L-Asparaginase is an antineoplastic agent that selectively decreases the level of L-asparagine in blood and diminishes the proliferation of the cancerous cells. L-Asparaginases from Escherichia coli are widely used for clinical application because of their high substrate specificity and limited glutaminase activity. L-Asparaginase II-encoding gene ansB was isolated by excluding the native signal from E. coli MTCC 739, cloned in frame with pelB leader sequence of prokaryotic expression vector pET20b and expressed in E. coli DE3 cells. Overexpression of recombinant protein was achieved with an optimized final concentration of 10 μM of isopropyl β-D-1-thiogalactopyranoside (IPTG). The protein was expressed as soluble protein. The recombinant protein contained hexahistidine tag at C-terminus and was purified using nickel-nitrilotriacetic acid chromatography. Enzymatic properties such as optimum temperature, pH and the effect of temperature on the stability of L-asparaginase II from E. coli MTCC 739 were determined and the purified protein showed an optimum activity at 37 °C and pH=6.L-asparaginaza je antineoplastični agens koji selektivno smanjuje razinu L-asparaginaze u krvi i sprečava proliferaciju kanceroznih stanica. L-asparaginaze iz bakterije Escherichia coli koriste se za klinička ispitivanja zbog njihove specifičnosti za supstrat i ograničene aktivnosti glutaminaze. Gen ansB za kodiranje L-asparaginaze II izoliran je iz E. coli MTCC 739, kloniran pomoću pelB sekvencije prokariotskog vektora pET20b i izražen u stanicama E. coli DE3. Pojačana je ekspresija rekombinantnog proteina postignuta pomoću 10 μM izopropil β-D-1-tiogalaktopiranozidaze, pri čemu je izraženi protein bio topljiv, a na C završetku imao je heksahistidinsku oznaku. Rekombinirani je protein pročišćen kromatografijom pomoću nikal(II)-nitrilotrioctene kiseline. Ispitani su ovi parametri: optimalna temperatura i pH-vrijednost za aktivnost enzima, te utjecaj temperature na njegovu stabilnost. Utvrđeno je da je pročišćeni protein imao optimalnu aktivnost pri 37 °C i pH=6

Ashok Pandey

Carlos Ricardo Soccol

Jalaja Vidya

Ushasree Mrudula Vasudevan

Hrčak - Portal of scientific journals of Croatia

ISSN 1330-9862 original scientific paper(FTB-2748)Cloning, Functional Expression and Characterization ofL-Asparaginase II from E. coli MTCC 739Jalaja Vidya1, Ushasree Mrudula Vasudevan1, Carlos Ricardo Soccol2and Ashok Pandey1*1Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST),695 019 Trivandrum, India2Bioprocess Engineering and Biotechnology Division, Federal University of Paraná (UFPR),P.O. Box 19011, CEP 81531-970 Curitiba, PR, BrazilReceived: September 13, 2010Accepted: January 11, 2011SummaryL-Asparaginase is an antineoplastic agent that selectively decreases the level of L-aspa-ragine in blood and diminishes the proliferation of the cancerous cells. L-Asparaginasesfrom Escherichia coli are widely used for clinical application because of their high substratespecificity and limited glutaminase activity. L-Asparaginase II-encoding gene ansB was iso-lated by excluding the native signal from E. coli MTCC 739, cloned in frame with pelB lea-der sequence of prokaryotic expression vector pET20b and expressed in E. coli DE3 cells.Overexpression of recombinant protein was achieved with an optimized final concentra-tion of 10 mM of isopropyl b-D-1-thiogalactopyranoside (IPTG). The protein was expressedas soluble protein. The recombinant protein contained hexahistidine tag at C-terminus andwas purified using nickel-nitrilotriacetic acid chromatography. Enzymatic properties suchas optimum temperature, pH and the effect of temperature on the stability of L-asparagi-nase II from E. coli MTCC 739 were determined and the purified protein showed an opti-mum activity at 37 °C and pH=6.Key words: functional expression, L-asparaginase II, enzymatic propertyIntroductionBacterial L-asparaginases have obtained considerableattention as a prerequisite for the treatment of acute lym-phoblastic leukaemia and lymphosarcoma for the past30 years following the first report in 1967 (1). Amongthe bacterial asparaginases, type II asparaginase (L-aspa-ragine aminohydrolase, EC 3.5.1.1) displays high speci-fic activity against L-asparagine and hence is used forclinical applications. After the catalysis, the enzyme con-verts L-asparagine to L-aspartate and ammonia via an acyl--enzyme intermediate. The chemotherapeutic nature ofthe enzyme relies on its ability to maintain low L-aspa-ragine level in blood and thus eliminate the source ofL-asparagine for tumour cells, which require a huge amountof amino acids for their protein synthetic machinery.To date numerous microbial genera have been screen-ed for L-asparaginase activity, and asparaginase with tu-mour inhibitory activity, encompassing reports from bothprokaryotic as well as eukaryotic sources. In spite of anumber of reports of L-asparaginase production from Pseu-domonas flourescens (2), Mycobacterium (3) Staphylococcus(4) and Serratia marcescens (5), their intrinsic glutaminaseactivity, which could result in serious side effects like neu-rotoxicity, hepatitis and other dysfunctions, restricts theirclinical applications. However, enzymes from Gram-ne-gative bacteria such as Escherichia coli (6) and Erwinia caro-tovora (7) were found as most effective, owing to less se-vere immunorelated side effects as they possess strongpreference to asparagine over glutamine. In order to im-prove the half life of L-asparaginase in blood during clin-ical trials, efforts have been made to modify the protein286 J. VIDYA et al.: L-Asparaginase II from E. coli MTCC 739, Food Technol. Biotechnol. 49 (3) 286–290 (2011)*Corresponding author; Phone: ++91 471 251 5279; Fax: ++91 471 249 1712; E-mail: pandey@niist.res.in, ashokpandey56@yahoo.co.inby replacement of certain amino acid residues by site--directed mutagenesis (8), attachment of some chemicalmoieties to the purified protein (9) or immobilization ofthe asparaginase in nanoparticles (10) or microparticlesof natural silk sericin protein (11).The aim of the current study is to construct an ex-pression cassette of L-asparaginase II gene from E. coliMTCC 739 bearing a C-terminal His6 tag, in frame withpelB leader sequence of pET20b under the control of aT7 inducible promoter. The ansB gene from E. coli MTCC739 was cloned and expressed in E. coli DE3 cells. Theexpressed protein was purified and studied for opera-tional properties.Materials and MethodsBacterial strains and plasmidsE. coli MTCC 739 was used as a source of the L-as-paraginase II gene. E. coli strains BL21(DE3) and DH5a,and pET-20b plasmid as an expression vector for ansBgene were obtained from Novagen (Milan, Italy).Construction of recombinant plasmid and cloning ofansB geneGenomic DNA of E. coli MTCC 739 (NII08131) wasused as template for polymerase chain reaction (PCR)amplification of L-asparaginase II (ansB) gene by exclud-ing the native signal sequence using AnsBF (forward, 5’--GCGGAATTCGTTACCCAATATCACCA-3’ and AnsBR(reverse, 5’-GGCGAAGCTTGTACTGATTGAAGA-3’) withEcoRI and HindIII restriction sites underlined respectiv-ely. The PCR conditions were as follows: initial dena-turation at 94 °C for 4 min, denaturation at 95 °C for 40s, annealing at 57.4 °C for 30 s, extension at 72 °C for 90s for 30 cycles, and a final extension at 72 °C for 10 min.The 981-bp amplicons, digested by EcoRI and HindIII, werecloned in pET20b vector having the same restriction ter-mini. The recombinant construct thus obtained (pET20b--M2His) bearing an N-terminal pelB leader and a C--terminal His6 tag was then transformed into chemicallycompetent E. coli DH5a cells. The clones were analyzedby a restriction digestion for an insert release of the re-combinant plasmid isolated from the confirmed clones.The plasmid DNA was isolated from the confirmed cloneof E. coli DH5a cells and used for transformation of E.coli DE3 competent cells for protein expression.Soluble expression and purification of the recombinantenzymeTo optimize the expression of L-asparaginase genein E. coli, the effect of various isopropyl b-D-1-thiogalac-topyranoside (IPTG) concentrations was studied. E. coliDE3 cells harbouring the expression plasmid pET20b--M2His was grown in Luria-Bertani broth containing 0.5 %glucose and 50 mg/mL of ampicillin and induced withdifferent IPTG concentrations starting from 10 to 400 mMat the A600 nm of 0.6. The culture was then allowed togrow for a postinduction period of 3 h at 37 °C and 200rpm. After the harvest, absorbance (at 600 nm) was mea-sured and all the samples were diluted to an absorbanceof 1 for a normalised SDS-PAGE and enzymatic assays.Periplasmic and cytosolic fractions were collected and ana-lysed to establish the location of expressed recombinantprotein in the cell compartments. For the preparation ofperiplasmic fractions, 50-mL culture was pelleted andresuspended in Tris-sucrose buffer (30 mM Tris, pH=8,20 % sucrose and 1 mM EDTA) followed by shaking for10 min and pelleting at 8000×g for 20 min. The pelletwas resuspended again in 10 mL of 5 mM MgSO4, keptin an ice bath and shaken for 10 min, and after centrifu-gation at 8000×g for 20 min the supernatant was collect-ed as periplasmic fraction. The cytosolic fraction wasprepared by sonicating the pellet collected from 50 mLof culture in lysis buffer (50 mM NaH2PO4, 300 mMNaCl and 10 mM imidazole). For recombinant proteinexpression analysis, total cell lysate made from all thesamples including induced and uninduced cultures wasanalysed on 12 % SDS-PAGE and then stained with Co-omasie brilliant blue R-250 (Fig. 1).The His6-tagged protein was purified by affinity chro-matography using nickel-nitrilotriacetic acid (Ni-NTA) spincolumn (Quiagen, Hilden, Germany) and the bound pro-tein was eluted in elution buffer (50 mM NaH2PO4, 300mM NaCl and 250 mM imidazole). The purified enzymewas dialysed over night against 50 mM Tris buffer, pH=7,and used for further characterization.Enzyme assayActivity analysis of L-asparaginase II was performedaccording to Imada et al. (12) comprising the followingsteps. The samples were mixed with 0.04 M L-aspara-gine in 0.05 M Tris buffer, pH=7, and 200 mL of assaymixture were incubated for 10 min at 37 °C for enzy-matic reaction. The reaction was interrupted with 50 mLof 1.5 M trichloroacetic acid and the samples were cen-trifuged before the addition of Nessler’s reagent to mea-sure the released ammonia after L-asparagine hydrolysis.All the measurements were done spectrophotometrically287J. VIDYA et al.: L-Asparaginase II from E. coli MTCC 739, Food Technol. Biotechnol. 49 (3) 286–290 (2011)Fig. 1. SDS-PAGE profile of total cell lysate from samples afterexpression. Lane 1: molecular mass marker (from top 200, 150,120, 100, 85, 70, 60, 50, 40, 30, 25, 20, 15 and 10 kDa), lane 2: to-tal cell lysate of host cell DE3, lane 3: total cell lysate of unin-duced sample, lanes 4–7: total cell lysate from induced sampleswith 10, 50, 100 and 400 mM IPTGat 450 nm. Experimental values were calculated as anaverage of three independent measurements. The enzymeactivity of recombinant protein was determined using anammonium sulphate calibration curve. One unit of en-zyme activity was defined as the amount of enzyme re-quired to release 1 mM of ammonia per minute.Functional features of purified enzymeOptimum conditions of temperature and pHThe analysis of optimum temperature and the effectof pH on the activity of purified enzyme was carriedout in the range of 40–70 °C and different pH from 4–10using appropriate buffer systems respectively. Optimumconditions were determined and maintained stable forfurther assays to determine the thermostability.ThermostabilityThe enzyme was incubated at 50 °C and the activi-ties were measured at various time intervals of 15, 30, 45and 60 min. Percentage of residual activities was calcu-lated based on the untreated control activity, which istaken as 100 %.Results and DiscussionCloning and overexpression of L-asparaginaseL-Asparaginase gene encoding asnB region was cloneddownstream to T7 promoter of pET20b vector allowingthe insertion of pelB sequence at the N-terminus andHis6 tag at the C-terminus and overexpressed in E. coliDE3 cells.The enzyme activity of the recombinant ansB in cy-tosolic and periplasmic fractions of E. coli DE3 was de-termined and found to be more in cytosol compared tothe collected periplasmic fraction. Previous reports havealready revealed an inhibitory effect of the C-terminalHis6 tag in targeting the recombinant protein to the E.coli periplasm (13), which may account for the limitedactivity of the recombinant protein in periplasm com-pared to the intracellular fraction. With an objective todetermine an optimized expression conditions for therecombinant construct, the effect of various inducer con-centrations on recombinant protein expression was stu-died. Of the different inducer concentration used (10, 50,100 and 400 mM) for expression studies, 10 mM IPTGwas optimum for the expression of L-asparaginase geneas determined by activity assay (Fig. 2). Furthermore, na-tive protein in the soluble fraction was decreased con-currently with the increase of IPTG concentration above50 mM. Hence, an inducer concentration of 10 mM wasselected for further expression and purification studies.Moreover, extracellular expression of L-asparaginase byE. coli K12 was reported with optimal IPTG concentra-tion of 100 mM from pET22b vector (14). SDS-PAGE pro-files distinguished the His6-tagged asparaginase in theinduced samples as 37 kDa band, which corresponds tothe expected molecular mass of E. coli L-asparaginase II.Purification and functional features of the enzymeExpression vector contained the gene for asparagi-nase fused to a C-terminal His6 tag, enabling the purifi-cation in a one-step procedure using affinity chromato-graphy. The bound recombinant protein was eluted fromthe nickel affinity column in 250 mM imidazole and theeluted fractions were analysed on native PAGE gel toreveal oligomeric molecular mass of 150 kDa (Fig. 3)and a simultaneous SDS-PAGE gel of 12 % showed asubunit molecular mass of 37 kDa (Fig. 4a).Effect of temperature and pH on enzymeThe effect of temperature on the activity of purifiedenzyme was checked from 37 to 70 °C. As shown in Fig.4b, the purified L-asparaginase showed a maximum activ-ity at 37 °C. However, the activity of the enzyme sharplydecreased above 37 °C. Optimum temperature of L-as-paraginase II from E. coli W3110 was at 40 °C for freeenzyme, and a 10-degree rise in temperature was ob-served for immobilized enzyme preparation (15).The enzyme activity profile was analyzed at variouspH from 4 to 9. The enzyme was optimally active atpH=6 and there was no significant reduction in the en-zyme activity in the alkaline range beyond the pH op-tima (Fig. 4c). Enzyme with similar activity, EC-2, wasreported from E. coli B, which showed a very little changeof activity over the pH range from 6.0 to 8.4, with an288 J. VIDYA et al.: L-Asparaginase II from E. coli MTCC 739, Food Technol. Biotechnol. 49 (3) 286–290 (2011)Fig. 2. L-Asparaginase activity in the soluble fractionsFig. 3. Molecular mass determination of the oligomeric L-aspa-raginase through native PAGE. Lane 1: purified L-asparaginasein tetrameric form having a molecular mass of around 150 kDa,lane 2: gene protein molecular mass marker for native PAGEoptimum at pH=7 (16). According to Ehrman (17), therelease of aspartate was high at pH=5.6 when b-aspar-tohydroxamate was used as substrate. The pH activityprofile of E. coli W3110 was reported at pH=7 and boththe free and immobilized enzymes were active at broadrange of alkaline pH, up to 10.Thermostability assaysThe thermal stability of the purified enzyme at 50°C was studied to find out the extent of temperature resist-ance of the enzyme. Around 26 % of the initial activitywas retained by the purified enzyme after 30 min of in-cubation at 50 °C. The activity was significantly reducedafter 45 min and decreased to less than 10 % in 1 h ofincubation at the same temperature (Fig. 4d). The earlierreports on the thermostability of different L-asparaginasepreparations indicate that the modified enzyme was morestable at elevated temperatures for a greater extent thanthe native enzymes (8,15) and the native enzyme fromE. coli W3110 retained about 22 % of its activity at 60 °Cafter an incubation of 30 min.ConclusionsAaparaginases are widely distributed in the micro-bial kingdom, and L-asparaginase from E. coli is wellknown in clinical application. This study comprises clon-ing and effective expression of asnB gene from E. coliMTCC 739 and determination of its functional proper-ties. Investigations to find out the potential of amino acidresidues for modification of this enzyme using computa-tional methods are currently underway and this enzymecould be used to improve the properties like thermosta-bility and substrate specificity in order to obtain the bestcharacteristics for therapeutic application.AcknowledgementsJalaja Vidya gratefully acknowledges the award ofJunior Research Fellowship by the Council of Scientificand Industrial Research (CSIR), New Delhi, India, to carryout this study.References1. J.M. Hill, J. Roberts, E. Loeb, A. Khan, A. Maclellan, R.W.Hill, L-asparaginase therapy for leukemia and other mali-gnant neoplasms. Remission in human leukemia, J. Am. Med.Assoc. 202 (1967) 882–888.2. A. Nilolaev, N.N. Sokolov, E.A. Kozlov, M.E. Kutsman, Iso-lation and properties of a homogeneous L-asparaginase pre-paration from Pseudomonas fluorescens AG, Biokhimiia, 40(1975) 984–989.3. I. Pastuszak, M. Szymona, Purification and properties ofL-asparaginase from Mycobacterium phlei, Acta Biochim. Pol. 23(1976) 37–44.289J. VIDYA et al.: L-Asparaginase II from E. coli MTCC 739, Food Technol. Biotechnol. 49 (3) 286–290 (2011)Fig. 4. Purification and functional features of His6-tagged L-asparaginase: a) lane 1: purified L-asparaginase through Ni-NTA spin col-umn, lane 2: protein molecular mass marker; b) temperature optimum for purified L-asparaginase; c) pH optimum for L-asparagina-se (buffer systems used were acetate buffer, pH=4–6, Tris-HCl, pH=7–8, and glycine NaOH, pH=9; d) thermal stability of theenzyme at 50 °C for various time intervals of 15, 30, 45 and 60 min, and residual activity compared to control4. J. Mikucki, J. Szarapinbska-Kwaszewska, Z. Krzeminbski, Fac-tors influencing L-asparaginase production by staphylo-cocci, Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. 132(1977) 135–142.5. J.W. Boyd, A.W. Phillips, Purification and properties ofL-asparaginase from Serratia marcescens, J. Bacteriol. 106(1971) 578–587.6. Y. Wang, S. Qian, G. Meng, S. Zhang, Cloning and expres-sion of L-asparaginase gene in E. coli, Appl. Biochem. Bio-technol. 95 (2001) 93–101.7. G.A. Kotzia, N.E. Labrou, Cloning, expression and charac-terization of Erwinia carotovora L-asparaginases, J. Biotech-nol. 119 (2005) 309–323.8. L.Z. Li, T.H. Xie, H.J. Li, C. Qing, G.M. Zhang, M.S. Sun,Enhancing the thermostability of Escherichia coli L-aspara-ginase II by substitution with Pro in predicted hydrogen--bonded turn structures, Enzyme Microb. Technol. 41 (2007)523–527.9. J.F. Zhang, L.Y. Shi, D.Z. Wei, Chemical modification of L-as-paraginase from Escherichia coli with a modified polyethyl-eneglycol under substrate protection conditions, Biotechnol.Lett. 26 (2004) 753–756.10. E. Teodor, S.C. Litescu, V. Lazar, R. Somoghi, Hydrogel--magnetic nanoparticle with immobilizes L-asparaginase forbiomedical applications, J. Mater. Sci: Mater. Med. 20 (2009)1307–1314.11. Y.Q. Zhang, M.L. Tao, W.D. Shen, Y.Z. Zhou, Y. Ding, Y.Ma, W.L. Zhou, Immobilization of L-asparaginase on themicroparticles of the natural silk sericin protein and itscharacters, Biomaterials, 25 (2004) 3751–3759.12. A. Imada, S. Igarasi, K. Nakahama, M. Isono, L-Asparagi-nase and glutaminase activities of micro-organisms, J. Gen.Microbiol. 76 (1973) 85–99.13. A. Khushoo, Y. Pal, K.J. Mukherjee, Optimization of extra-cellular production of recombinant asparaginase in Esche-richia coli in shake-flask and bioreactor, Appl. Microbiol. Bio-technol. 68 (2005) 189–197.14. A. Khushoo, Y. Pal, B.N. Singh, K.J. Mukherjee, Extracellu-lar expression and single step purification of recombinantEscherichia coli L-asparaginase II, Protein Expr. Purif. 38 (2004)29–36.15. M.M. Youssef, M.A. Al-Omair, Cloning, purfication, char-acterization and immobilization of L-asparaginase II fromE. coli W3110, Asian J. Biochem. 3 (2008) 337–350.16. H.A. Campbell, L.T. Mashburn, E.A. Boyse, L.J. Old, TwoL-asparaginases from Escherichia coli B. Their separation,purification, and antitumor activity, Biochemistry, 6 (1967)721–730.17. M. Ehrman, H. Cedar, J.H. Schwartz, L-Asparaginase II ofEscherichia coli. Studies on the enzymatic mechanism ofaction, J. Biol. Chem. 246 (1971) 88–94.290 J. VIDYA et al.: L-Asparaginase II from E. coli MTCC 739, Food Technol. Biotechnol. 49 (3) 286–290 (2011)

Kloniranje, funkcionalna ekspresija i karakterizacija L-asparaginaze II iz E. coli MTCC 739

Abstract

Similar works

Full text

Available Versions

Hrčak - Portal of scientific journals of Croatia