme. The covalent attachment of dansylcadaverine to the N,N-dimethylcasein was monitored fluorometrically (␭exc, 280 nm; ␭em, 538 nm) at 37⬚C in a 96-well plate fluorometer (SpectraMax Gemini, Molecular Devices, Sunnyvale, CA). The blank lacked rhtTGase. After an initial lag of about 5 min, the reaction was linear for at least 1 h. Although the fluorometric assay is very sensitive and well suited for multiple, routine analyses, the relative increase in fluorescence intensity is not easy to convert to a quantitative measure of enzyme turnover/specific activity. For this reason, two alternative methods were used to determine the specific activity of rhtTGase (26): (a) a [14C]putrescine-binding assay and (b) a hydroxamate assay. In the [14C]putrescine-binding assay, which was modified from the procedure of Lorand et al. (27), the reaction mixture (100 ␮l) contained 100 ␮g of N,N-dimethylcasein (acyl donor), 200 mM Tris–HCl (pH 7.5), 10 mM DTT, 10 mM CaCl2, 0.5 mM putrescine, and 2 ␮l of [1,414 C]putrescine (0.45 ␮mol/ml; 111 ␮Ci/mmol; Amersham). The reaction was initiated by addition of rhtTGase. After incubation at 37⬚C for 20 min, the reaction was terminated by the addition of 25 ␮l of 50% w/ v trichloroacetic acid (TCA). After 30 min at 0⬚C, the precipitated protein was pelleted by centrifugation at 10,000g for 15 min. The supernatant was removed and the pellet was washed twice with 10% w/v TCA. The pellet was then dissolved in 100 ␮l of 1 M NaOH. The NaOH solution containing N,N-dimethylcasein covalently linked to labeled putrescine was transferred to a 10-ml liquid scintillation vial containing 5 ml of SigmaFluor High Performance liquid scintillation cocktail. Each sample was counted in a liquid scintillation counter (Beckman). The blank was a reaction mixture containing water in place of enzyme. Assays were carried out in duplicate. The hydroxamate assay is based on the ability of tTGase to catalyze the nucleophilic attack of hydroxylamine (acyl acceptor) on CGG (acyl donor) to form a ␥ -glutamyl hydroxamate. ␥ -Glutamyl hydroxamates yield a stable brown color in the presence of acidic FeCl3 (Emax 535 nm; 850 M⫺1; ref. 28). The method is derived

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from that of Folk and Cole (1) as modified by Cooper et al. (26) for multiple well plate analyses. Protein was measured by the bicinchoninic acid procedure using bovine serum albumin as a standard according to the instructions of the manufacturer (Pierce). RESULTS AND DISCUSSION Expression and Purification of His6-rhtTGase After induction with IPTG and centrifugation, the bacterial cell pellet could be frozen and stored at ⫺80⬚C for at least several months without apparent loss in enzyme activity. SDS–PAGE analysis, Western blotting, and enzymatic analyses of the supernatant and cell debris after thawing of the bacterial pellet and disruption of the cellular envelope showed that the majority (⬎90%) of the his6-tagged rhtTGase was in the form of inactive, insoluble inclusion bodies (data not shown). In an attempt to minimize the formation of inclusion bodies and increase the yield of soluble, active enzyme, we carried out several experiments in which the temperature of the incubation was decreased from 37⬚ to 30⬚C after addition of IPTG. However, this procedure did not result in any significant improvement in yield of active enzyme or decrease of inclusion bodies. Different concentrations of IPTG (0.5, 1, 2, and 5 mM; 3 h exposure) were also compared for their effectiveness in inducing maximal expression of soluble, active His6rhtTGase. Induction with 1 mM IPTG gave the highest expression of active enzyme (data not shown). Because the majority of the enzyme was present in the cells as inactive, inclusion bodies we attempted various procedures to convert inclusion bodies to soluble, active enzyme. However, all attempts (e.g., treatment with concentrated urea followed by slow dialysis, incubation with Ca2+, as well as ATP) failed to generate active enzyme from the inclusion bodies. While our work was in progress, Ambrus and Fe´su¨s (29) reported that polyethylene glycol enhanced the refolding of recombinant human tissue transglutaminase in inclusion bodies. These authors also mentioned the roles of Ca2+ and nucleotides in the early phase of structural reconstitution. In future studies we will attempt to use the polyethylene glycol procedure to reconstitute active rhtTGase from the inactive inclusion bodies. Three procedures for disruption of the cellular envelop were compared to obtain the best yield of rhtTGase from the bacterial pellets, namely (a) treatment with lysozyme, (b) sonication, and (c) treatment with CelLyticB Bacterial Cell Lysis/Extraction Reagent. Empirically, it was found that treatment with CelLyticB Bacterial Cell Lysis/Extraction Reagent gave the best yield of soluble, active rhtTGase. In a representative experiment, the relative yields from lysozyme treatment, sonication, and treatment with CelLyticB Bacterial Cell Lysis/Extraction Reagent were about

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1:2.5:3.5, respectively. Typically, a 200-ml bacterial suspension yielded about 1 g of packed cells. Purified enzyme was obtained from the cell pellet by treatment with CelLyticB Bacterial Cell Lysis/Extraction Reagent and IMAC as described under Materials and Methods. We evaluated several elution protocols for IMAC, including increasing the concentration of imidazole in the eluting buffer in a stepwise fashion or as a gradient. In addition we tried using a Ni-NTA column only, a HisTrap column only, or a combination of the two. It was found empirically that addition of the bacterial homogenate directly to a Ni-NTA column, followed by isochratic elution with a buffer containing 10 mM imidazole, followed by buffer containing 100 mM imidazole gave a preparation of rhtTGase of the highest specific activity from the first column. Western blotting of an aliquot (10 ␮l) of the protein eluted with 100 mM imidazole from the first IMAC (i.e., Ni-NTA) column showed a protein band with the Mr expected for hrtTGase (⬃80,000). However, protein staining showed that although the specific activity of the rhtTGase in the 100 mM imidazole wash was considerably greater (⬎25-fold) than that in the starting homogenate, several protein contaminants were still present. In order to remove these contaminants, the enzyme in the 100 mM imidazole fraction was rapidly dialyzed and then applied to a different IMAC (i.e., HisTrap) column. After eluting the second column with buffer A containing successively increasing concentrations of imidazole, the fraction eluting with 100 mM imidazole was shown to contain pure rhtTGase by both SDS–PAGE and Western blotting analyses. A summary of a typical purification procedure is shown in Table 2. SDS–PAGE followed by protein staining or Western blotting of aliquots of the bacterial suspension and purified protein are shown in Fig. 1. There are examples in the literature where a single IMAC step is insufficient to remove bacterial protein and/or endotoxins (e.g., 30, 31).

Comparison of the Activity of Highly Purified His6Tagged rhtTGase with Other Preparations of tTGase The His6-tagged rhtTGase activity was determined by three methods (Table 2). The specific activity of highly purified His6-tagged rhtTGase was ⬃76 nmol/ min/mg ([14C]putrescine-binding assay), ⬃98,000 RFU/ min/mg (fluorometric assay) and ⬃730 nmol/min/mg (hydroxamate assay), respectively. The purification was about 100- to 140-fold compared to the activity in the supernatant from the lysed bacteria and recovery of pure enzyme from the bacterial homogenate was about 20–30%. The specific activity of the highly purified rhtTGase obtained with the hydroxamate assay (⬃0.7 ␮mol/min/

TABLE 2 Purification of Recombinant His6-Tagged Human Tissue Transglutaminase (rhtTGase) from an E. coli Expression Systema Purification step Total protein (mg): Total activity 14 C assay (nmol/min) Fluorometric assay (RFU/min) Hydroxamate assay (nmol/min) Specific activity 14 C assay (nmol/min/mg) Fluorometric assay (RFU/min/mg) Hydroxamate assay (nmol/min/mg) Relative yield 14 C assay Fluorometric assay Hydroxamate assay Purification (fold) 14 C assay Fluorometric assay Hydroxamate assay

S 63.8

E1 2.2

E2 0.128

33.3 29.4 9.72 59,800 41,900 12,500 328 209 93 0.52 13.4 75.8 937 19,000 97,900 5.1 95 727 [100] [100] [100] 1 1 1

88 70 64

30 21 28

26 20 17

146 104 142

a

From 200 ml of E. coli culture. S, E1, and E2 represent the supernatant from lysed bacterial cells, the first IMAC step, and the second IMAC step, respectively.

mg, Table 2) is surprisingly low. Semipurified commercial guinea pig liver tTGase (Sigma) is reported to have a specific activity of about 1.5–3.0 ␮mol/min/mg in the hydroxamate assay, which we have verified (26). The specific activity of highly purified guinea pig liver tTGase has previously been reported to have a specific activity of 18 ␮mol/min/mg (17). The dissimilarity between the highly purified human enzyme and the highly purified guinea pig enzyme in regard to activity with CGG (hydroxamate assay) is most likely due to a difference in substrate specificity rather than to the purification of a partially denatured rhtTGase. Thus, the specific activity we obtained for highly purified rhtTGase using the [14C]putrescine-binding assay [76 nmol/min/ mg (⫽ 4.6 ␮mol/h/mg)] is not significantly different from that obtained by Lee et al. of 5.3–6.2 ␮mol/h/mg for their preparation of highly purified human rhtTGase (24), despite different protein assay procedures and slightly different [14C]putrescine-binding assays between the two studies. Moreover, we have found that the specific activities of the commercial guinea pig liver enzyme using the [14C]putrescine-binding assay (⬃25 nmol/min/mg) and the fluorometric assay (⬃49000 RFU/min/mg) are only about 30 and 50%, respectively, of that obtained in the present work with highly purified rhtTGase (Table 2). Lee and colleagues have obtained highly purified


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FIG. 1. SDS–PAGE of fractions obtained during the purification of his6-rhtTGase. (A) Protein staining with Gelcode Blue reagent; (B) Western blotting. Lane M, prestained protein standards; lane 1, 10 ␮l of supernatant from a whole cell lysate (⬃64 ␮g of protein); lane 2, 10 ␮l of the active fraction eluted from the Ni-NTA column (⬃3 ␮g of protein); lane 3, 10 ␮l of the active fraction eluted from the HisTrap column (⬃0.3 ␮g of protein).

rhtTGase (24) using E. coli transfected with a pEThtTGase expression vector. Although our purified preparation of rhtTGase has a similar specific activity to that obtained by Lee et al., it is difficult to evaluate the relative merits of the two procedures. Lee et al. used their procedure to generate a site-directed mutant (C277S) to show that Cys-277 is necessary for the transglutaminase activity of human tTGase but not for its GTPase activity (24). Purification of the rhtTGase from the bacterial homogenate was effected by means of a novel reusable immunoaffinity column prepared from a monoclonal antibody (32). However, beyond stating that expression was at a low level, no details were given of the yield per liter of culture, the yield after purification, or stability on long-term storage (24). Our procedure for obtaining highly purified rhtTGase may be of more general use because it does not require the use of a specialized immunoaffinity column. Ambrus and Fe´su¨s (29) have also expressed human tTGase in E. coli using the vector PTRC 99A. These authors stated that the activity in the soluble fraction of the E. coli homogenates was low but gave no details on specific activity, yield, or stability to long-term storage of active enzyme in the soluble fraction of the E. coli homogenate. They also noted that most of the activity was in inclusion bodies. As noted above, however, the authors were able to obtain some active enzyme by treating the inclusion bodies with a regime that included exposure to polyethylene glycol.

Stability of His6-Tagged rhtTGase under Different Storage Conditions. We have found that once the lyophilized commercial guinea pig liver tTGase is suspended in buffer it loses activity over hours to days at 0–4⬚C. The commercial enzyme also loses complete activity on freezing (data not shown). We determined the stability of the highly purified His6-rhtTGase obtained by the present procedure when stored at 4⬚C or frozen at ⫺80⬚C. Our preparation of purified human enzyme was dialyzed for 2–3 h at 4⬚C against 50 mM Tris-acetate buffer (pH 8.0). Stock solutions of 400 mM EDTA and 100 mM DTT were added to give final concentrations of 0.5 and 10 mM, respectively. Addition of 1% (w/v) BSA slowed, but did not prevent, the loss of activity of His6-rhtTGase at 4⬚C. Addition of 30% (v/v) glycerol did not prevent the loss of activity at 4⬚C. (The enzyme may catalyze aggregation with itself as substrate on prolonged storage at 4⬚C.) However, storage at ⫺80⬚C resulted in no significant loss of activity even after several months. It is curious that the highly purified His6-tagged rhtTGase is stable to freezing at ⫺80⬚C, whereas the commercial guinea pig liver enzyme is not. Possibly, the addition of the His6 tag stabilizes the enzyme. The stability of rhtTGase under the various storage conditions is shown in Fig. 2. Based on these findings, we recommend storage of purified His6-rhtTGase at ⫺80⬚C in Tris-acetate buffer (pH 8.0) containing 0.5 mM EDTA, 10 mM DTT.

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FIG. 2. Stability of highly purified his6-rhtTGase under different storage conditions. The relative activities of the enzyme were determined by the fluorometric assay as described under Materials and Methods. Buffer I contained 25 mM Tris-acetate (pH 8.0), 0.5 mM EDTA, and 10 mM DTT; buffer II contained 25 mM Tris-acetate (pH 8.0), 1% (w/v) bovine serum albumin, and 30% (v/v) glycerol; buffer III contained 25 mM Tris-acetate (pH 8.0) and 30% (v/v) glycerol. The relative activity of the enzyme at day 0 was assigned 100%. The data are the average of determinations on two preparations. The enzyme is stable stored at ⫺80⬚C in buffer I, with no loss of specific activity over a period of at least 3 months.

Conclusion In conclusion, we have described an efficient, easily reproducible procedure for the expression and purification of His6-rhtTGase in moderate yield (⬃0.6 mg/liter of bacterial culture) and conditions for stable storage. ACKNOWLEDGMENT This work was supported by Grant P01 AG14930 from the NIH.

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