Cloning, expression and characterization of the recombinant Yersinia pseudotuberculosis l -asparaginase

Protein Expression and Purification 82 (2012) 150–154

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Cloning, expression and characterization of the recombinant Yersinia pseudotuberculosis L-asparaginase M.V. Pokrovskaya a, S.S. Aleksandrova a, V.S. Pokrovsky a,b,⇑, N.M. Omeljanjuk a, A.A. Borisova a, N.Yu. Anisimova b, N.N. Sokolov a a b

Institute of Biomedical Chemistry, RAMS, Moscow, Russia N.N. Blokhin Cancer Research Center, RAMS, Moscow, Russia

a r t i c l e

i n f o

Article history: Received 9 November 2011 and in revised form 19 December 2011 Available online 29 December 2011 Keywords: L-Asparaginase Enzyme therapy Yersinia pseudotuberculosis Antiproliferative effect

a b s t r a c t We have cloned ansB (YPTB1411) gene from Yersinia pseudotuberculosis Q66CJ2 and constructed stable inducible expression system that overproduce L-asparaginase from Y. pseudotuberculosis (YpA) in Escherichia coli BL21 (DE3) cells. For purification of YpA we used Q-Sepharose and DEAE-Toyopearl column chromatography. We examined kinetics of the enzyme reaction, catalytic activity as a function of pH, temperature and ionic strength, thermostability and other enzyme properties. Biochemical properties of YpA are similar with those of E. coli type II L-asparaginase. Km for L-asparagine is 17 ± 0.9 lM and pI 5.4 ± 0.3. Enzyme demonstrates maximum activity at pH 8.0 and 60 °C. YpA L-glutaminase activity is relatively low and more than 15 times less than specific activity towards L-asn. We evaluated also the antiproliferative effect of YpA in vitro and in vivo with E. coli L-asparaginase (EcA) as the reference substance at similar conditions. Ó 2012 Published by Elsevier Inc.

Introduction L-Asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) primarily catalyzes the conversion of L-asparagine to L-aspartate and ammonia. A number of L-asparaginases from different sources show antileukemic activity [1–3]. Enzymes isolated from Escherichia coli type II (EcA)1 and Erwiniachrysantemi (ErA) L-asparaginases use as therapeutic agents for acute lymphoblastic leukemia treatment for over 30 years [4]. Recently it has been reported that L-asparaginases are effective in treatment of NK/T-cell and cutaneous T-cell lymphoma [5,6]. However, overall treatment efficacy is limited by antibody response and by various side-effects which include in some cases severe allergic reactions. A number of drawbacks are partially attributed to relatively high intrinsic L-glutaminase activity and immunogenic properties of explored enzymes [7–9]. Side effects associated with immune response can be circumvented by sequential therapy with serologically unrelated L-asparaginases. Therefore, search of new enzymes with different serological properties, high therapeutic activity and low L-glutaminase activity is important for biotechnology and clinical oncology.

⇑ Corresponding author at: Institute of Biomedical Chemistry, RAMS, Moscow, Russia. E-mail address: [email protected] (V.S. Pokrovsky). 1 Abbreviations used: EcA, Escherichia coli type II; YpA, Yersinia pseudotuberculosis; ErA, Erwinia chrysantemi; LB, Luria–Bertani broth; OD, optical density; IU, international units; BSA, bovine serum albumin; MTT, methylthiazolyldiphenyl–tetrazolium bromide. 1046-5928/$ - see front matter Ó 2012 Published by Elsevier Inc. doi:10.1016/j.pep.2011.12.005

The requirements for clinical efficient L-asparaginase are optimal activity under physiological conditions, high substrate specificity, and prolonged stability in circulating blood. Our objective was to explore the biochemical and anticancer properties of the new L-asparaginase from Yersinia pseudotuberculosis (YpA) and development of simple and fast enzyme purification method suitable for production of medical quality asparaginase preparation. Materials and methods Chemicals L-Asparagine (‘‘Reanal’’, Hungary), glycine, KH2PO4, Na2HPO4, KCl (‘‘Serva’’, Germany), Nessler reagent, trichloroacetic acid, bactotryptone, yeast extract (‘‘Fluka’’, Switzerland); NaOH, Na2B4O7  10H2O (‘‘Merck’’, Germany); TRIS (‘‘BIO-Rad’’, USA); HCl, CH3COOH, CH3COONa (‘‘Reakhim’’, Russia).

Bacterial strain E. coli BL-21 B F dcm omp ThsdS(rB- mB-) galk (DE3) was received from «Stratagene», USA. Medium and bacterial growth We grew bacterial cells aerobically on a rotary shaker at 150–180 rpm in 1000 ml flasks, which contained 200 ml of

M.V. Pokrovskaya et al. / Protein Expression and Purification 82 (2012) 150–154

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Luria–Bertani broth (LB) supplemented with ampicillin (100 lg/ ml) at 37 °C. We monitored cell growth by measuring medium optical density (OD) at 600 nm (OD600) with ‘‘Aquarius 7000’’ spectrophotometer and made induction by the addition of 0.02–0.50% arabinose when the cells had reached an OD600 of approximately 0.5–2.0. During cell growth we periodically collected medium samples for analysis.

#SM0671). We measured MALDI TOF-MS spectra using a Bruker Reflex II mass spectrometer (‘‘BrukerDaltonics’’, Germany). Ions were formed by a pulsed UV laser beam (Nd:YAG laser, 355 nm). Calibration was done using bovine serum albumin (BSA) as an external standard with software Bruker Flex Control 2.4.

Gene manipulation

We monitored the dependence of YpA activity at 37 °C in the pH range 3.0–11.0 with the standard buffers. We studied the influence of temperature on activity over the range 30–80 °C in 0.0125 M borate buffer, pH 8.0. In both cases, the relative activity was measured by the standard assay. We determined the rates of thermoinactivation by 1 min preincubation the enzyme at various temperatures (40–80 °C). The residual activity was measured under the standard assay conditions described above.

The pET23a vector bearing ansB (YPTB1411) gene from Y. pseudotuberculosis Q66CJ2 was kindly provided by K.V. Sidoruk (State Scientific Center ‘‘GosNIIgenetika’’, Moscow, Russia). AnsB gene was isolated from pET23a by digestion with endonucleases HindIII and XbaI (‘‘Fermentas’’, Lithuania), purified from agarose gel (‘‘Qiagen’’, Germany) and placed under control of the arabinose-inducible araBAD promoter on the vector pBAD24. We named the resulting recombinant plasmid pBad24/YpA and used it to transform the E. coli cells. Expression and purification of YpA The protein was expressed in E. coli BL-21 (DE3) transformed with pBad24/YpA and induced with 0.2% arabinose in exponential growth phase (OD600 = 1.5). We examined the effect of inducer concentration by using 0.02%, 0.05%, 0.1%, 0.2%, 0.5% arabinose. After about 12 h of incubation the cells (30–35 g) were harvested by centrifugation for 15 min at 2500 g, suspended in buffer A (10 mM potassium phosphate, 1 mM glycine, 1 mM EDTA, pH 7.5) and sonicated for 10 min (1 min pulse on, 1 min pulse off). After sonication we centrifuged the cells 15000 g for 60 min. We applied soluble fraction of cell-free extract onto a Q-Sepharose column (1.5  16.0 cm) equilibrated with buffer A. After washing with buffer A, the protein was eluted by application of a 0.0–1.0 M linear KCl gradient. The pooled enzyme fractions were loaded onto a DEAE Toyopearl column (1.0  12.5 cm) equilibrated with the same buffer. The active fractions were eluted with KCl in a linear gradient (0.0–1.0 M) in buffer A. Fractions were analyzed by SDS–PAGE and concentrated (‘‘Amicon, Millipore NMWL 30.000’’). The all purification steps were carried out at 4 °C. We evaluated asparaginase activity by measuring released ammonia with Nessler’s reagent. Protein concentration was determined by the modified Lowry method with bovine serum albumin as a standard [10]. Enzyme assay Portions (1–100 ll) of YpA solution were added to 0.0125 M borate buffer (pH 8.0) to give a final volume of 0.8 ml. The reaction was initiated by the addition of 0.2 ml of 0.04 M L-asparagine (or L-glutamine) in the same buffer and the reaction mixture was incubated for 2–5 min at 37 °C. We stopped the reaction by addition of 0.5 ml of 20% trichloroacetic acid solution. The ammonia liberated was determined by direct nesslerization [11]. L-asparaginase activity was expressed in generally accepted international units (IU). One IU was determined as the amount of enzyme catalyzing the production of 1 lmol of ammonia per min under optimal assay conditions. We defined the specific activity as the number of IU per milligram of protein. The pI value was determined using Servalyt-Precotes (‘‘Serva’’, Germany) with a pH range of 3.0–10.0 and compared with standard markers (‘‘BioRad’’). Measurement of molecular mass We determined the purity of YpA by SDS–PAGE using 12.5% acrylamide PhastGels by the method of Laemmli [12]. The Mr of YpA was compared with that of 17–170 kDa Mr markers (‘‘Fermentas’’,

Effects of pH and temperature on L-asparaginase activity

Kinetic analysis The standard reaction mixture contained 12.5 mM borate buffer, pH 8.0, and variable concentrations of L-asparagine or L-glutamine in a final volume of 1 ml. Reaction was started by adding the YpA or EcA solution (0.5–10.0 mg/ml). We determined the enzyme activity spectrophotometrically at 480 nm and 37 C using Nessler reagent to detect ammonia release as described above. The plot of Lineweaver–Burk was used to determine Vmax and Km values. Cell culture and cell viability Human lung adenocarcinoma A549 and human breast adenocarcinoma MDA-MB-231 were provided by the American Type Culture Collection (Rockville, USA) and were used as the in vitro model in this study. Cancer cells were routinely grown in RPMI-1640 (‘‘Sigma’’, USA) containing 10% heat-inactivated (56 °C, 30 min) fetal bovine serum (‘‘Hyclone Laboratories’’, Logan, UK), 2 mM L-glutamine and antibiotics (100 lg/ml penicillin sodium salt and 100 lg/ml streptomycin sulfate (‘‘Sigma’’, USA)). To evaluate the in vitro cytotoxicity, we placed cells in 5  105 cells/ml concentrations in 96-well culture plates (‘‘Nunc’’, USA) for 24 h under 5% CO2 and saturated humidity at 37 °C. They were then incubated for another 72 h together with varying concentrations of YpA in fresh medium, or respective volume of RPMI-1640. We measured the YpA-induced cytotoxicity by the conversion capacity of the viable cells from methylthiazolyldiphenyl–tetrazolium bromide—MTT (‘‘Sigma’’, USA) to its formazan, and then expressed as relative survival (%), i.e., relative survival, % = A/B  100, where A—optical density in the vessel containing cells coincubated with YpA, B—optical density in the vessel containing intact cells. Optical density value measured by tray photometer (‘‘Multiscan MS’’, Finland), k = 540 nm. Then we evaluated the index of cytotoxicity—IC50 represented the concentration of YpA that is required for 50% inhibition of cancer cells viability in vitro. Survival of tumor-bearing mice model We inoculated i.p. female DBA/2 mice (18–23 g) with 0.3 ml of suspension ascitic fluid, containing L5178y lymphoma cells, 1  106 cells/mouse, on day 0. One day after tumor implantation, we divided mice into four groups: control group (n = 11) given 0.3 ml saline only, YpA, ErA and EcA group (n = 13). L-asparaginases 1000 IU/kg was administered i.p. in 50 IU/ml saline solution on days 1–10. ErA and EcA were used as positive controls to YpA. We daily observed mice groups to evaluate their general conditions. Mice surviving for 60 days without evidence of disease were considered cured. Sixty-day survivors were not included for calculation of mean survival time but were considered independently.

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We performed all animal experiments in accordance with the EU (86/609/EEC and 2010/63/EU) guidelines. Results of experiments were analyzed using Kaplan–Meier estimation of survival and log–rank test. A p-value of less than 0.05 was considered significant. Results Cloning, expression and purification of YpA The sequence of the cloned ansB gene consisted of 1055 bp showed more than 99% identity with similar sequences of different Y. pseudotuberculosis strains: A7FJX3, B1JRE3, B2KA19 and was therefore considered the actual Y. pseudotuberculosis ansB gene. The deduced amino acid sequence composed of 345 amino-acid residues showed high similarity with Yersinia pestis (Q1CGG1, Q1CA77, A6BSN7) and E. coli (B7MZQ6, B7U1041) L-asparaginases (99% and 74%, respectively), Fig. 1. It was revealed that created recombinant strain has stable protein production for at least 20 passages in vitro. The maximum specific activity of YpA was found to be attained 8 h after induction, after which a plateau was observed. The rate of YpA production after induction was about 5–10 times higher that achieved in the absence of inducer. However, changes in the inducer concentration from 0.02% to 0.50% did not result in any significant improvement in enzyme production. We obtain the highest levels of enzyme activity when arabinose was added to the medium at the exponential phase. The summary of purification procedure is given in Table 1. L-asparaginase activity in cell-free extract was 8.7 IU/mg, YpA accounts for about 5–7% of total protein in extract. More than 80% of the L-asparaginase activity bound to Q-Sepharose and DEAEToyopearl and was eluted in a single peak by a KCl gradient (at 0.3 M KCl). After chromatographic steps of purification the specific activity increased to 62 IU/mg, which was 7 times higher as compared with cell-free extract. The purification procedure described yielded an 82% recovery of enzyme. SDS–PAGE electrophoresis indicated the presence of only one major band with the molecular weight of 36.4 kDa when the gels were stained with Coomassie Blue (Fig. 2). MALDI-TOF mass spectrum of purified recombinant protein shows a mass-peak at near

Table 1 Purification of YpA. Treatment

Cell-free extract Q-Sepharose pooled peak DEAE-Toyopearl pooled peak

Volume

Total protein

Total activity

Specific activity

Overall yield

ml

mg

IU

IU/mg

%

54 29

2260 336

19700 16400

8.7 47.8

100 85

26

257

16100

62.7

82

Note: we used 35 g of wet weight cells.

36.5 kDa, in agreement with the calculated mass of recombinant protein. Physical and chemical properties of YpA Isoelectric point of YpA is 5.4 ± 0.3, corresponds to that of EcA [12]. It was found that YpA is stable at a wide range of pH and retains about 91–100% of its maximal activity at physiological pHs. The pH-rate profile of the asparaginase activity approximated a sigmoid curve, with a maximum activity at a pH value of 8.0 and a lack of activity below pH 5.0 (Fig. 3). More than 80% of maximum activity was measured between pH 6.0 and 9.0. A similar pH value was obtained for E. coli, Pseudomonas aeruginosa and many other microbial L-asparaginases [13,14]. We measured the reaction rate of L-asparaginase at various temperatures. Maximum reaction rate was obtained at 60 °C but enzyme was also active over a wide temperature range, retaining >80% of its activity at 57–73 °C. The catalytic activity of YpA does not vary significantly as a function of ionic strength (100– 3000 mM KCl). The purified enzyme preparation showed no loss of activity when stored at 70. . . 20 °C (pH 5.0–9.0) for weeks. At room temperature under sterile conditions the enzyme activity is preserved for weeks also. After 3 min incubation at 60 °C remains only 15% of YpA activity, and after 5 min—only 6% (Fig. 4). The incubation at 80 °C for 10 min leads to almost full loss of activity, less than 1% of initial.

Fig. 1. Amino acid sequences of YpA and EcA. 1—YpA. 2—native Yersinia pseudotuberculosis Q66CJ2 L-asparaginase. 3—EcA. The amino acid replacements in recombinant YpA are marked.

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Fig. 4. Activity of YpA ( temperatures.

) or EcA (

153

) after 3 min incubation at various

corresponds to data received earlier [15]. Direct nesslerization method showed that YpA hydrolyses L-glutamine at a rate of 6% compared to the hydrolysis of L-asparagine. Antiproliferative effect

Fig. 2. SDS–PAGE electrophoresis. 1—Fermentas, #SM0671: 170, 130, 95, 72, 55, 43, 34, 26, 17 kDa. 2—cell-free extract without induction. 3—cell-free extract after induction. 4—YpA after chromatographic steps of purification.

We found that IC50 of YpA towards A549 was 2.23 U/ml, MDAMB-231—10 U/ml. These results are identical to that for EcA as the reference substance at similar conditions (unpublished data). The L5178y tumor cells were sensitive to YpA. Treatment with YpA prolonged the lives of all mice and cured 2 of 13 mice. Compared to control group, the animals treated with YpA showed significant increase in mean survival time, almost 36%, from 17.5 ± 1.8 days to 23.7 ± 3.1 days, as shown in Fig. 5. In the EcA group there were 4 cured mice, the mean survival time was 32.2 ± 5.4. The difference between YpA and EcA groups was not statistically significant (p > 0.05). ErA in the same dose was quite ineffective, increase in mean time survival less than 7%. Discussion

Kinetic properties YpA showed hyperbolic dependence of L-asn and L-gln hydrolysis rate. Km for L-asparagine was found to be 17 lM, Vmax 4.8 mM/ min (at concentration of enzyme in a reaction mixture 21 lM), kcat = 0.22 c 1. Km for EcA measured similarly was also 17 lM that

In this paper we have described procedures for expression and purification of YpA, as well as its biochemical characterization. YpA has been purified to near homogeneity (62 IU/mg of protein) as determined SDS–PAGE electrophoresis. The purification procedure described yielded an 82% recovery of enzyme.

Fig. 3. Activity dependence on pH.

YpA,

EcA.

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tion. However, the close similarity in physical and biochemical properties of YpA and EcA described here as well as the moderate effect of YpA by the side of EcA does not point to clear explanation for future clinical advantages of YpA. It would be of interest to make further immunological comparisons of YpA and EcA as the practical aspect of obtaining antileukemic enzymes with minimal cross-reactivities for clinical use. References

Fig. 5. Kaplan–Meier survival curve for DBA/2 mice bearing L5178y lymphoma cells. 1—YpA group, 2—control group.

Table 2 Physical and kinetic properties of YpA and EcA.

Monomer molecular mass, kDa Number of amino-acid residues pH optimum pI Km for L-asn, lM Activity towards L-gln, % of L-asn activity

YpA

EcA

36.27 345 8.0–8.5 5.4 ± 0.3 17 ± 0.9 5.7

36.85 348 7.0–7.5 4.8 ± 0.2 [12] 17 ± 0.9 5

In common with EcA, the YpA shows some degree of thermostability, and possesses optimum activity over a wide range of pH values including physiological pHs. Both enzymes have pH optimum at 7.0– 8.0, similar isoelectric points, and molecular weights. In addition, the amino acid composition of both enzymes is 74% identical (Table 2). Finally, this study gives promising data on the possible application of YpA in oncology. YpA has the necessary characteristics of chemotherapeutically active enzyme and is much more effective than clinical approved ErA. Several properties of enzyme, such as activity at the physiological pH range, thermostability and high efficacy in vivo, make it extremely valuable for further investiga-

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