تعیین توالی ژن، کلونینگ و بیان آل- آسپاراژیناز نوترکیب از سودوموناس آیروجینوزا سویه SN4 در اشریشیاکلی

Introduction

Bacterial L- asparaginase has been broadly used as an actual therapeutic agent against lymphosarcoma and acute lymphoblastic leukemia (1- 3). Numerous cases of current research are accessible regarding the use of L- asparaginase in melanoma therapy (4- 6). The use of anti- tumor therapy is based on its capacity to hydrolase L- asparagine to L- aspartic acid and ammonia in serum and cerebro- spinal fluid. Since tumor cells are unable to make endogenous L- asparagine, starvation for this amino acid hints to death of these cells (7). L- asparaginase received enlarged care in current years as food handling helps to diminish the development of acrylamide in carbohydrate-based foods that were fried, baked or roasted (8 & 9). L- asparaginase has also been considered for presentation in the L- asparagine biosensor for leukemia (10). Meanwhile, the statement that E. coli L- asparaginase has an anticancer activity similar to that of guinea pig serum, there has been extensive attention in L- asparaginase from numerous sources particularly microorganisms. Many plants, mammalian and bacterial species can produce L- asparaginases. Although other bacteria, such as Thermococcus gammatolerans (11), Pectobacterium carotovorum MTCC 1428 (12 & 13), Cladosporium sp. (14), Streptomyces (15), Pseudomonas aeruginosa (16), and Actinomycetes (17), Escherichia coli (18)have a latent for L- asparaginase making, but the enzymes from Erwinia sp. and E. coli (1, 7 & 19) has been used as an anti- cancer agent.

Up to now, only L- asparaginase from Erwinia and E. coli has been used in medical application. Because of various side effects of these enzymes, finding other bacterial L- asparaginase received more attention. So, isolation of L- asparaginase gene from Pseudomonas aeruginosa strain SN4, cloning, expression in E. coli is the aim of this study.

Materials and methods

Chemical materials: L- asparagine and trichloroacetic acid (TCA) were obtained from Sigma Chemicals Co. L- asparaginase- specific primers used in this study were synthesized by Bioneer Co. (Soft Korea). Taq polymerase and dNTPs were procured from Sinaclone (Iran).

Cloning of PaA gene: Pseudomonas aeruginosa strain SN4 was previously isolated and identified in our lab (20). A BLAST search of P. aeruginosa genome sequence using as a quire, the gene encoding for L- asparaginase from P. aeruginosa yielded a single 1041- bp ORF that was hypothesized to code for L- asparaginase. This strain was grown- up at 30^◦C in a medium containing 0.5% yeast extract, 1% peptoneand 1% NaCl. After 16 h, cells were harvested by centrifugation (10,000× g 10 min, at 4^◦C) and genomic DNA was isolated according to a standard method (21). Genomic DNA isolated from P. aeroginosa strain SN4 was used as the template for amplification of L- asparaginase gene using the primers: pCFP (5- GGAATTCCATATGATGGCCCTGATGCTC- 3) and pCRP

(5- CCGCTCGAGGTATTCCCAGAAGAT- 3), where, the italic sequences are the restriction sites of Nde1 and Xho1 enzymes, respectively. The DNA amplification was approved in a total volume of 25 µl, containing 2.5 µl PCR Buffer, 1 µl of DNA templet, 0.5 µl lµl dNTP mix, 1 µl each of both primers, 0.2 µl Taq polymerase, and 18.7 µl Milli q water. The PCR method included 33 cycles of 45 s at 94^◦C, 60 s at 55^◦C and 45 s at 72^◦C. A final extension time at 72^◦C for 10 min was done after 33 cycles (22 & 23). The PCR product was sequenced and the L- asparaginase gene was exposed to restriction digestion with NdeI and XhoI enzymes and then ligated into pET21a plasmid (Novagen, Inc.) which was previously having the restriction sites (Fig. 1).

The ligated sample was transformed into competent E. coli (DE3) pLysS DH5a cells, according to CaCl₂ method. The transformed E. coli cells were grown into LB agar plate containing 100 µg/ml ampicillin, IPTG (1mM) and X- gal (1mM). The recombinant clones were grown up in 50 ml LB medium containing 100 µg/ml ampicillin at 37^◦C. The plasmid was isolated from E. coli DH5a and was used to transform competent E. coli BL21 cells, which were screened with blue/white selection. Colony PCR, double digestion and sequencing were performed to further approve the presence of L- asparaginase gene and also to check the orientation of integration in the plasmid.

Fig. 1- Map of pET21a vector

L- asparaginase production and purification: The E. coli BL21 cells were grown into LB agar plate containing ampicillin (100 µg/ml). 2 ml of an overnight cultures inoculated into 20 ml of LB medium in Erlenmeyer flasks (100 ml) and grown for 24 h at 37^◦C in a 180 rpm shaker incubator. L- asparaginase synthesis was induced by the addition of 1mM IPTG (isopropyl- β- D- thiogalactopyranoside) after 9 h of growth. 9 hours after induction, cells were pelleted by centrifugation at 5000 g and 4^◦C for 15 min, resuspended in sodium phosphate buffer (10 mM, pH 7.5), sonicated and centrifuged at 10,000×g for 15 min. The supernatant was gathered and used for purification.

The lysate of E. coli BL21 containing pET21aASN was applied to a Q- Sepharose column, an anion exchanger, pre- equilibrated with 45 mM Tris- HCl buffer, pH 8.5. The proteins were eluted (1 ml/min) with NaCl gradient (0.1- 0.5 M) and 45 mM Tris- HCl buffer (pH 8.5). The active fractions were concentrated with ammonium sulphate (80%) and dialyzed. The purity of the enzyme was valued by SDS- PAGE. SDS- PAGE was done according to the method of Laemmli (24) on a slab gel comprising 10.0% (w/v) polyacrylamide (running gel) and 5.0% (w/v) (stacking gel). The molecular size of the purified L- asparaginase was estimated using molecular markers and the proteins were stained with Coomassi Brilliant Blue R- 250. The purified L- asparaginase was dialyzed to remove salt.

L- asparaginase assay: The L- asparaginase activity was investigated by the technique of Imada, utilizing the Nessler reaction (25). In this method, the ammonia liberated from L- asparagine in the enzyme reaction measured by the Nessler reaction. The reaction was started by adding 100 µl cell suspension into the 900 µl of 100 mM L- asparagine prepared in 50 mM Tris/HCl buffer, pH 8.0 and incubated for 20 min at 37^◦C. The reaction was stopped by the addition of 100 µl 1.5 M TCA. The reaction mixture was pelleted by centrifugation at room temperature (10,000 g for 10 min) to remove the precipitate and the ammonia released into the supernatant was determined colorimetrically (A₄₈₀) by adding 0.2 ml Nessler reagent into tubes containing 0.3 ml supernatant and 1.5 ml H₂O. The content in the tubes was vortexed and incubated at room temperature for 10 min, and the A₄₈₀ values were measured against the blank that received TCA before the addition of cell suspension. One L- asparaginase unit (U) is defined as the amount of enzyme that liberates 1 µmole of ammonia per min at 37^◦C. Ammonium sulphate was used as the standard for enzyme activity calculations. For rapid colorimetric assay the recombinant bacteria were screened using the modified M9medium (1 l: 3.0 g KH₂PO₄; 6.0 g Na₂HPO₄·2H₂O; 0.5 g NaCl; 5.0 g L- asparagine; 0.5 gMgSO₄.7H₂O; 0.014 g CaCl₂.2H₂O; 2.0% (w/v) glucose, and 15.0 g agar) combined with a pH indicator(phenol red). Development of pink color was considered as a positive result for L- asparaginase production (20).

Results

Cloning PaA: A BLAST search of P. aeruginosa SN4genome sequence in NCBI database (http://www.ncbi.nlm.nih.gov/) using the amino acid sequence of L- asparaginase from P. aeruginosa DSM 50071 (accession number: CP012001) as a query sequence, generated a high- probability match to a single 1041- bp ORF, encoding a predicted protein of 346 amino acids. To define whether the supposed P. aeruginosa protein was actually an active L- asparaginase, we cloned and expressed the full- length gene in E. coli and confirmed for its capacity to hydrolysis L- asparagine.

PCR was used to amplify the full- length gene from genomic DNA of P. aeruginosa SN4 (Fig. 2). The PCR product was cloned into the pET21a vector. This plasmid was used to transform the E. coli BL21 (DE3) pLysS as an expression host.
L- asparaginase from Erwinia carotovora has beencloned in E. coli BL21 (26).

L- asparaginase expression: E. coli BL21 containing pET21aASN was grown up in LB medium supplemented with 1 mM IPTG. After 9 h of induction, 2 ml of this culture was added to 2 ml of phosphate buffer (20 mM, pH 8.0) containing L- asparagine (10 mM) and phenol red (0.001%). After 10 min incubation at room temperature, a tube containing E. coli BL21 with 9h induction, showed a dense pink color against the other (Fig. 3). These results indicated high activity of recombinant L- asparaginase about 93.4 IU/ml.

After induction with 1 mM IPTG, sample was picked up at different time incubation (3, 6 and 9 h). Results showed that the maximum L-asparaginase production was echived after 6 h of incubation (Fig 4b).

Purification results showed that active fraction was obtained in 15% of NaCl concentration. After dialysis, active fraction was loaded on SDS–PAGE gel and then the gel stained with Coomassie Blue. Results showed the attendance of only one band with the molecular weight of 35.0 kDa (Fig. 4a).

Gene sequence analysis: The sequence of recombinant L- asparaginase gene consisted of 1041 bp showed more than 99% identity with similar sequences of different P. aeruginosa strains(Fig. 5 and Fig. 6). Results in Fig. 5, showed the multiple sequence alignment of H. pylori CCUG 17874 (Hepy; EIE30409.1), B. subtilis W168 (Basu; AAA22243.1), E. coli (Esco; NP 417432.1), and Er. chrysanthemi 3937 (Erch; AAS67028.1), B. licheniformis (BaLi, WP_044789473), K. pneumoniae JM45 (Klpn, AGT24790), P. aeruginosa SN004 (Pasn) andP. aeruginosa (Paea, WP_023099587).

Fig. 3- L- asparaginase activity in medium supplemented with phenol red and L- asparagine. Lane 1; E. coli BL21 containing pET21aASN without induction and Lane 2; extract of E. coli BL21 containing pET21aASN after 9 h of incubation induction with 1mM IPTG.

PaSn      -MAL--MLLLPQAAQAKEVAPQQKLSNVVILATGGTIAGAGASAANSATYTAAKVPVDQL

Erch      MERWFKSLFIIAFFFISTANAADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTL

Klpn      --------------MSSLAFSETRLPHIVILATGGTIAGSAASNTQTTGYKAGAIGVQTL

Hepy      --------------------MAQNLPTIALLATGGTIAGSGASAS-SDSYKSGELGIKEL

Cifr      -MEFFKRTAL-AALVMGFSGAALALPNITILATGGTIAGGGDSAT-KSNYTAGKVGVENL

Bali      ------------------------MKKLLLLTTGGTIASVEGENGLAPGVKAEEL-----

Basu      ------------------------MKKLLMLTTGGTIASVEGENGLAPGVKADEL-----

Paae      ---------------------MLPVKNLFVLYTGGTIGMLQTPQGLAPAGGFEARMRDHL

Esco      ----------------------MQKKSIYVAYTGGTIGMQRSEQGYIPVSGHLQRQLALM

: :  *****.

PaSn      LASVPQLKDIANVRGEQVFQIASESFTNENLLELGKTVAKLADSDDVDGIVITHGTDTLE

Erch      INAVPEVKKLANVKGEQFANMASENMTGDVVLKLSQRVNALLARDDVDGVVITHGTDTLE

Klpn      INAVPEMSKIAHVEGEQVANIGSENMTSDIILQLSKRVNALLARDDVDGVVITHGTDTLD

Hepy      LKAIPSLNKIARIQGEQVSNIGSQDMNEEIWFKLAKRAQELLDDSRIQGVVITHGTDTLE

Cifr      VEAVPQLKDIAVVKGEQVVNIGSQDMNDDVWLTLAKKINTEC--DKTDGFVVTHGTDTME

Bali      LSYLSDDHSNYTIDCQSLMDIDSTNMQPEHWVMMAEAVYENYS--RYDGFVITHGTDTMA

Basu      LSYVSKLDNDYTMETQSLMNIDSTNMQPEYWVEIAEAVKENYD--AYDGFVITHGTDTMA

Paae      QSLGDAPDLRWRFA-ELQPPLDSANMTQGNWLAMRDAIVAALD-AGHDGVLLLHGTDTLA

Esco      PEFHRPEMPDFTIH-EYTPLMDSSDMTPEDWQHIAEDIKAHYD--DYDGFVILHGTDTMA

.  :    : * .:       : .           :*.:: *****:

PaSn      ETAYFLTL-VEHTEKPIVVVGSMRPGTAMSAGGMLNLYNAVAVAGDKSARGKGVLITMND

Erch      ESAYFLHL-TVKSDKPVVFAAAMRPATAISADGPMNLLEAVRVASDKQSRGRGVLVVLND

Klpn      ETPYFLNL-TVKSNKPVVFTAAMRPATAISADGPMNLLEAVTVAADPDARGRGVMVVLND

Hepy      ESAYFLNL-VLRSTKPVVLVGAMRNAASLSADGALNLYNALSVALNEKSANKGVLVVMDD

Cifr      ETAYFLDL-TVKCNKPVVLVGAMRPSTGMSADGPFNLYNAVVTAADKASANRGVLVVMND

Bali      YTSAALSYMLQNIDKPVAITGSQVPITFKKTDAKKNIKDAVRFACDG---IGGVYVVFDG

Basu      YTSAALSYMLQHAKKPIVITGSQIPITFQKTDAKKNITDAIRFACEG---VGGVYVVFDG

Paae      YSAAALSFLLLDLPIPVLLTGSMLPAGAPGSDAWDNLVGALRVLQEGH--ARGVQVYFND

Esco      YTASALSFMLENLGKPVIVTGSQIPLAELRSDGQINLLNALYVAANYP--INEVTLFFNN

:   *         *: ...:        : .  *:  *:    :       * : ::

PaSn      EILSGRDASKMVNIKTEAFKSP-WGPLGMVVEGKSYWFRAPVK--RHTVNSEFDIKQI-S

Erch      RIGSARYITKSNASTLDSFRANEEGYLGVVIGNHIYYQNRLDK--LHTNRSVFDVRGL-A

Klpn      RIGAARFVTKTNATSLDTFRAPEEGYLGVVVGGKPQFETRVDK--IHTLRSVFDVRQL-K

Hepy      TIFSAREVVKTHTTHTSTFKALNSGAIGSVYYGKVRYYMQPLR--KHTTESEFSILELKT

Cifr      TVMDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPAR--KHTTSTPFDVSKL-T

Bali      RVILGTRAIKLRTKSYDAFESINYPYIAFIHDKEIEYNKRVPEVKPGA----LKLDT--S

Basu      RVIQGTRAIKLRTKSYDAFESINYPYIAFINEDGIEYNKQVTEPENDT----FTVDT--S

Paae      ALLHGARVSKLRSDAFDAFAELPRPRHAEH------------APVPAAL---GYRQP--R

Esco      RLYRGNRTTKAHADGFDAFASPNLPPLLEA---GIHIRRLNTPPAPHGE-GELIVHP--I

:  .    *       :*

PaSn      ALAPVEIAYSYGNVSDTAYKALAQAGAKAIIHAGTGNGSVPA---RVVPTLQELRKQGVQ

Erch      SLPKVDILYGYQDDSEYLYDAAISHGVKGIVYAGMGAGSVSV---RGIAGMRKAQDKGVV

Klpn      VLPKVVIIYGYQDDPEYMYDAAIAHHADGIIYAGTGAGSVSV---RSAAGIKKAQQAGIV

Hepy      PLPKVDIIYTHASMTSDLFQASLKSHAKGVVIAGVGNGNVSA---GFLKTMQEAGQMGVV

Cifr      ELPKVGIVYNYANASDLPAKALVDAGYAGIVSAGVGNGNLYK---TIFDTLATAAHKGTV

Bali      LNTDVCLVKLHPGLKPEFFD-CLKGSYKGIVIESYGSGGIPFEKRNILEKVNELIDSGMV

Basu      LCTDVCLLKLHPGLKPEMFD-ALKSMYRGIVIESYGSGGVPFEGRDILSKVNELIESGIV

Paae      REVNLAVLPLYPGLRAAHLRGLLDSGVEALLLECYGSGTGPSDDEELLALLREAHARGVL

Esco      TPQPIGVVTIYPGISADVVRNFLRQPVKALILRSYGVGNAPQNK-AFLQELQEASDRGIV

: :   :                 .::    * *            :      *

PaSn      IIRSSHVNAGGFVLRNAEQPD--DKNDWIVAHDLNPQKARILAAVAMTKTQDSKELQRIF

Erch      VMRSSRTGNGIV-PPDEAL-------PGLVADSLNPAHARILLMLALTRTSDPAVIQDYF

Klpn      VVRASRTGSGVV-PPDDSQ-------PGLVADSLNPAKARILLMTALTQTKDPQLIQQYF

Hepy      IVRSSRVGSGEI-TSGE-ID----DKTFITSDNLNPQKARVLLQLALTKTNDKAKIQEMF

Cifr      VVRSSRVPTGAT-TQDAEVDD--AKYGFVASGSLNPQKARVLLQLALTETKDPKQIQEMF

Bali      VAITTQCLEEGEDMSIYEVGRKVNQDAIIRSRNMNTEAIVPKLMWALGKNGQQAEVKKIM

Basu      VVITTQCLEEGEDMSIYEVGRRVNQDLIIRSRNMSTEAIVPKLMWALGQSSDLPVVKRIM

Paae      LAAVSQCAYGQVEFGVYAAGSRLRDAGLLSAGGMTREAALGKLFGLLGAGLGRDEAQRWF

Esco      VVNLTQCMSGKVNMGGYATGNALAHAGVIGGADMTVEATLTKLHYLLSQELDTETIRKAM

:   ::                      : .  :.           :         :  :

Fig. 6- Phylogenetic analysis of the L- asparaginase protein of Pseudomonas aeruginosa strain SN4. Bootstrap values and scale bar depicting substitution rate per site are indicated. The phylogenetic tree constructed by the neighbor- joining method showing the position of L- asparaginase of of Pseudomonas aeruginosa strain SN4. Protease from Escherichia coli was used as outgroup.