High-Level Expression and Purification of a Human “Mini”-hexokinase

PROTEIN EXPRESSION AND PURIFICATION

7, 58–66 (1996)

Article No. 0009

High-Level Expression and Purification of a Human ‘‘Mini’’-hexokinase Marzia Bianchi, Giordano Serafini, Dario Corsi, and Mauro Magnani Institute of Biological Chemistry ‘‘G. Fornaini,’’ University of Urbino, Italy

Received May 26, 1995, and in revised form September 5, 1995

Human hexokinase type I is a 100-kDa enzyme with the catalytic site located in the C-terminal domain. We had previously expressed this domain in Escherichia coli, however only a small amount of the recombinant enzyme was catalytically active. To overcome this problem we have now expressed the ‘‘mini’’-hexokinase using the pET expression system. An average of 1000 U of enzyme per liter of culture was obtained. The recombinant enzyme was purified to homogeneity by a combination of ion-exchange chromatography, affinity chromatography, and dye-ligand chromatography. The enzyme was unstable under ultrafiltration; thus, a multicolumn purification procedure was developed in order to avoid the ultrafiltration steps. The recombinant ‘‘mini’’-hexokinase was found to have the same kinetic properties as the entire enzyme. Using the method described, the enzyme can be obtained in sufficient quantities for biophysical and biochemical investigations. q 1996 Academic Press, Inc.

Hexokinase type I (HK; ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) commits glucose to catabolism by catalyzing its phosphorylation to glucose 6-phosphate (Glc 6-P) with MgrATP as phosphate donor. In mammals, there are four isozymes of hexokinase, named types I, II, III, and IV, which vary in their tissue distribution and kinetic properties (1). Type I, II, and III isozymes consist of single polypeptide chains with a molecular mass of 100 kDa and all three are inhibited by the product Glc 6-P (2,3). Type IV hexokinase (also known as glucokinase), similar to yeast hexokinase isozymes A and B, is about 50 kDa and is insensitive to inhibition by Glc 6-P (2). These properties led several authors to suggest that the 100-kDa Glc 6-P-sensitive hexokinases evolved through the duplication and fusion of a gene encoding a 50-kDa Glc 6-P-insensitive hexokinase, similar to that found in yeast (4–7). Ac-

cording to this hypothesis, one of the duplicated catalytic sites (that present in the C-terminal domain) retained its catalytic function (8–11), while the other (in the NH2-terminus) evolved to take on a regulatory function, becoming an allosteric site for the binding of Glc 6-P (12). However, this evolutionary scenario is incompatible with the finding that an isolated catalytic C-terminal domain of rat brain hexokinase retained sensitivity to inhibition by Glc 6-P (10). Based on these considerations we decided to express the C-terminal domain of HK type I (which we named ‘‘mini’’-hexokinase or ‘‘mini’’-HK) in Escherichia coli in order to obtain this truncate form of the enzyme purified to homogeneity and in a catalytically active form. This work was aimed at expressing sufficient amounts of ‘‘mini’’HK in bacterial hosts in order to better understand the structure/function relationships in hexokinase and also to engineer the purified recombinant enzyme for other potential biotechnological applications (i.e., biosensors, encapsulation in erythrocytes). In a previous work (13) we had already expressed the C-terminal domain of HK type I in E. coli: the corresponding 2155-bp-long cDNA was ligated into the pJLA expression vector (14). This prokaryotic expression system allowed the production of a great amount of recombinant ‘‘mini’’-HK, but only a small percentage of the enzyme seemed to be catalytically active. Using this expression system we were able to show that the recombinant ‘‘mini’’-hexokinase retained the catalytic site and was also sensitive to inhibition by hexose 6-phosphates. The main limitation of the pJLA expression system was, as stated above, the reduced amount of catalytically active enzyme obtained. To overcome this problem we investigated other prokaryotic expression systems. In this paper we report the expression in E. coli of the human recombinant ‘‘mini’’-hexokinase by using the pET expression system and we compare the results obtained with those already obtained with the pJLA vector (13). We also present details of the chromatographic proce-

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1046-5928/96 $12.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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dures carried out for the purification of the ‘‘mini’’-HK to homogeneity and some of its kinetic properties. MATERIALS AND METHODS Materials

pET-3d expression vector and E. coli strain BL21 (DE3) were purchased from Novagen (Madison, WI). Restriction enzymes, T4 DNA ligase, and the protease inhibitors phenylmethylsulfonyl fluoride, leupeptin, and pepstatin were from Boehringer-Mannheim Biochemicals. Tris base, acrylamide, TEMED, SDS, and all other reagents and equipment for electrophoresis and immunoblotting techniques were from Bio-Rad as was also the IPTG (isopropyl-2-D-thiogalactopyranoside) used for induction experiments. Coenzymes, enzymes, and substrates for enzymatic assays were obtained from Boehringer-Mannheim Biochemicals. Ultrafiltration membranes (microsep 30 K) were from Filtron and PM 30 membranes were from Amicon. DEAE Sepharose Fast Flow was purchased from Pharmacia LKB Biotechnology. Matrex Gel Blue A was from Amicon. The ECL detection system was from Amersham. The sonicator used was a Labsonic 1510 B. Braun. All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO) and Carlo Erba unless otherwise indicated. Methods

Construction of the Expression Vector pET-3d HK The C-terminal coding region of human hexokinase type I cDNA was isolated as a 2155-bp NcoI–EcoRI fragment by digestion of the pJLA502 HK vector with the two restriction enzymes (13). The cDNA coding for the ‘‘mini’’-hexokinase was gel-purified and then inserted into the pET-3d expression plasmid (Novagen) previously cut with NcoI and EcoRI, generating the construct pET-3d HK. The maintenance of the correct reading frame for the partial HK I cDNA was verified by nucleotide sequence analysis with the T7 primer. The scheme used for construction of the plasmid for expression of the C-terminal domain of HK I is shown and detailed in Fig. 1. For the expression of the ‘‘mini’’hexokinase we used the vector pET-3d containing unique NcoI and EcoRI sites, the former of which allowed the insertion of the cDNA into the ATG start codon. The recombinant plasmid obtained (pET-3d HK) was transformed into the E. coli strain BL21 (DE3). The induction experiments were initially performed according to the instructions of the manufacturers. Briefly, cells were grown in LB broth plus ampicillin (at a concentration of 50 mg/ml) at 377C to early log phase (A600 nm Å 0.4). IPTG was then added to a final concentration of 0.4 mM to induce the T7 RNA polymer-

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ase gene and cultures were further incubated at 377C for different periods of time ranging from 1 to 5 h. The plasmid stability test, carried out just before induction (according to the instructions of the manufacturers), also revealed that almost all the cells retained the target plasmid in a functionally active form. Purification of the Recombinant ‘‘Mini’’-HK Step 1: Preparation of bacterial extracts. Cells (derived from 1 liter of postinduction culture) were harvested by centrifugation at 8500g for 20 min at 47C. The resulting pellet was resuspended in 1/10 culture volume of lysis buffer containing 15 mM Tris–HCl, pH 8.0, 1 mM EDTA, 50 mM glucose, 0.5% (v/v) Triton X100, 3 mM mercaptoethanol, 9% (v/v) glycerol, lysozyme (2 mg/ml), 1 mM phenylmethylsulfonyl fluoride, leupeptin (15 mg/ml), and 0.1 mM pepstatin. The cell suspension was stored at 07C for 30 min and then sonicated five times for 30 s at 200 W. Sonicated extracts were then centrifuged for 40 min at 30,000g and 47C. The supernatants were then used for hexokinase assay, protein assay, and purification procedures (see below). As controls for expression experiments, E. coli cells BL21 (DE3) transformed with the plasmid pET-3d alone (lacking the ‘‘mini’’-HK-coding cDNA) and the uninduced cells were used. Step 2: DEAE Sepharose Fast Flow anion-exchange column chromatography. The supernatant obtained from step 1 was applied to a DEAE Sepharose Fast Flow (Pharmacia) column (3.5 1 30 cm) equilibrated in 5 mM sodium potassium phosphate buffer, pH 7.55, containing 1 mM glucose, 3 mM mercaptoethanol, and 9% (v/v) glycerol and operated at 200 ml/h. The column was first washed with 400 ml of 25 mM KCl in the equilibrating buffer and then hexokinase activity was eluted with a linear gradient from 0 to 0.5 M KCl (1000 ml in each chamber) in the equilibrating buffer. Fractions (10 ml each) were collected and assayed for hexokinase activity. Step 3: Ammonium sulfate fractionation. Solid ammonium sulfate was slowly added to the pooled fractions containing HK activity from the previous step to achieve a 35% saturation. The suspension was gently stirred for 30 min and then centrifuged at 31,000g for 30 min. The supernatant solution was removed and brought to 70% saturation with ammonium sulfate. After 30 min the suspension was centrifuged as above. The 35 to 70% ammonium sulfate precipitate was dissolved in 10 ml of affinity buffer (3 mM sodium potassium phosphate buffer, pH 7.55, containing 3 mM mercaptoethanol) and then dialyzed three times (1 h each) against 200 vol (2 liters) of affinity buffer (see above) to remove glucose. The dialyzed enzyme solution was then centrifuged at 31,000g for 30 min.

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FIG. 1. Construction of the plasmid for the expression of the C-terminal half of hexokinase type I. The steps performed to construct the pET-3d HK expression vector are illustrated (see Methods for details).

Step 4: Affinity chromatography. The dialyzed enzyme solution from step 3 was applied to a (1.4 1 14 cm) column of Sepharose-N-aminohexanoylglucosamine, prepared as in Ref. 15, equilibrated in the affinity buffer reported above, and operated at 90 ml/h. After

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the sample application the column was washed with 25 bed volumes of 25 mM KCl in the equilibrating buffer until the A280 nm of the eluate reached baseline. Elution from the affinity chromatography column was obtained by 50 mM glucose added to the wash buffer.

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Step 5: Blue A chromatography. After affinity chromatography, the hexokinase solution was concentrated by ultrafiltration through a microsep 30 K Filtron membrane to about 10 ml and then centrifuged for 30 min at 31,000g. This sample was loaded onto a (1.8 1 14 cm) Blue A column equilibrated in 20 mM Hepes buffer, pH 7.55, containing 10 mM glucose, 3 mM mercaptoethanol, and 9% (v/v) glycerol and operated at 90 ml/h. For the washings the pH of the equilibrating buffer was raised to 8.5 and 25 mM KCl was added. Elution was obtained by adding 1.5 mM Glc 6-P to the wash buffer. The pooled fractions containing hexokinase activity were concentrated by ultrafiltration as above and then used for specific activity determination, kinetic studies, and electrophoretic analysis. Hexokinase Assay Hexokinase activity was measured at 377C spectrophotometrically in a system coupled with glucose-6phosphate dehydrogenase (G6PD; EC 1.1.1.49) or, in the Glc 6-P inhibition studies, in a coupled enzyme system with pyruvate kinase (PK; EC 2.7.1.40) and lactate dehydrogenase (LDH; EC 1.1.1.28) as described in (16). One unit of hexokinase activity is defined as the amount of enzyme which catalyzes the formation of 1 mmol of Glc 6-P or ADP/min at 377C. Kinetic studies were performed in 80 mM Tris–HCl, pH 7.2 (16). Determination of Protein Protein concentration was determined using the Bradford method (17) with bovine serum albumin as standard or spectrophotometrically by measuring the absorbance of solutions at 280 nm against appropriate blanks. SDS–PAGE and Immunoblotting SDS–PAGE of the expressed C-terminal domain of human HK I was performed in 10% polyacrylamide gels according to the method of Laemmli (18), while Western blot analysis was carried out exactly as described in (13). RESULTS

Optimization of Recombinant Human ‘‘Mini’’hexokinase Expression The aim of the present study was the optimization of the recombinant human ‘‘mini’’-HK expression in prokaryotic cells. In particular, the results reported in this paper concern the use of the pET expression system. The cDNA coding for the C-terminal half of human hexokinase type I was inserted into the pET-3d vector (generating the expression plasmid pET-3d HK), under

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the control of the T7 RNA polymerase promoter as detailed under Materials and Methods. Induction of T7 RNA polymerase in cells containing pET-3d HK resulted in accumulation of a 52-kDa protein (the expected molecular weight for this truncate form of hexokinase) which was not present in host cells transformed with pET-3d alone or in the uninduced control cells (not shown). Our early efforts were directed toward the optimization of the induction conditions in order to obtain the expression of the greatest amount of recombinant enzyme in a catalytically active form. In particular, the influence of length and temperature of induction, as well as bacterial densities before and after induction, were examined. The first experiments were performed exactly according to the instructions of the manufacturers, which means that cells were grown with shaking at 377C until A600 nm reached 0.4. IPTG was then added to obtain a final concentration of 0.4 mM and the incubation at 377C was continued for different periods of time ranging from 1 to 5 h. To study the time course of the ‘‘mini’’-HK expression after induction, 10-ml aliquots of the growing culture were taken at 60-min intervals, following IPTG addition. Bacterial cells, collected at each time point, were harvested by centrifugation and then processed as described under Materials and Methods for the preparation of bacterial extracts. The supernatants were used for hexokinase and protein assays and for specific activity determination. Under these conditions, the highest specific activity (1.5 U/mg of protein) and the highest amount of catalytically active protein (4.5 HK U/ml) were obtained after 4 h of induction at a bacterial density lower than 1.2 OD. When induction was performed at higher cell densities or for a period of time longer than 4 h the specific activity was lower, suggesting that high bacterial densities and high temperatures favor intracellular inactivation of the recombinant hexokinase. Based on these preliminary results, we decided to investigate the effects of temperature after IPTG addition on the ‘‘mini’’-HK specific activity. For this purpose the induction temperature was lowered to 30 or 227C, respectively. When bacterial cells were grown at 377C and induced at a lower temperature (307C), the recovery of the catalytically active ‘‘mini’’-HK increased. A further reduction of the induction temperature to 227C for 15 h provided a specific activity of 2.0 U/mg of protein corresponding to a concentration of 8.7 HK U/ml. These conditions were selected for all the large-scale experiments performed later to purify the recombinant ‘‘mini’’-hexokinase. In 10 different experiments an average of 1000 { 90 U of recombinant ‘‘mini’’-HK per liter of culture was obtained. These values of HK activity calculated after induction were considerably high compared to those already obtained with the pJLA502 expression vector previously described (13). E. coli cells

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transformed with this expression vector and induced for 90 min at 427C contained a biologically active hexokinase with a specific activity of 0.125 U/mg of protein and the total HK activity obtained with 1 liter of induced culture was around 50–100 U. In fact the pJLA502 vector allowed the production of a large amount of recombinant protein, but the major part of the expressed ‘‘mini’’-HK was catalytically inactive. This was probably due to the induction mechanism based on the temperature shift to 427C rather than to the instability of the protein itself in the bacterial cells. Purification of the Recombinant Human ‘‘Mini’’hexokinase The recombinant human ‘‘mini’’-hexokinase was purified to homogeneity from induced E. coli cells containing pET-3d HK, following the purification procedure already described for human placenta hexokinase type I (19). This consists of a three-step purification comprising DEAE ion-exchange chromatography followed by affinity chromatography on Sepharose-Naminohexanoylglucosamine and dye-ligand chromatography on a Blue A column. Details of these procedures are described under Materials and Methods. All operations were performed at 47C. In a typical induction experiment, starting from 1 liter of induced bacterial cells the average total enzyme obtained was about 1000 U with a specific activity of approximately 2.0 U/mg of protein. This sample was loaded onto the DEAE column. After washings, the enzyme was eluted with a linear gradient from 0 to 0.5 M KCl in the equilibrating buffer. Two peaks of activity were obtained (Fig. 2); however, the first peak appeared to be the human recombinant glucose phosphorylating activity, according to the criteria described in Ref. 13 (immunoinactivation by a specific anti-human HK antibody and inhibition by phosphorylated sugars). The second peak, also present in the control uninduced cultures, was concluded to be the endogenous bacterial glucose phosphorylating enzyme. The fractions containing HK activity of the first peak were pooled. With this purification step the specific activity increased five to seven times ranging from 10 to 14.3 U/mg of protein (with an average purification of about 6-fold) and the yield was approximately 70%. The enzyme pool obtained after DEAE chromatography was concentrated with solid ammonium sulfate as described under Materials and Methods, leading to an average recovery of 77%. The enzyme solution from the previous step was applied to an affinity column of Sepharose-N-aminohexanoylglucosamine. Elution was obtained with 50 mM glucose (Fig. 3A). The average recovery with this affinity chromatography method was approximately 91% and the specific activity of the pooled enzyme was 80 U/mg of protein.

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FIG. 2. DEAE Sepharose Fast Flow ion-exchange chromatography of the C-terminal domain of human hexokinase type I expressed in E. coli using the pET system. Sonicated and centrifuged E. coli extracts (see Materials and Methods) prepared from induced E. coli BL21 (DE3) cells (containing the cDNA coding for the C-terminal half of human hexokinase) were loaded onto a (3.5 1 30 cm) DEAE Sepharose Fast Flow column equilibrated in 5 mM sodium potassium phosphate buffer, pH 7.55, containing 1 mM glucose, 3 mM mercaptoethanol, and 9% (v/v) glycerol and operated at 200 ml/h. Hexokinase activity was eluted with a linear gradient (0–0.5 M) of KCl in the equilibrating buffer and 10-ml fractions were collected. The total hexokinase activity loaded was 1000 { 90 U. The first peak was not present in uninduced cells or in E. coli cells containing the pET-3d plasmid lacking the cDNA insert.

After the affinity chromatography step, the enzyme solution was concentrated by ultrafiltration through a microsep 30 K Filtron membrane: this step caused a considerable loss of enzyme activity with a recovery of around 40%. The residual hexokinase activity was loaded onto a Blue A column. After elution by 1.5 mM glucose 6-phosphate (Fig. 3B), all active fractions were pooled and an average yield of 88% was typical for this step. The specific activity was around 90 U/mg and the overall purification at this stage was about 40- to 50fold. The pooled enzyme was concentrated again by ultrafiltration as before and this step resulted again in a great loss of enzyme activity, yielding a recovery of only 40%. Samples from various steps in the purification protocol were analyzed by SDS–PAGE and the Western blotting technique (data not shown) which revealed that the ‘‘mini’’-HK was purified to homogeneity after the three chromatographic steps, but problems concerning the inactivation of the recombinant enzyme during the ultrafiltration steps remained to be solved. To prevent the consistent loss of enzyme activity found after the ultrafiltration procedure we added different compounds to the hexokinase solution to be concentrated (i.e., 3 mM sodium potassium phosphate buffer and/or 20% (v/v) glycerol and/or 0.15 M NaCl and/or 3 mM mercaptoethanol) but none of them was able to overcome the inactivation of the enzyme when it was concentrated by ultrafiltration. The use of a different

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three chromatographic steps were connected one to the other in an obvious sequence. Briefly, a typical purification experiment was performed as described below. The bacterial extract, starting from 1 liter of induced E. coli culture, contained around 1000 U of HK activity. This sample was loaded onto the DEAE column. After elution with a linear gradient of KCl, the first peak of HK activity was concentrated by ammonium sulfate fractionation, dialyzed, and then applied to the affinity column. The procedure up to this stage was exactly as described above. Then, the enzyme solution eluted from the affinity chromatography with 50 mM glucose was directly loaded onto the Blue A dye-ligand chromatography column because these two columns were con-

FIG. 3. Affinity chromatography (A) and Blue A chromatography (B) of the post-DEAE ‘‘mini’’-hexokinase, obtained using the pET expression system. (A) The enzymatic activity eluted with the first peak of the DEAE column was subjected to ammonium sulfate fractionation as described under Materials and Methods and then loaded onto the affinity column of Sepharose-N-aminohexanoylglucosamine (1.4 1 14 cm) equilibrated in 3 mM sodium potassium phosphate buffer, pH 7.55, containing 3 mM mercaptoethanol and operated at 90 ml/h. Hexokinase activity was eluted by adding 25 mM KCl and 50 mM glucose to the equilibrating buffer. (B) The enzymatic activity from the affinity column was first concentrated by ultrafiltration as described under Materials and Methods and then loaded onto a (1.8 1 14 cm) Blue A column equilibrated in 20 mM Hepes buffer, pH 7.55, containing 10 mM glucose, 3 mM mercaptoethanol, and 9% (v/ v) glycerol and operated at 90 ml/h. The buffer used for the elution of the ‘‘mini’’-HK was the same as the equilibrating buffer with the addition of 25 mM KCl, 1.5 mM glucose 6-phosphate and the pH value was raised to 8.5. In each case, fractions of 10 ml were collected.

ultrafiltration apparatus (Amicon PM 30) and pretreatment of the membranes as suggested by the manufacturers to avoid adsorption losses did not improve the hexokinase activity recovery. Multicolumn Procedure for the Purification of the ‘‘Mini’’-hexokinase To avoid problems related to the inactivation of the recombinant enzyme during the ultrafiltration steps performed before and after the Blue A column and to improve the total yield of HK activity at the end of the purification, we used a multicolumn procedure in which

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FIG. 4. Flow chart of the multicolumn procedure used for the purification of recombinant ‘‘mini’’-hexokinase. After DEAE chromatography (see text), the dialyzed enzyme solution was loaded onto a (1.4 1 14 cm) affinity column. The eluate from this column was loaded onto the (1.8 1 14 cm) Blue A column. The enzyme solution was finally loaded onto a (1.4 1 5 cm) DEAE column to concentrate the enzyme. Elution was obtained with 0.5 M KCl in equilibrating buffer. The columns were operated at 90 ml/h at 47C.

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Purification of Human ‘‘Mini’’-hexokinase Expressed in E. coli

Step I II III

Total activity (units)

Specific activity (units/mg)

Recovery (%)

Purification (fold)

1000 600 320

2.0 12.0 165.0

100 60 32

1 6 82

Cell-free extract Ammonium sulfate (35 to 70%) of DEAE peak Multicolumn stepa

a The values refer to the HK eluted from the last DEAE chromatography step in the multicolumn purification procedure. All values are means of approx 10 different experiments.

nected; thus, the first ultrafiltration step was avoided. Washing and elution conditions for the Blue A column were as described above, the only difference being that the hexokinase activity eluted from Blue A with 1.5 mM Glc 6-P was directly applied onto a DEAE Sepharose Fast Flow column (1.4 1 5 cm) previously equilibrated as described for the first DEAE chromatography. Elution of HK activity from the last DEAE column was obtained using 0.5 M KCl in the equilibrating buffer. This step allowed a yield of 100% with respect to the hexokinase activity eluted from the Blue A column, overcoming the inactivation of the enzyme found during the second ultrafiltration step. The final concentrated enzyme preparation had a specific activity of 165 U/mg of protein. Details of this multicolumn purification procedure are shown in the scheme reported in Fig. 4. Table 1 summarizes the purification steps and indicates the average yield from approx 10 preparations. Samples from various steps in the purification protocol were also analyzed by SDS–PAGE and the Western blotting technique as shown in Fig. 5. Properties of the Purified Enzyme The purity of the final enzyme preparation was initially assessed by SDS–PAGE on 10% polyacrylamide gels. The enzyme obtained at the end of the purification procedure (Fig. 5A, lane 6) produced one single band of 52 kDa (the expected molecular weight for this truncate form of hexokinase) with no relevant traces of minor bands after Coomassie blue staining, indicating that the purified enzyme was homogeneous. Determinations of the KM for glucose (60 mM) and ATP (1.06 mM) suggested that the recombinant ‘‘mini’’-HK interacts with the substrates glucose and ATP with apparent affinities very similar, and in most cases nearly identical, to those of the intact enzyme purified from human placenta (19). The optimum pH for catalytic activity was 8.0. The highest catalytic activity was obtained at saturating glucose (5 mM) and ATP (5 mM) concentrations in the presence of 5 mM mercaptoethanol or 3 mM reduced glutathione. KCl was found to be an inhibitor of

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HK activity: 50% inhibition was found at a concentration of KCl of 200 mM. Moreover, we found that the stability of the purified recombinant ‘‘mini’’-HK depends on the presence of glucose (1 mM), mercaptoethanol (3 mM), and glycerol (9%, v/v). It was also observed that the enzyme activity was well maintained when the sample was frozen at 0807C. DISCUSSION

One of the most important properties of the 100-kDa mammalian hexokinase type I, in contrast with the 50-kDa yeast hexokinase and liver glucokinase, is its inhibition by Glc 6-P (3). Given the remarkable amino acid similarity between the N- and C-terminal halves of the 100-kDa hexokinases, some authors (7,20) have suggested that these 100-kDa Glc 6-P-sensitive enzymes evolved by duplication and fusion of a gene encoding a 50-kDa Glc 6-P-insensitive hexokinase. According to this hypothesis one of the duplicated catalytic sites (namely the one present in the C-terminal domain) retained its catalytic function (10,11,21), while the other, in the N-terminal half, evolved to take on a regulatory function, becoming an allosteric site for the binding of the Glc 6-P (12). This evolutionary scenario could be reconsidered given the evidence that 50-kDa hexokinases found in various marine organisms (10,22–24) and in Schistosoma mansoni (25) are inhibited by glucose 6-P. Furthermore, we (13) and others (11) have previously shown that the recombinant Cterminal half of HK is inhibited by Glc 6-P. The aim of the present study was the functional expression of the C-terminal half of human hexokinase type I in E. coli in large amounts for crystallographic studies in order to verify whether it contains a regulatory site in addition to the catalytic one and to produce quantities of protein sufficient for use in future biophysical characterizations. To this end, we expressed this truncate form of enzyme (referred to as ‘‘mini’’HK) in bacterial hosts which was then purified to homogeneity. The pJLA expression vector, described in a previous study (13), allowed the production of a great

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FIG. 5. (A) SDS–PAGE of human ‘‘mini’’-hexokinase expressed with the pET system at different purification steps and (B) Western blot analysis with an anti-human hexokinase type I IgG. (A) Lane 1, 4 mg of human placenta hexokinase; lane 2, 2.5 mg of rabbit brain hexokinase; lane 3, 10 mg of total postinduction cell proteins of bacterial cells transformed with the pET-3d lacking the hexokinase cDNA insert; lane 4, 10 mg of total postinduction cell proteins of bacterial cells transformed with the pET-3d HK plasmid; lane 5, 7 mg of ammonium sulfate precipitate of the DEAE Fast Flow pool; lane 6, 1.5 mg of the sample at the end of the multicolumn procedure; lane 7, low-molecular-weight protein standards. (B) Protein samples, similar to those shown in A, were run on a 10% SDS–polyacrylamide gel and electroblotted as described in Ref. 13. The blot was probed with an immunoaffinity-isolated rabbit anti-human hexokinase type I IgG and antibody binding was visualized with the enhanced chemiluminescence Western blotting detection system (ECL).

amount of recombinant ‘‘mini’’-HK (equal to 7.5% of total cell proteins as revealed by laser-scanning densitometric analysis of the SDS–polyacrylamide gels). However, only a small percentage of this recombinant enzyme seems to be catalytically active. In this paper we report the use of an expression vector for the ‘‘mini’’hexokinase based on the pET system. The pET plasmid directs high-level expression in E. coli of the human ‘‘mini’’-hexokinase in a catalytically active form, confirming that the inactivation found with the pJLA system was due to the induction mechanism based on a temperature shift to 427C rather than to the instability of the protein itself. In fact, the C-terminal domain of human hexokinase type I expressed in E. coli by the pET system exhibits significant catalytic activity. The specific activity of the purified ‘‘mini’’-HK was 165 U/mg, a value almost identical to that reported for the intact 100-kDa enzyme, purified to homogeneity from human placenta (19). Nevertheless, the recombinant enzyme showed a slightly reduced stability compared to that of the full-length enzyme, especially when subjected to high hydrostatic pressure during the ultrafiltration steps initially performed during the purification procedure to concentrate the enzymatic activity.

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To avoid this inactivation during the ultrafiltration steps, we developed a multicolumn purification procedure. A specific activity of 165 U/mg was obtained for the purified ‘‘mini’’-HK (as stated above), comparable to that reported for the entire enzyme purified from human placenta (180 U/mg; Ref. 19) but only half the value obtained for the ‘‘mini’’-HK expressed with the pJLA system. Taken together these results raise the question of how many catalytic sites are present in the intact 100-kDa mammalian hexokinase type I. Based on the assumption that the catalytic site is in the Cterminal of HK, for the ‘‘mini’’-HK we expected a specific activity double that obtained for the intact enzyme, which is twice the size of the C-terminal half. However, these considerations were not confirmed by the results obtained with the pET system. It is possible that the HK C-terminal half, expressed and purified to homogeneity, is a mixture of catalytically active and inactive enzyme and that other expression systems may provide an even higher specific activity. Similar results were obtained by Arora et al. (11). Therefore, the evolutionary relationships among the 100-kDa mammalian hexokinases have yet to be explained, also considering the salient data showing that sensitivity to inhibition by

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Glc 6-P is intrinsic to the catalytic C-terminal half of the type I isozyme (10,13). In conclusion, this paper describes an effective expression system which allows the production of great amounts of recombinant enzyme (around 1000 U per liter of induced culture) in a biologically active form. The multicolumn purification procedure has thus been optimized and can be considered a very effective method for purification of the ‘‘mini’’-HK to homogeneity with good recovery yields. Using this system, purified ‘‘mini’’-hexokinase can be obtained in sufficient quantities to perform biophysical and biochemical studies, to further investigate the structure/function relationships, and also to engineer this purified recombinant enzyme for other potential biotechnological applications.

11.

12.

13.

14.

15.

ACKNOWLEDGMENT This work was partially supported by funding from MURST (60 and 40%).

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