This article was downloaded by: [University of Connecticut] On: 07 October 2014, At: 04:57 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Animal Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/labt20
Cloning, E. coli Expression, and Characterization of Heart Lactate Dehydrogenase B From River Buffalo (Bubalus bubalis) Muhammad Shahid Nadeem b
a b
c
, Jenny Moran , Bibi Nazia Murtaza
, Khushi Muhammad & Habib Ahmad
a
a
Department of Genetics , Hazara University, Garden Campus , Mansehra , Pakistan b
School of Biological Sciences , University of the Punjab, Quaid-eAzam Campus , Lahore , Pakistan c
Institute of Science and Technology in Medicine (ISTM), School of Life Sciences , Keele University Staffordshire , Keele , United Kingdom Published online: 03 Dec 2013.
To cite this article: Muhammad Shahid Nadeem , Jenny Moran , Bibi Nazia Murtaza , Khushi Muhammad & Habib Ahmad (2014) Cloning, E. coli Expression, and Characterization of Heart Lactate Dehydrogenase B From River Buffalo (Bubalus bubalis), Animal Biotechnology, 25:1, 23-34, DOI: 10.1080/10495398.2013.804832 To link to this article: http://dx.doi.org/10.1080/10495398.2013.804832
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Animal Biotechnology, 25: 23–34, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1049-5398 print=1532-2378 online DOI: 10.1080/10495398.2013.804832
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CLONING, E. COLI EXPRESSION, AND CHARACTERIZATION OF HEART LACTATE DEHYDROGENASE B FROM RIVER BUFFALO (BUBALUS BUBALIS) Muhammad Shahid Nadeem1,2, Jenny Moran3, Bibi Nazia Murtaza2, Khushi Muhammad2, and Habib Ahmad1 1
Department of Genetics, Hazara University, Garden Campus, Mansehra, Pakistan 2 School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan 3 Institute of Science and Technology in Medicine (ISTM), School of Life Sciences, Keele University Staffordshire, Keele, United Kingdom Lactate dehydrogenase is an enzyme of glycolytic pathway which catalyzes the interconversion of pyruvate and lactate. The present study describes cDNA cloning, E. coli expression and characterization of lactate dehydrogenase B (LDH-B) from the heart ventricles of river buffalo (Bubalus bubalis). Total RNA was isolated from the heart tissue, a 1005bp cDNA encoding complete polypeptide chain of 334 amino acids was generated by reverse transcriptase reaction and analyzed for nucleotide sequence. The consensus sequence obtained from both strands has shown 84% to 98% homology with that of different mammalian species. The attributed gene was cloned, expressed in BL21 (DE3) RIPL Codon Plus strain of E. coli using pET21a (þ) plasmid. The purified recombinant enzyme displayed a KM value of 50 lM for pyruvate, an optimum activity at 35 C and pH 7.0. The enzyme was found as a homotetramer of 140 kDa on FPLC based gel-filtration column. Molecular weight of a subunit of enzyme as determined by mass spectrometric analysis was 36530.21 Da. The present study describes the first ever report about the cDNA sequence and characteristics of recombinant LDH-B from River buffalo. Keywords: Lactate dehydrogenase B; Heart ventricles; River buffalo; E. coli expression; Gel-filtration; Mass spectrometric analysis
INTRODUCTION Lactate dehydrogenase (LDH; EC 1.1.1.27) catalyzes the interconversion of pyruvate and lactate. The enzyme is found in five tetrameric isozymes in mammalian tissues, these isozymes are combination of LDH-A and LDH-B subunits. A homotetrameric LDH-C4 isozyme is found only in mature testis and spermatozoa (1, 2). Three families of mammalian LDH genes (LDH-A, LDH-B, and LDH-C) and their Address correspondence to Muhammad Shahid Nadeem, Department of Genetics, Hazara University, Garden Campus, Mansehra 21300, Pakistan. E-mail: [email protected] 23
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related polypeptides have been studied in detail (3). According to phylogenetic studies, the LDH-C gene was originated by independent gene duplication events during the evolution of vertebrates, with separate LDH-B gene duplications in fish and birds (pigeon) (4–8). The isozyme LDH-A which is predominantly found in skeletal muscle, converts pyruvate to lactate under anaerobic conditions, whereas the LDH-B isozyme converts lactate to pyruvate in well oxygenated cardiac muscles (8). A subunit of each tetramer has a molecular weight of 35 to 36 kDa (9, 10). The isozymes have significant differences in the kinetic properties such as their thermal stability and sensitivity to inhibitors (11, 12). Lactate dehydrogenase has been isolated and characterized from a wide range of organisms including animals, plants, and bacteria (13–16). Nucleotide sequences of DNA encoding the subunit proteins of enzyme have been cloned and analyzed from a variety of organisms including birds and reptiles (7), fungi (17), bacteria (18), silk worm (19), and protozoans (16, 20). The characteristics of recombinant enzymes have also been studied. Distribution of LDH isozymes in different tissues and organs has clinical significance. Changes in the activity of specific isozymes in blood plasma are used as diagnostic markers for many diseases (21–23). Clinical assays usually involve enzymes from porcine and bovine sources. River buffalo (Bubalus bubalis) is a species of tremendous importance in livestock and rural economy in Pakistan as it contributes 27 million tons of milk and 0.68 million tons of meat annually (24, 25). We have already described the purification and characteristics of heart LDH-B from river buffalo (15). It was assumed that the recombinant of LDH-B with properties similar to that of native enzyme will present a potential alternate for the clinical assays. Present study was aimed at the cloning of cDNA, nucleotide sequence analysis, expression in E. coli, and characterization of purified recombinant enzyme from the species, which is unexplored for most of its genes and proteins.
MATERIALS AND METHODS Materials and Reagents Nicotinamide Adenine Dinucleotide (NADH), Sodium pyruvate, and other related reagents were obtained from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Trizol reagent kit was obtained from Invitrogen (Invitrogen, Carlsbad, CA, USA). E. coli expression strains and plasmid vectors were obtained from Novagen and Invitrogen. Restriction enzymes and cloning reagents were obtained from Fermentas (Thermo Fisher Scientific Inc., USA). Fresh B. bubalis heart tissue, used as raw material, was obtained from the main slaughter house in Lahore, Pakistan. Synthesis of cDNA Total RNA was isolated from fresh heart tissue by Trizol reagent kit. Procedure provided by the manufacturers was followed. The complementary strand of mRNA (cDNA) encoding a subunit of heart lactate dehydrogenase b was generated by Moloney Murine Leukaemia Virus reverse transcriptase (MoMLV) using gene-specific reverse primer 50 -AGCCTTGCAGCCGGAAGTCACAGGTCC-30 and incubation
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of reaction mixture at 37 C for 50 min. The reverse transcriptase was inactivated by incubation at 94 C for 5 min after the completion of reaction. DNA with two strands was synthesized by classic Polymerase Chain Reaction (PCR) using forward with nucleotide sequence as 50 -CTTTCTCTCCCCTGGGCCATATGGCAAC-30 and the aforementioned reverse primer. Primers were selected from the gene sequence encoding the enzyme in Bos taurus heart. The PCR product was analyzed on1.2% agarose gel, purified and used for cloning.
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Molecular Cloning of LDH-B Gene DNA purified from the agarose gel was ligated into pTZ57R=T plasmid which was used for the transformation of DH5a strain of E. coli. The transformed cells were selected for the presence of recombinant plasmid LB-agar medium containing 100 mg of ampicillin, 120 mg each of isopropyl b-D-1-thiogalactopyranoside (IPTG), and 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) per mL of medium. White colonies were selected, grown in LB broth containing same concentrations of ampicillin, IPTG, and X-gal. The plasmids were isolated using standard protocol and subjected to restriction with EcoRI and SalI. One of the positive clones was analyzed for nucleotide sequence for both strands. The consensus sequence was submitted to DDBJ=EMBL=GenBank, which is available under the accession number AB750686. Homology of newly sequenced gene was determined by the BLAST tool (26), alignment by ClustalW (27), and a phylogenetic tree was constructed for some closely related mammalian species. Production of Recombinant LDH-B in E. coli The site for NdeI restriction endonuclease was introduced at the 50 end of the gene by PCR. The nucleotide sequence obtained in the present study has a restriction site for NdeI enzyme at 435 bp. The recombinant pTZ57RT was separately restricted with EcoRI and NdeI and fragments of 435 bp and 570 bp were added in the ligation mixture containing pET21a (þ) restricted with the same enzymes. Ligation mixture was used for the transformation of E. coli BL21 (DE3) RIPL CodonPlus competent cells and successful transformations were confirmed by isolation of plasmids and restriction analysis. Conditions for the production of recombinant enzyme were optimized. Enzyme was produced by growing the recombinant cells at 25 C in a shaking incubator adjusted at 100 rpm in the presence of 0.5 mM of IPTG for 6–7 hrs. Bacterial culture was harvested by centrifugation, suspended in ice cold buffer (20 mM Potassium phosphate pH 7.0). Measurement of Enzyme Activity The activity of recombinant LDH-B was measured using the conditions optimized in our previous studies for native heart enzyme (18). Spectrophotometer (SHIMADZU BioSpec-1601) was adjusted at 340 nm and 35 C. The experimental and control cells were added with 220 mM NADH and 200 mM Sodium pyruvate in a reaction mixture of 3.0 mL prepared in phosphate buffer pH 7.0. The reagents were mixed and incubated for 5 min to record the change in absorbance. The enzyme
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solution was mixed with the reaction components and a change in the absorbance at 340 nm was monitored for 5 min. Activity was calculated by using Beer-Lambert law, using the value of extinction coefficient for NADH as 6220 M1 cm1.
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Purification of Recombinant Enzyme Bacterial cell pellet was suspended in ice-cold 20 mM Potassium phosphate buffer and passed through a French Press device at 22000 psi. The homogenate was centrifuged at 12000 g and 4 C for 15 min. Active fraction of recombinant enzyme was transferred to a clean glass beaker, precipitated by 80% (NH4)2SO4 saturation, and dialyzed against 20 mM Potassium phosphate buffer pH 7.0 and purified by Resource Q column (GE Healthcare) on FPLC (AKTA Purifier). Enzyme bound to the column was eluted by linear gradient of NaCl at a flow rate of 0.5 mL=min. A considerable proportion of expressed LDH-B was found as misfolded inclusion bodies. The inclusion bodies were washed thrice with 20 mM phosphate buffer pH 7.0 and the pellet obtained by final centrifugation was dissolved in 8 M urea solution containing 2 mM DTT (Dithiothretol) and folded by adding a pulse of solubilized solution in 2 M urea solution containing 2 mM DTT (sink solution) adjusted at 4 C in a stirrer. Each pulse of LDH-B was added slowly after two hours so that it may increase 0.5 units of absorbance of sink solution at 280 nm. After the batch-wise addition of whole solubilized inclusion bodies, the sink solution was kept at 4 C, stirring for 10 hours and subjected to dialysis in 10 volumes of 20 mM Phosphate buffer. The dialyzate was concentrated with using Kvick start cassette device. The activity of enzyme was measured in folded LDH-B sample and it was purified by the aforementioned procedure. Determination of Molecular Weight Purified recombinant enzyme was analyzed on SDS-PAGE to determine purity and molecular weight (28). The quaternary structure was determined by FPLC based gel-filtration chromatography which was carried out using AKTA Purifier and Superdex 200 10=300 GL column (GE Healthcare) in 50 mM phosphate buffer, pH 7.0, containing 100 mM NaCl. More accurate molecular weight of a subunit of enzyme was determined by MALDI-TOF analysis. Purified enzyme sample (1.0 mL) containing 3 mg of enzyme was mixed with 20 mL of matrix-B (5 mg sinapinic acid in 1 mL of 30% acetonitrile containing 0.1% trifluroacetic acid), 5 mL of this mixture was spotted on mass spectrometric plate, and allowed to dry for 30 min. Mass spectrum of enzyme was recorded with Bruker Autoflex MALDI-TOF (Bruker Daltonics Inc. MA 01821 USA-Billerica). Enzyme Kinetics The effect of temperature on the enzyme activity was determined by incubation of reaction mixture in water bath adjusted at different temperatures for 5 min. Enzyme activity was measured by the addition of suitable dilution of purified LDH-B. Thermostability of recombinant enzyme was studied by the incubation of enzyme aliquots at different temperatures in 50 mM Potassium phosphate buffer
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adjusted at pH 7.0. Residual enzyme activity was examined as described in the aforementioned assay method. To determine the effect of pH on enzyme activity, various buffer solutions adjusted at pH 4.5 to 10.5 were used and activity was measured. All other assay conditions were kept constant. For the calculation of KM value for pyruvate, the concentration of substrate was increased from 10 mM to 200 mM in the presence of 240 mM of NADH at pH 7.0.
RESULTS
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Cloning and Sequence Analysis Reverse transcriptase reaction resulted in the generation of a cDNA fragment that was converted to a double stranded 1005 bp DNA by PCR. Sequencing results indicated that the amplified DNA fragment consisted of a complete open reading frame coding for 334 amino acids (Fig. 1). The nucleotide sequence encoding B. bubalis LDH-B subunits has shown considerable homology with that of Bos taurus and Bos grunniens (98.1% each), Equus caballus (92.9%), Homo sapiens (92.2%), Sus scrofa (91.94%), Mus musculus (84.77%), Pongo abelii (92.33%), Canis familiaris (91.5%),
Figure 1 The nucleotide sequence encoding complete polypeptide chain of LDH-B from river buffalo (Bubalus bubalis) heart and amino acid sequence deduced from it.
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Felis catus (90.5%), and Oryctolagus cuniculus (87.8%) (Fig. 2a). The phylogenetic tree was constructed on the basis of homology in the nucleotide sequences encoding LDH-B in different species (Fig. 2b).
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Production and Purification of Recombinant Enzyme LDH-B gene was cloned into the expression plasmid pET21a (þ) using NdeI and EcoRI restriction sites. E. coli colonies with recombinant pET21a (þ) having LDH-B gene were confirmed by restriction analysis with EcoRI and NdeI enzymes. Two fragments of DNA restricted out with size of 570 bp and 435 bp (Fig. 3). When the DE3 RIPL cells of BL21 strain of E.coli transformed with pET-LDH-B recombinant plasmid were grown at 37 C, harvested after expression induction with 0.5 mM IPTG (final concentration) and analyzed by SDS-PAGE, most of the recombinant LDH-B were found as inclusion bodies. However, the recombinant protein was found
Figure 2 Comparison of nucleotide sequence, a) Percentage homology of LDH-B from the heart of river buffalo with that of some mammalian species, b) Phylogenetic tree indicating the association of different mammalian species on the basis of nucleotide sequence homology in the genes encoding heart LDH-B.
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Figure 3 Agarose gel electrophoresis photograph of expression plasmid pET21a (þ) cloned with LDH-B. Lane C: The uncut control plasmid. Lane E: The experimental plasmid ligated with LDH-B gene and cloned in E. coli. Lane M: DNA marker (Fermentas Cat. No. SM0403).
in both soluble form and as inclusion bodies when the incubation temperature was adjusted at 25 C and the induction time was increased to 6–7 hours (Fig. 4). The recombinant LDH-B constituted about 30–40% of the total E. coli proteins. During the purification, recombinant LDH-B was precipitated with 80% (NH4)2SO4 saturation along with some host cells proteins. Final yield of purified supernatant enzyme after Resource Q column was 46.8% and that from folded inclusion bodies was 47.94%. Enzyme from both fractions was purified up to 41.7 and 22.0 folds, respectively (Table 1).
Figure 4 SDS-PAGE photograph. Lane M: Protein marker. Lane A: Purified recombinant LDH-B. Lane B: LDH-B expression as inclusion bodies. Lane C: The expression of LDH-B as soluble form. Lane D: Expression of control pET21a (þ) proteins.
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Table 1 Recombinant LDH-B activity, specific activity, percentage yield, and fold purification at different stages of purification of enzyme from supernatant and inclusion bodies
Purification stages Crude Supernatant 80% Ammonium Sulphate PPT Resource Q Column Folded Inclusion Bodies Resource Q Column
Activity U=mL
Protein mg=mL
Specific activity
Total units
Percentage recovery
Fold purification
180.0 298.0 578.0 97.0 465.0
17.00 25.50 1.32 6.50 1.45
10.50 11.70 437.80 14.90 320.70
36000 29800 16850 9700 4650
100.00% 82.80% 46.80% 100.00% 47.94%
1.00 1.11 41.70 1.00 22.00
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One unit of enzyme is defined as the amount of enzyme which converts one micromole of substrate to product per minute under our assay conditions.
Figure 5 Molecular weight determination of lactate dehydrogenase B, a) Elution profile of LDH-B on FPLC based superdex-G-200 column for gel-filtration, b) MALDI-TOF analysis of purified heart lactate dehydrogenase. X-axis indicates the protein MW in Daltons and the Y-axis indicates the intensity of laser beam. The MW of purified recombinant LDH-B is 36530.21 Da.
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Molecular Weight of Recombinant LDH-B Purified recombinant enzyme provided a single band on SDS-PAGE with a molecular weight of 35 kDa. The retention volume of on gel-filtration chromatography column was 12.4 mL (Fig. 5a) and calculated molecular weight of active enzyme was about 140 kDa. Mass spectrometric analysis of enzyme displayed the molecular weight of a subunit of LDH-B as 36530 Da (Fig. 5b).
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Enzyme Characteristics The enzyme was stable and active at a wide temperature range. It displayed maximum activity at 35 C retaining approximately 30% activity at both 20 and 60 C. Thermostability experiments revealed that the activity decreased rapidly with the increase in temperature above 45 C and was completely lost by incubation at 75 C for 5 min. The enzyme has an optimum activity at pH 8.0, although it was active in a broad pH range. The KM value of recombinant enzyme was determined by a linear increase in the substrate concentration. Under our assay conditions, LDH-B displayed a KM value of 50 mM for sodium pyruvate.
DISCUSSION River buffalo (B. bubalis) is tremendously important in agriculture and livestock in south Asia. However, it remained unexplored for the characteristics of its enzymes and related gene sequences in the past. The present study describes the synthesis of cDNA encoded complete polypeptide chain of a subunit of heart LDH-B, the analysis of its nucleotide sequence, production of recombinant enzyme in E. coli, and its purification and properties. A 1005 bp cDNA molecule was synthesized using primers selected from the Bos taurus cDNA sequence encoded LDH-B (NCBI accession No. BC142006.1). B. taurus sequence was used for the selection of primers because of its higher similarity with the nucleotide sequences of river buffalo genes for aspartate aminotransferase and mitochondrial malate dehydrogenase reported in our previous studies (29, 30). The phylogenetic tree constructed on the basis of nucleotide sequence homology of LDH-B genes of different mammalian species has shown that B. bubalis is closely related to B. taurus and B. grunniens (Fig. 2b). Amino acid sequence deduced from cDNA has 97% homology with that of both species. However, the second two species have more than 99% amino acid homology with each other. Recombinant pET21a (þ) plasmid was ligated with LDH-B gene and used for the transformation of E. coli cells. When the production of recombinant enzyme was induced at 37 C the entire product appeared as inclusion bodies. However, a considerable proportion was found as active supernatant enzyme fraction while induced at 25 C for longer duration. Enzyme was purified from both fractions with simple ion-exchange chromatography (Table 1). The procedure adopted in the present study is more simple and economical as compared to the affinity chromatography methods reported in the literature for the purification of LDH from different species (31, 32). The purified enzyme has shown electrophoretic homogeneity on SDS-PAGE with a molecular weight of 35 kDa (33, 9). The tetrameric molecular structure and molecular weight of active enzyme was confirmed by gel-filtration
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chromatography results. Mass spectrometric analysis provided a molecular weight (36530 Da) closer to the theoretical molecular weight of enzyme (36547.4 Da) as calculated by ProtParam software of EXPASY. Purified enzyme has shown activity at a wide-range of temperature and pH with maximum activity at 35 C and pH 7.0, which is comparable with that of human enzyme (34) and different from ovine and lizard lactate dehydrogenase (35, 10). KM value of enzyme for sodium pyruvate is 50 mM, which is similar to that reported in the literature (36, 37). The present study provides useful information for cataloguing the river buffalo on the basis of its genetic make-up and characteristics of encoded enzymes.
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15. Nadeem MS, Nissar A, Shahid S, Imtiaz A, Mahfooz M, Asghar MT, Shakoori AR. Purification and Characterization of Lactate Dehydrogenase from the Heart Ventricles of River Buffalo (Bubalus bubalis). Pak J Zool 2011; 43:315–319. 16. Singh V, Kaushal DC, Rathaur S, Kumar N, Kaushal NA. Cloning, overexpression, purification and characterization of Plasmodium knowlesi lactate dehydrogenase. Prot Exp Purification 2012; 84:195–203. 17. Skory CD. Isolation and expression of lactate dehydrogenase genes from Rhizopus oryzae. Appl Env Microbiol 2000; 66:2343–2348. 18. Weekes J, Yuksel GU. Molecular characterization of two lactate dehydrogenase genes with a novel structural organization on the genome of Lactobacillus sp. strain MONT4 Appl Env Microbiol 2004; 70:6290–6295. 19. Xia H, Wu C, Xu Q, Shi J, Feng F, Chen K, Yao Q, Wang Y, Wang L. Molecular cloning and characterization of lactate dehydrogenase gene 1 in the silkworm, Bombyx mori. Mol Biol Reproduct 2011; 38:1853–1860. 20. Erdemir A, Aktas M, Dumanli N, Turgut-Balik D. Isolation, cloning and sequence analysis of the lactate dehydrogenase gene from Theileria annulata may leadto design of new antitheilerial drugs. Veterin Med 2012; 57:559–567. 21. Bien E, Balcerska A. Clinical significance of erythrocyte sedimentation rate, C-reactive protein and serum lactate dehydrogenase levels in the diagnosis, prognosis and treatment monitoring of children suffering from cancer. Med Wiek Rozwoj 2004; 8:1081–1089. 22. Kato GJ, McGowan V, Machado RF, Little JA, Taylor J, Morris CR, Nichols JS, Wang X, Poljakovic M, Morris SM Jr, Gladwin MT. Lactate dehydrogenase as a biomarker of hemolysis-associated nitric oxide resistance, priapism, leg ulceration, pulmonary hypertension, and death in patients with sickle cell disease. Blood 2006; 107:2279–2285. 23. Duan Y, Li G, Hu HX. Clinical significance of serum lactate dehydrogenase, b2-microglobulin and vascular endothelial growth factor level detection in patients with non-Hodgkin’s lymphoma. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2012; 20:608–610. 24. Bilal MQ, Suleman M, Raziq A. Buffalo: black gold of Pakistan. Livest Res Rural Develop 2006; 18:128–135. 25. Khan B, Iqbal A. The water buffalo: An underutilized source of milk and meat: A review. Pak J Zool 2009; 9:517–521. 26. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215:403–410. 27. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994; 22:4673–4680. 28. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nat 1970; 227:680–685. 29. Nadeem MS, Rashid N, Iqbal M, Gardner QA, Akhtar M. First cloning and characterization of aspartate aminotransferase from river buffalo (Bubalus bubalis). Biologia 2011; 66=6:1202–1210. 30. Nadeem MS, Ahmad H, Murtaza BN, Rauf A. cDNA Cloning, Nucleotide sequence analysis and characterization of Bubalus bubalis heart mitochondrial malate dehydrogenase. Rom Biotech Lett 2012; 17:7504–7514. 31. Ghose S, Mattiasson B. A two-step displacement and affinity chromatography purification in lactate dehydrogenase recovery from beef heart extract. Biotech Tech 1993; 7:615–620. 32. Labrou NE, Clonis YD. Biomimetic dye affinity chromatography for the purification of bovine heart lactate dehydrogenase. J Chromatogr A 1995; 7:35–44. 33. Allison WS, Admiraal J, Kaplan NO. The subunits of dogfish M4 lactic dehydrogenase. J Biol Chem 1969; 244:4743–4749.
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Animal Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/labt20
Cloning, E. coli Expression, and Characterization of Heart Lactate Dehydrogenase B From River Buffalo (Bubalus bubalis) Muhammad Shahid Nadeem b
a b
c
, Jenny Moran , Bibi Nazia Murtaza
, Khushi Muhammad & Habib Ahmad
a
a
Department of Genetics , Hazara University, Garden Campus , Mansehra , Pakistan b
School of Biological Sciences , University of the Punjab, Quaid-eAzam Campus , Lahore , Pakistan c
Institute of Science and Technology in Medicine (ISTM), School of Life Sciences , Keele University Staffordshire , Keele , United Kingdom Published online: 03 Dec 2013.
To cite this article: Muhammad Shahid Nadeem , Jenny Moran , Bibi Nazia Murtaza , Khushi Muhammad & Habib Ahmad (2014) Cloning, E. coli Expression, and Characterization of Heart Lactate Dehydrogenase B From River Buffalo (Bubalus bubalis), Animal Biotechnology, 25:1, 23-34, DOI: 10.1080/10495398.2013.804832 To link to this article: http://dx.doi.org/10.1080/10495398.2013.804832
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Animal Biotechnology, 25: 23–34, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1049-5398 print=1532-2378 online DOI: 10.1080/10495398.2013.804832
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CLONING, E. COLI EXPRESSION, AND CHARACTERIZATION OF HEART LACTATE DEHYDROGENASE B FROM RIVER BUFFALO (BUBALUS BUBALIS) Muhammad Shahid Nadeem1,2, Jenny Moran3, Bibi Nazia Murtaza2, Khushi Muhammad2, and Habib Ahmad1 1
Department of Genetics, Hazara University, Garden Campus, Mansehra, Pakistan 2 School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan 3 Institute of Science and Technology in Medicine (ISTM), School of Life Sciences, Keele University Staffordshire, Keele, United Kingdom Lactate dehydrogenase is an enzyme of glycolytic pathway which catalyzes the interconversion of pyruvate and lactate. The present study describes cDNA cloning, E. coli expression and characterization of lactate dehydrogenase B (LDH-B) from the heart ventricles of river buffalo (Bubalus bubalis). Total RNA was isolated from the heart tissue, a 1005bp cDNA encoding complete polypeptide chain of 334 amino acids was generated by reverse transcriptase reaction and analyzed for nucleotide sequence. The consensus sequence obtained from both strands has shown 84% to 98% homology with that of different mammalian species. The attributed gene was cloned, expressed in BL21 (DE3) RIPL Codon Plus strain of E. coli using pET21a (þ) plasmid. The purified recombinant enzyme displayed a KM value of 50 lM for pyruvate, an optimum activity at 35 C and pH 7.0. The enzyme was found as a homotetramer of 140 kDa on FPLC based gel-filtration column. Molecular weight of a subunit of enzyme as determined by mass spectrometric analysis was 36530.21 Da. The present study describes the first ever report about the cDNA sequence and characteristics of recombinant LDH-B from River buffalo. Keywords: Lactate dehydrogenase B; Heart ventricles; River buffalo; E. coli expression; Gel-filtration; Mass spectrometric analysis
INTRODUCTION Lactate dehydrogenase (LDH; EC 1.1.1.27) catalyzes the interconversion of pyruvate and lactate. The enzyme is found in five tetrameric isozymes in mammalian tissues, these isozymes are combination of LDH-A and LDH-B subunits. A homotetrameric LDH-C4 isozyme is found only in mature testis and spermatozoa (1, 2). Three families of mammalian LDH genes (LDH-A, LDH-B, and LDH-C) and their Address correspondence to Muhammad Shahid Nadeem, Department of Genetics, Hazara University, Garden Campus, Mansehra 21300, Pakistan. E-mail: [email protected] 23
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related polypeptides have been studied in detail (3). According to phylogenetic studies, the LDH-C gene was originated by independent gene duplication events during the evolution of vertebrates, with separate LDH-B gene duplications in fish and birds (pigeon) (4–8). The isozyme LDH-A which is predominantly found in skeletal muscle, converts pyruvate to lactate under anaerobic conditions, whereas the LDH-B isozyme converts lactate to pyruvate in well oxygenated cardiac muscles (8). A subunit of each tetramer has a molecular weight of 35 to 36 kDa (9, 10). The isozymes have significant differences in the kinetic properties such as their thermal stability and sensitivity to inhibitors (11, 12). Lactate dehydrogenase has been isolated and characterized from a wide range of organisms including animals, plants, and bacteria (13–16). Nucleotide sequences of DNA encoding the subunit proteins of enzyme have been cloned and analyzed from a variety of organisms including birds and reptiles (7), fungi (17), bacteria (18), silk worm (19), and protozoans (16, 20). The characteristics of recombinant enzymes have also been studied. Distribution of LDH isozymes in different tissues and organs has clinical significance. Changes in the activity of specific isozymes in blood plasma are used as diagnostic markers for many diseases (21–23). Clinical assays usually involve enzymes from porcine and bovine sources. River buffalo (Bubalus bubalis) is a species of tremendous importance in livestock and rural economy in Pakistan as it contributes 27 million tons of milk and 0.68 million tons of meat annually (24, 25). We have already described the purification and characteristics of heart LDH-B from river buffalo (15). It was assumed that the recombinant of LDH-B with properties similar to that of native enzyme will present a potential alternate for the clinical assays. Present study was aimed at the cloning of cDNA, nucleotide sequence analysis, expression in E. coli, and characterization of purified recombinant enzyme from the species, which is unexplored for most of its genes and proteins.
MATERIALS AND METHODS Materials and Reagents Nicotinamide Adenine Dinucleotide (NADH), Sodium pyruvate, and other related reagents were obtained from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Trizol reagent kit was obtained from Invitrogen (Invitrogen, Carlsbad, CA, USA). E. coli expression strains and plasmid vectors were obtained from Novagen and Invitrogen. Restriction enzymes and cloning reagents were obtained from Fermentas (Thermo Fisher Scientific Inc., USA). Fresh B. bubalis heart tissue, used as raw material, was obtained from the main slaughter house in Lahore, Pakistan. Synthesis of cDNA Total RNA was isolated from fresh heart tissue by Trizol reagent kit. Procedure provided by the manufacturers was followed. The complementary strand of mRNA (cDNA) encoding a subunit of heart lactate dehydrogenase b was generated by Moloney Murine Leukaemia Virus reverse transcriptase (MoMLV) using gene-specific reverse primer 50 -AGCCTTGCAGCCGGAAGTCACAGGTCC-30 and incubation
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of reaction mixture at 37 C for 50 min. The reverse transcriptase was inactivated by incubation at 94 C for 5 min after the completion of reaction. DNA with two strands was synthesized by classic Polymerase Chain Reaction (PCR) using forward with nucleotide sequence as 50 -CTTTCTCTCCCCTGGGCCATATGGCAAC-30 and the aforementioned reverse primer. Primers were selected from the gene sequence encoding the enzyme in Bos taurus heart. The PCR product was analyzed on1.2% agarose gel, purified and used for cloning.
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Molecular Cloning of LDH-B Gene DNA purified from the agarose gel was ligated into pTZ57R=T plasmid which was used for the transformation of DH5a strain of E. coli. The transformed cells were selected for the presence of recombinant plasmid LB-agar medium containing 100 mg of ampicillin, 120 mg each of isopropyl b-D-1-thiogalactopyranoside (IPTG), and 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) per mL of medium. White colonies were selected, grown in LB broth containing same concentrations of ampicillin, IPTG, and X-gal. The plasmids were isolated using standard protocol and subjected to restriction with EcoRI and SalI. One of the positive clones was analyzed for nucleotide sequence for both strands. The consensus sequence was submitted to DDBJ=EMBL=GenBank, which is available under the accession number AB750686. Homology of newly sequenced gene was determined by the BLAST tool (26), alignment by ClustalW (27), and a phylogenetic tree was constructed for some closely related mammalian species. Production of Recombinant LDH-B in E. coli The site for NdeI restriction endonuclease was introduced at the 50 end of the gene by PCR. The nucleotide sequence obtained in the present study has a restriction site for NdeI enzyme at 435 bp. The recombinant pTZ57RT was separately restricted with EcoRI and NdeI and fragments of 435 bp and 570 bp were added in the ligation mixture containing pET21a (þ) restricted with the same enzymes. Ligation mixture was used for the transformation of E. coli BL21 (DE3) RIPL CodonPlus competent cells and successful transformations were confirmed by isolation of plasmids and restriction analysis. Conditions for the production of recombinant enzyme were optimized. Enzyme was produced by growing the recombinant cells at 25 C in a shaking incubator adjusted at 100 rpm in the presence of 0.5 mM of IPTG for 6–7 hrs. Bacterial culture was harvested by centrifugation, suspended in ice cold buffer (20 mM Potassium phosphate pH 7.0). Measurement of Enzyme Activity The activity of recombinant LDH-B was measured using the conditions optimized in our previous studies for native heart enzyme (18). Spectrophotometer (SHIMADZU BioSpec-1601) was adjusted at 340 nm and 35 C. The experimental and control cells were added with 220 mM NADH and 200 mM Sodium pyruvate in a reaction mixture of 3.0 mL prepared in phosphate buffer pH 7.0. The reagents were mixed and incubated for 5 min to record the change in absorbance. The enzyme
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solution was mixed with the reaction components and a change in the absorbance at 340 nm was monitored for 5 min. Activity was calculated by using Beer-Lambert law, using the value of extinction coefficient for NADH as 6220 M1 cm1.
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Purification of Recombinant Enzyme Bacterial cell pellet was suspended in ice-cold 20 mM Potassium phosphate buffer and passed through a French Press device at 22000 psi. The homogenate was centrifuged at 12000 g and 4 C for 15 min. Active fraction of recombinant enzyme was transferred to a clean glass beaker, precipitated by 80% (NH4)2SO4 saturation, and dialyzed against 20 mM Potassium phosphate buffer pH 7.0 and purified by Resource Q column (GE Healthcare) on FPLC (AKTA Purifier). Enzyme bound to the column was eluted by linear gradient of NaCl at a flow rate of 0.5 mL=min. A considerable proportion of expressed LDH-B was found as misfolded inclusion bodies. The inclusion bodies were washed thrice with 20 mM phosphate buffer pH 7.0 and the pellet obtained by final centrifugation was dissolved in 8 M urea solution containing 2 mM DTT (Dithiothretol) and folded by adding a pulse of solubilized solution in 2 M urea solution containing 2 mM DTT (sink solution) adjusted at 4 C in a stirrer. Each pulse of LDH-B was added slowly after two hours so that it may increase 0.5 units of absorbance of sink solution at 280 nm. After the batch-wise addition of whole solubilized inclusion bodies, the sink solution was kept at 4 C, stirring for 10 hours and subjected to dialysis in 10 volumes of 20 mM Phosphate buffer. The dialyzate was concentrated with using Kvick start cassette device. The activity of enzyme was measured in folded LDH-B sample and it was purified by the aforementioned procedure. Determination of Molecular Weight Purified recombinant enzyme was analyzed on SDS-PAGE to determine purity and molecular weight (28). The quaternary structure was determined by FPLC based gel-filtration chromatography which was carried out using AKTA Purifier and Superdex 200 10=300 GL column (GE Healthcare) in 50 mM phosphate buffer, pH 7.0, containing 100 mM NaCl. More accurate molecular weight of a subunit of enzyme was determined by MALDI-TOF analysis. Purified enzyme sample (1.0 mL) containing 3 mg of enzyme was mixed with 20 mL of matrix-B (5 mg sinapinic acid in 1 mL of 30% acetonitrile containing 0.1% trifluroacetic acid), 5 mL of this mixture was spotted on mass spectrometric plate, and allowed to dry for 30 min. Mass spectrum of enzyme was recorded with Bruker Autoflex MALDI-TOF (Bruker Daltonics Inc. MA 01821 USA-Billerica). Enzyme Kinetics The effect of temperature on the enzyme activity was determined by incubation of reaction mixture in water bath adjusted at different temperatures for 5 min. Enzyme activity was measured by the addition of suitable dilution of purified LDH-B. Thermostability of recombinant enzyme was studied by the incubation of enzyme aliquots at different temperatures in 50 mM Potassium phosphate buffer
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adjusted at pH 7.0. Residual enzyme activity was examined as described in the aforementioned assay method. To determine the effect of pH on enzyme activity, various buffer solutions adjusted at pH 4.5 to 10.5 were used and activity was measured. All other assay conditions were kept constant. For the calculation of KM value for pyruvate, the concentration of substrate was increased from 10 mM to 200 mM in the presence of 240 mM of NADH at pH 7.0.
RESULTS
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Cloning and Sequence Analysis Reverse transcriptase reaction resulted in the generation of a cDNA fragment that was converted to a double stranded 1005 bp DNA by PCR. Sequencing results indicated that the amplified DNA fragment consisted of a complete open reading frame coding for 334 amino acids (Fig. 1). The nucleotide sequence encoding B. bubalis LDH-B subunits has shown considerable homology with that of Bos taurus and Bos grunniens (98.1% each), Equus caballus (92.9%), Homo sapiens (92.2%), Sus scrofa (91.94%), Mus musculus (84.77%), Pongo abelii (92.33%), Canis familiaris (91.5%),
Figure 1 The nucleotide sequence encoding complete polypeptide chain of LDH-B from river buffalo (Bubalus bubalis) heart and amino acid sequence deduced from it.
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Felis catus (90.5%), and Oryctolagus cuniculus (87.8%) (Fig. 2a). The phylogenetic tree was constructed on the basis of homology in the nucleotide sequences encoding LDH-B in different species (Fig. 2b).
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Production and Purification of Recombinant Enzyme LDH-B gene was cloned into the expression plasmid pET21a (þ) using NdeI and EcoRI restriction sites. E. coli colonies with recombinant pET21a (þ) having LDH-B gene were confirmed by restriction analysis with EcoRI and NdeI enzymes. Two fragments of DNA restricted out with size of 570 bp and 435 bp (Fig. 3). When the DE3 RIPL cells of BL21 strain of E.coli transformed with pET-LDH-B recombinant plasmid were grown at 37 C, harvested after expression induction with 0.5 mM IPTG (final concentration) and analyzed by SDS-PAGE, most of the recombinant LDH-B were found as inclusion bodies. However, the recombinant protein was found
Figure 2 Comparison of nucleotide sequence, a) Percentage homology of LDH-B from the heart of river buffalo with that of some mammalian species, b) Phylogenetic tree indicating the association of different mammalian species on the basis of nucleotide sequence homology in the genes encoding heart LDH-B.
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Figure 3 Agarose gel electrophoresis photograph of expression plasmid pET21a (þ) cloned with LDH-B. Lane C: The uncut control plasmid. Lane E: The experimental plasmid ligated with LDH-B gene and cloned in E. coli. Lane M: DNA marker (Fermentas Cat. No. SM0403).
in both soluble form and as inclusion bodies when the incubation temperature was adjusted at 25 C and the induction time was increased to 6–7 hours (Fig. 4). The recombinant LDH-B constituted about 30–40% of the total E. coli proteins. During the purification, recombinant LDH-B was precipitated with 80% (NH4)2SO4 saturation along with some host cells proteins. Final yield of purified supernatant enzyme after Resource Q column was 46.8% and that from folded inclusion bodies was 47.94%. Enzyme from both fractions was purified up to 41.7 and 22.0 folds, respectively (Table 1).
Figure 4 SDS-PAGE photograph. Lane M: Protein marker. Lane A: Purified recombinant LDH-B. Lane B: LDH-B expression as inclusion bodies. Lane C: The expression of LDH-B as soluble form. Lane D: Expression of control pET21a (þ) proteins.
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Table 1 Recombinant LDH-B activity, specific activity, percentage yield, and fold purification at different stages of purification of enzyme from supernatant and inclusion bodies
Purification stages Crude Supernatant 80% Ammonium Sulphate PPT Resource Q Column Folded Inclusion Bodies Resource Q Column
Activity U=mL
Protein mg=mL
Specific activity
Total units
Percentage recovery
Fold purification
180.0 298.0 578.0 97.0 465.0
17.00 25.50 1.32 6.50 1.45
10.50 11.70 437.80 14.90 320.70
36000 29800 16850 9700 4650
100.00% 82.80% 46.80% 100.00% 47.94%
1.00 1.11 41.70 1.00 22.00
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One unit of enzyme is defined as the amount of enzyme which converts one micromole of substrate to product per minute under our assay conditions.
Figure 5 Molecular weight determination of lactate dehydrogenase B, a) Elution profile of LDH-B on FPLC based superdex-G-200 column for gel-filtration, b) MALDI-TOF analysis of purified heart lactate dehydrogenase. X-axis indicates the protein MW in Daltons and the Y-axis indicates the intensity of laser beam. The MW of purified recombinant LDH-B is 36530.21 Da.
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Molecular Weight of Recombinant LDH-B Purified recombinant enzyme provided a single band on SDS-PAGE with a molecular weight of 35 kDa. The retention volume of on gel-filtration chromatography column was 12.4 mL (Fig. 5a) and calculated molecular weight of active enzyme was about 140 kDa. Mass spectrometric analysis of enzyme displayed the molecular weight of a subunit of LDH-B as 36530 Da (Fig. 5b).
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Enzyme Characteristics The enzyme was stable and active at a wide temperature range. It displayed maximum activity at 35 C retaining approximately 30% activity at both 20 and 60 C. Thermostability experiments revealed that the activity decreased rapidly with the increase in temperature above 45 C and was completely lost by incubation at 75 C for 5 min. The enzyme has an optimum activity at pH 8.0, although it was active in a broad pH range. The KM value of recombinant enzyme was determined by a linear increase in the substrate concentration. Under our assay conditions, LDH-B displayed a KM value of 50 mM for sodium pyruvate.
DISCUSSION River buffalo (B. bubalis) is tremendously important in agriculture and livestock in south Asia. However, it remained unexplored for the characteristics of its enzymes and related gene sequences in the past. The present study describes the synthesis of cDNA encoded complete polypeptide chain of a subunit of heart LDH-B, the analysis of its nucleotide sequence, production of recombinant enzyme in E. coli, and its purification and properties. A 1005 bp cDNA molecule was synthesized using primers selected from the Bos taurus cDNA sequence encoded LDH-B (NCBI accession No. BC142006.1). B. taurus sequence was used for the selection of primers because of its higher similarity with the nucleotide sequences of river buffalo genes for aspartate aminotransferase and mitochondrial malate dehydrogenase reported in our previous studies (29, 30). The phylogenetic tree constructed on the basis of nucleotide sequence homology of LDH-B genes of different mammalian species has shown that B. bubalis is closely related to B. taurus and B. grunniens (Fig. 2b). Amino acid sequence deduced from cDNA has 97% homology with that of both species. However, the second two species have more than 99% amino acid homology with each other. Recombinant pET21a (þ) plasmid was ligated with LDH-B gene and used for the transformation of E. coli cells. When the production of recombinant enzyme was induced at 37 C the entire product appeared as inclusion bodies. However, a considerable proportion was found as active supernatant enzyme fraction while induced at 25 C for longer duration. Enzyme was purified from both fractions with simple ion-exchange chromatography (Table 1). The procedure adopted in the present study is more simple and economical as compared to the affinity chromatography methods reported in the literature for the purification of LDH from different species (31, 32). The purified enzyme has shown electrophoretic homogeneity on SDS-PAGE with a molecular weight of 35 kDa (33, 9). The tetrameric molecular structure and molecular weight of active enzyme was confirmed by gel-filtration
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chromatography results. Mass spectrometric analysis provided a molecular weight (36530 Da) closer to the theoretical molecular weight of enzyme (36547.4 Da) as calculated by ProtParam software of EXPASY. Purified enzyme has shown activity at a wide-range of temperature and pH with maximum activity at 35 C and pH 7.0, which is comparable with that of human enzyme (34) and different from ovine and lizard lactate dehydrogenase (35, 10). KM value of enzyme for sodium pyruvate is 50 mM, which is similar to that reported in the literature (36, 37). The present study provides useful information for cataloguing the river buffalo on the basis of its genetic make-up and characteristics of encoded enzymes.
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