嗜酸氧化亚铁硫杆菌谷胱苷肽还原酶基因的克隆表达

Cloning and expression of the glutathione reductase genefrom Acidithiobacillus ferrooxidansCheng-gui Zhang *, Jin-lan Xia **, Zhen-yuan Nie, Guan-zhou QiuKey Laboratory of Biometallurgy of Ministry of Education, School of Resources Processing andBioengineering, Central South University, Changsha 410083, ChinaAbstractThe glutathione reductase (GR) from Acidithiobacillus ferrooxidans ATCC 23270was cloned and expressed in E. coli BL21. The in vitro enzymatic activity assay ofthe expressed product showed that the recombinant protein has the GR enzymaticproperties and its optimal activity at pH 7.5 suggests that GR is located in thecytoplasm. Alignment of GRs shows that the GR from A. ferrooxidans also containsthe highly conserved segments and in which the highly conserved NADPH bindingsite sequence one arginine residue was replaced by an asparagine residue.Key words: Glutathione reductase; Acidithiobacillus ferrooxidans; Geneexpression1 IntroductionGlutathione reductase (GR, Glutathione: NADP+ oxidoreductase, E.C.1.8.1.7) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) using an NADPH as the electron source. The enzyme is important in maintaining a reducing environment within the cell, and it belongs to the family of FAD-dependent disulfide oxidoreductases (12).Glutathione reductase (GR) plays a crucial role in maintaining a high intracellular concentration ratio of [GSH]/[GSSG] that is required in most living organisms to maintain the functionally important thiol groups of cellular proteins in the reduced form, as well as cellular growth and differentiation, cell signaling (5) and the detoxification of many electrophiles (3) encountered under both normal and oxidation stress conditions, that usually modify the expression of cellular glutathione reductase (GR) gene.GSH is identified to play a catalytic role to accelerate the bioxidation of the element sulfur (S8) in Acidithiobacillus.(14). Acidithiobacillus is a very important genus for the industrial bioleaching of sulfides minerals as well as the pretreatment of sulfides-embedded gold ores. Researches on the sulfur oxidation process in Acidithiobacillus spp. showed that the bacterial oxidation of the sulfur particles is believed to be initiated by the thiol groups of cysteine residues. Consequently, elemental sulfur are oxidized with catalysis of a periplasmic sulfur dioxygenase (SDO ：EC 1.13.11.18) to sulfite. This sulfur dioxygenase was described absolutely requiring low-molecular-mass thiol compounds for activity(15,16). In vitro assays showed that GSSH is the actual substrate of sulfur dioxygenase, a non-enzymatic reaction product from glutathione (GSH) and elemental sulfur, as shown in equations 1-3.89S +GSH (GS H)GSnH+(9-n)/8S→→8 (1)Foundation Item : Project (50321402) supported by Nature Science Foundation of China for innovation research group; Project (2004CB619204) supported by National major basic research item, China.Corresponding author : Xia Jin-lan, Ph.D., Professor; Tel & Fax: +86-731-8830544; E-mail: jlxia@2GS H+GS H GS G+H S >1, 1x+y -1x y x y →≥ (2)S D O 2-+223G S S H +O +H O G S H +S O +2H ⎯⎯⎯→ (3)The bio-oxidation processes from sulfur to sulfite were GSH-dependent disulfide-reductions to reproduce GSH, but also a uniquely reactive with GSH-mixed disulfides.Glutathione (GSH) present in millimolar concentrations is generally responsible for the low redox potential and high free SH (thiol groups) level inside of cells and kept reducing environments by NADPH and glutathione reductase (1, 6). This mechanism of thiol redox control is emerging as a major regulatory mechanism in signal transduction(8).In this study, the gene of GR protein was cloned from A. ferrooxidans and expressed in E. coli BL21, and the expressed product was purified by one-step affinity chromatography and characterized for its enzymatic and basic structural properties to understand the actual role of GR in biooxidation of sulfur.2 Materials and methods2.1 Bacterial strains and growth conditionsA. ferrooxidans ATCC 23270 was obtained from the American Type Culture Collection, and it was cultivated in ferrous iron-containing 9K medium (pH 2.0), which contains (in g liter-1): (NH 4)2S04, 3.0; MgSO 4 · 7H 20, 0.5; KC l, 0.1; K 2HPO 4, 0.5; Ca (NO 3)2, 0.01 and FeSO 4 · 7 H 20, 44.5 or sulfur pills, 5.Escherichia coli strain BL21 (DE3) was grown in Luria-Bertani medium.2.2 DNA manipulationsThe cultures were filtered to reduce the amount of jarosite and elemental sulfur. The cells were pelleted and washed sequentially twice with 5mmol/L H 2SO 4 and TE buffer (10 mmol/L Tris-Cl pH 8.0, 1 mmol/L EDTA), respectively, and the pelleted cells was then re-suspended in 400μL TE, to which 80μL 20% SDS, 3μL 20 g/L of Proteinase K were added. The suspended cells were mixed and incubated for 1 hour at 55°C. After that 100μL 5 mol/L NaCl and 80μL CTAB-NaCl solution (10% cetyltrimethylammol/Lonium bromide in 0.7mol/L NaCl) were sequentially added and mixed thoroughly, and the mixture was incubated for another 10 min at 65°C. The samples were then extracted with an equal volume of chloroform/isopropyl-alcohol, mixed well by shaking and vortexing and centrifuged for 15 min at 12,000 rpm. The supernatant was transferred to a new tube, to which two-three times of volume of isopropanol was added to precipitate DNA, and the DNA precipitate was centrifuged for 10 min at 12,000rpm, and then washed twice with 70% ethanol. The pellet was dried under vacuum and dissolved in 100μL of sterilized distilled water.2.3 Primers and PCR conditionsThe oligonucleotide primers were purchased from Bio Basic Inc. (Shanghai, China), and their sequences were deduced from the sequences of the open reading frames (ORFs) found in the available DNA genomic sequence of A. ferrooxidans ATCC 23270 (). gr gene was cloned fromthe total DNA of A. ferrooxidans ATCC 23270 using PCR amplification with primers: TGR-NH2-Bam H I 5′-GGCCTCCCATATGACACAATCCTACGATCTCATC-3′GR-CERT-Hin d III 5′-GGTGGTTGCTCTTTCAGCGCATGGTGACGAATTCCTC-3′GR-NH2- Bam H I and GR-CERT-Hin d III primers corresponding to the N-terminal and C-terminal end sequences of GR contain Bam H I and Hin d III restriction sites, respectively.All amplification reactions were done by using 10ng of DNA and controls were made by replacing DNA with water. The following components were added to a sterilized 0.2-mL microcentrifuge tube. The reaction mixtures (50μL) contained 1.0μL (20umol) of the primer, 1.5 units (1.5μL) of Taq DNA Polymerase (Fermentas), 5μL of 10×supplied PCR buffer, 1μL (10mmol/Lol) of each dNTP, 4μL (25 mmol/Lol) of MgCl2, and 1μL of DNA template. Distilled water was added to make up the volume to50μL.The DNA amplifications were carried out in a Perkin-Elmer DNA Thermal Cycler under heating at 93 ℃ for 3 min followed by 29-cycle of programmol/Led (93℃, 30 s denaturation step; 58 ℃, 30 s annealing step; 72℃, 30 s extension step) and by a final DNA extension step at 72℃ for 7 min. The amplified product was maintained at 4℃ after cycling.A low number of amplification cycles were used to decrease the errors in bases of sequence.2.4 Construction of the expression vector for GR protein and SDS-PAGE of GR proteinThe DNA fragments separated by electrophoresis in 1% agarose gels were recovered, purified with Wizard PCR Prep (Promega). The purified DNA fragments were ligated to pET28a (+) vector (Novagen) with T4 ligase (BioLabs), both previously digested with Bam H I and Hin d III. The ligation products (pET28-gr) were used to transform E. coli BL21 (DE3). The recombinant clones were selected onLuria-Bertani (LB, 0.5% yeast extract, 1% Bactotryptone, and 1% NaCl) agar plates containing 40μg/mL of kanamycin.The positive clones were analyzed by using colony PCR. Pure plasmids with inserts were obtained by using the Wizard Plus Minipreps DNA purification system (Promega) and digested with BamH I and Hind III, the recombinant vector was checked for the DNA sequence by sequencing in both directions to identify the correct of sequence without frameshift mutation.The induction expression analysis was done in the presence of 0.5 mmol/Lol/L or 1 mmol/Lol/L IPTG, which was added when the cultures reached an optical density of 0.6 at 600 nm, i.e. abbreviated OD600 of 0.6. The expressed recombinant GR was analyzed on total cell extracts by 15% of SDS-PAGE.2.5 The purification of GR proteinGR was purified under denaturing conditions as follows:The E. coli strain BL21 (DE3) cells with GR-pET28 plasmid was grown at 30°C in 500ml of LB medium containing Kanamycin (30µg/ml) to an OD600 of 0.6. At this point, the cells were incubated at 30°C with the addition of 0.5 mmol/L IPTG about 6 hours at 180rpm. The cells were harvested by centrifugation and the cell pellet was washed with an equal volume of sterile water. The cells were again harvested by centrifugation, suspended in start buffer (20 mmol/L potassium phosphate, pH 7.4, 0.5mol/L NaCl), incubated with 5mg lysozyme at room temperature for half an hour. The cells were lysed by sonication four times for 30 s each time using a 150-W Autotune Series High Intensity Ultrasonic sonicator equipped with an 8 mmol/L-diameter tip.After centrifugation (at 20,000 g for 15 min), the pellet was resuspended in 15 mL of binding buffer containing 8mol/L Urea and incubated for 1 h in an ice bath. The sample was centrifuged (20,000 g for 20 min), and the supernatant,was used for protein purification.The Hi-Trap column（Amersham Bioscience）was first equilibrated with 0.1 mol/L nickel sulfate to charge the column with nickel ions followed by 5 column volumes of MiliQ water to remove unbound nickel ions from the column, and then by 5 column volumes of start buffer (20 mmol/L potassium phosphate, pH 7.4, 0.5 mol/L NaCl, 8M Urea) to equilibrate the column. The clarified sample was applied to the Hi-Trap column after filtering it through a 0.45 μm filter.The column was washed with 5 column volumes of start buffer followed with 5 column volumes of wash buffer (20 mmol/L potassium phosphate, pH 7.4, 0.5 mol/L NaCl, 50 mmol/L imidazole, 8mol/LUrea), and subsequently the protein was eluted with an elution buffer (20 mmol/L potassium phosphate, pH 7.4, 0.5 mol/L NaCl, 500 mmol/L imidazole, 8M Urea).The collected fractions (1.0 ml) were analyzed by SDS-PAGE. Finally, GR protein containing fractions, which were essentially free from other proteins, were pooled and renatured by removal of the urea in five sequential dialysis steps with 50 mmol/L phosphate (pH 7.6) buffer containing 8, 4, 1, and 0.5mol/L Urea and no urea, respectively.The method of Bradford (10) was used to determine the protein content with bovine serum albumin as the standard. The eluted fractions were analyzed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE). The gels were stained with Coomassie Brilliant Blue R-250.2.6 Assay of recombinant glutathione reductaseGR activity was measured according to the method of Bhattacharya A and Bhattacharya S (2) using the principle that GR utilizes one molecule of NADPH to catalyze the conversion of one molecule of substrate (GSSG) into two molecules of GSH.An assay mixture was constituted with 50mmol/L phosphate buffer (pH 7.6), 1mmol/L EDTA, 0.1mmol/L NADPH, 1mmol/L GSSG, and 0.1% BSA. The activities were assayed at 20℃in a final volume of 3mL. The mixture was preincubated for 5 min at room temperature, the sample (cytosol) was added to it and mixed, and the decrease in absorbance was monitored at 340nm in a Beckman DU 640△spectrophotometer. The increases in O.D. /min were calculated using a blank, which contained all the components of the assay mixture except GSSG. The activity of the enzyme was calculated using the molar extinction coefficient of NADPH (ε340=6.22×103 (mol/L)-1 cm-1). One unit of GR activity was defined as the amount of enzyme that catalyzes the oxidation of 1 µmol of NADPH per min. All assays were performed on triplicate samples, and means ± SD are reported.3Results3.1 Agarose gel electrophoresis of the PCR product of gr geneCloning of the GR protein gene from Acidithiobacillus ferrooxidans ATCC 23270 and construction of expression vector PCR product was analyzed by using 6% agarose gel electrophoresis and the band with correct size was clearly shown on the electrophoresis [Fig. 1(A)]. The resulting full-length gr genewas 1350 bp encoding 449 aa.bp 8000100060005000200015001500bp M 1M 1(A) (B) Figure 1 (A) lane 1, Agarose gel electrophoresis of the PCR product of gr gene cloned from A. ferrooxidans ATCC 23270 genome by PCR; lane M, DNA marker; (B) lane 1, Agarose gel electrophoresis the gr gene cut out from pET28-gr plasmid by BamH I and Hind III double enzyme digestion; lane M, DNA marker. The recombinant vector pET28a-gr was used to transform E. coli BL21 to propagate. The pET28a-gr was extracted and again double-enzyme digested with BamH I and Hind III, the result confirms that the PCR product has been cloned into pET28a (+), as seen from the appeared band with correct size [Fig. 1(B)]. 3.2 SDS-PAGE of expressed product E. coli BL21 was transformed with the verified pET28a-gr express the gr gene. The total proteins of the cell were analyzed with SDS-PAGE after an IPTG induction. A clearly visible band is observed with the same molecular weight approximately 49.6 kDa corresponding to those estimated from deduced amino acid sequence (Fig. 2A), the product accounts for about 60% of total cell proteins, measured with the software of Quantity One. We found that the protein was localized in the insoluble inclusion bodies. To resolubilize recombinant GR protein, we sonicated the IPTG-induced bacteria, centrifuged them at 12,000×g, and purified the protein using a His-tag bound purification procedure.The subunit molecular mass of the 6×His fusion proteinwas determined to be approximately 50 kDa using 15% SDS-PAGE (Fig. 2B).Figure 2 （A）Identification of the GR protein expression on 15% SDS–PAGE and stained with Coomassie blue: lane 1, protein molecular mass markers; lane 2, the total E. coli BL21 cell proteins transformed with pET28a-gr without the absence of IPTG; lane 3 and lane 4, The total cell proteins in the absence of 0.5 mM and 1 mM IPTG; lane 4, purified GR protein after Hi-Trap chromatography. The arrowhead indicates the migrating position of GR protein。