Molecular cloning and characterization of a thioredoxin from Taiwanofungus camphorata
© Chen et al.; licensee Springer. 2014
Received: 11 July 2014
Accepted: 21 November 2014
Published: 4 December 2014
Thioredoxin (Trx) is reduced by thioredoxin reductase. Trx is used in ribonucleoide reduction, assimilatory sulfate reduction, in modulation of protein sulfhydryl groups, and refolding proteins.
A TcTrx (Tc: Taiwanofungus camphorata) cDNA (640 bp, GenBank AY838902.1) encoding a putative thioredoxin (Trx) of 135 amino acid residues with calculated molecular mass of 16.17 kDa was cloned from Taiwanofungus c amphorata. The deduced amino acid sequence containing a motif (Cys-Gly-Pro-Cys) that is highly conserved among the reported Trxs. A three dimensional structural model of the TcTrx has been created based on the known structure of Malassezia sympodialis Trx (MsTrx, PDB ID: 2j23). To characterize the TcTrx, the codon optimized coding region was subcloned into an expression vector and transformed into Saccharomyces cerevisiae. The recombinant His8-tagged TcTrx was expressed and purified by Ni affinity chromatography. The purified enzyme showed a band of approximately 32 kDa (expected dimeric form) on a 12% SDS-PAGE. The molecular mass determined by MALDI-TOF is 33.16 kDa which suggests that the purified enzyme is a dimeric enzyme. Furthermore, the enzyme exhibited TcTrx activity via insulin assay. The Michaelis constant (K M ) value for insulin was 3.78 × 10−2 mM. The enzyme’s half-life of deactivation was 13 min at 45°C. The enzyme was most active at pH 7.
A three dimensional structural model of T. camphorata Trx based on its TcTrx cDNA sequence. The active form of the TcTrx has been successfully expressed in yeast. The enzyme possesses Trx activity and is capable of reduction of disulfide bonds during the formation of newly synthesized proteins.
KeywordsTaiwanofungus camphorata Thioredoxin (Trx) Three-dimension structural model Insulin
Taiwanofungus camphorata (T. camphorata) is a valued mushroom found only in the forests of Taiwan. It has been used for centuries as health food, among others (Ao et al. ). T. camphorata has been shown to have anti-inflammatory properties (Hsieh et al. ). The active compounds identified from the fruiting bodies of T. camphorata in a submerged culture are benzenoids, diterpenes, maleic and succinic acid derivatives, polysaccharides, steroids, and triterpenoids (Ao et al. ). It can be obtained as health supplements formulated from the mass of T. camphorata from the artificial cultivation by an Asian Nova Biotechnology Inc (http://www.asian-bio.com/) at a high market value. Many studies were aimed to find the exact bioactive compounds of the mushroom (Ao et al. ). In order to search for physiologically active components including antioxidant enzymes, we have established an expressed sequence tag (EST) from the fruiting bodies of T. camphorata. Based on the established EST, several antioxidant enzymes including a 2-Cys peroxiredoxin (Huang et al. ), a catalase (Ken et al. ), a dithiol glutaredoxin (Ken et al. ), a 2-Cys peroxiredoxin isozyme (Liau et al. ), a monothiol glutaredoxin (Ken et al. ), a nitroreductase (Chen et al. ), a peroxiredoxin (Huang et al. ) and an aryl-alcohol dehydrogenase (Ken et al. ) have been cloned and characterized. This encourages us further to search for active components from the established EST of T. camphorata for potential health food applications.
Thioredoxin (Trx) is reduced by thioredoxin reductase with NADPH as the hydrogen donor. Trx is used in refolding proteins ((Li and Churchich ), in modulation of protein sulfhydryl groups (Buchanan et al. ), and phage assembly (Russel and Mode ). Here, we report a three dimensional structural model of Taiwanofungus camphorata thioredoxin based on its sequence. The coding sequence of the TcTrx cDNA was introduced into an S. cerevisiae expression system and the active enzyme purified and characterized its properties.
Isolation of TcTrx cDNA
We have previously established an EST database from fruiting bodies of T. camphorata and sequenced all clones with insert size greater than 0.4 kb (data not shown). The identity of a Trx cDNA clone was assigned by comparing the inferred amino acid sequence in various databases using the basic local alignment search tool (BLAST) (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). The Trx cDNA fragment was subcloned into pCR®4-TOPO® (invitrogen, CA) vector and transformed into E. coli TOPO10. The nucleotide sequence of the insert was determined in both strands. Sequence analysis revealed that the Trx cDNA covered an open reading frame of a putative Trx cDNA (640 bp, GenBank AY838902.1).
Bioinformatics analysis of TcTrx
The BLAST program was used to search homologous protein sequences in the nonredundant database (NRDB) at the National Center for Biotechnology Information, National Institutes of Health (http://www.ncbi.nlm.nih.gov/). Multiple alignments were constructed using ClustalW2 program. Protein secondary structure was predicted by SWISS-MODEL program and represented as α helices and β strands. A three dimensional structural model of TcTrx was created by SWISS-MODEL (Arnold et al. ) (http://swissmodel.expasy.org/) based on the known crystal structure of Malassezia sympodialis Trx (MsTrx, PDB ID: 2j23).
Subcloning of TcTrx cDNA into an expression vector
Expression and purification of the recombinant TcTrx
The yeast transformant containing the TcTrx gene was grown at 30°C, 170 rpm in 100 mL of YPD medium for 18 h. The cells were harvested and the soluble proteins extracted in PBS (phosphate buffer saline) with glass beads as described previously (Ken et al. ). The recombinant TcTrx was purified by Ni-NTA affinity chromatography (elution buffer: 1× PBS/5% glycerol/1 mM DTT containing 20–250 mM imidazole) according to manufacturer’s instruction (Qiagen). The purified protein was analyzed by a 12% SDS-PAGE followed by staining with Coomassie Brilliant Blue R-250 and destaining with 10% acetic acid/10% methanol. The purified protein was pooled, desalted, and buffer exchanged using Amicon ultra centrifugal filter unit (5000 MWCO). The exchanged buffer exchanged was 0.01 × PBS/0.01 mM DTT/2.5 mM imidazole/0.1% glycerol. The final recombinant TcTrx protein concentration was determined by a Bio-Rad Protein Assay Kit (Richmond, CA). For storage, one volume of glycerol was added to the purified protein and stored at −20°C for further analysis.
Molecular mass analysis via JOEL MALDI-TOF
The purified recombinant TcTrx (0.82 μg/μL) was prepared in 0.01 × PBS containing 0.01 mM DTT, 2.5 mM imidazole and 0.1% glycerol. The sample (5 μL) was used for molecular mass determination using an MALDI-TOF mass spectrometer (JMS-S3000, Japan).
TcTrx activity assay
Trx activity was assayed by the method of Holmgren (Holmgren and Reichard ; Holmgren [1979a], [b]) using the insulin precipitation assay which was monitored by a spectrophotometric assay of the increase in turbidity at 650 nm. The reaction mixture (200 μL) at 25°C contained 0.1 M potassium phosphate (pH 7.0), 2 mM EDTA, 0.33 mM DTT, 15 mM NADPH, 0.025 mM insulin and 1.0 μg TcTrxR (thioredoxin reductase from T. camphorata, Huang et al. ). The reaction was started by the addition of 1.0 μg TcTrx (0.41 μg/μL). The reaction was followed by the increase in A 650 due to insulin precipitation on reduction.
The kinetic properties of the TcTrx (1.0 μg) was determined by varying the concentrations of insulin (0.025 ~ 0.055 mM). The change in absorbance at 650 nm was recorded between 30 and 60 sec. The KM, Vmax and kcat were calculated from Lineweaver-Burk plots.
The TcTrx enzyme was tested for stability in terms of its activity under various conditions. Aliquots of the TcTrx sample (1.0 μg) were treated as follows: (1) Thermal effect. Each enzyme sample (1.0 μg) was heated at 40, 50, or 60°C for 5 min. Then samples were checked for TcTrx activity: 80% residual activity at 40°C treatment, 40% residual activity at 50°C treatment. Therefore, we choose at 45°C heating to this enzyme effect, each enzyme sample (1.0 μg) was heated at 45°C for 2, 4, 8, 16 min. (2) pH effect. Each enzyme sample (1.0 μg) was adjusted to desired pH by adding a half volume of buffer with different pHs: 0.2 M citrate buffer (pH 4.0), 0.2 M phosphate buffer (pH 6.0, 7.0 or 8.0) or 0.2 M CAPS buffer (pH 10.0). Each sample was incubated at 37°C for 30 min. At the end of each treatment, samples were checked for TcTrx activity by insulin precipitation assay at pH 7.
Results and discussion
A three dimensional structural model of Taiwanofungus camphorata thioredoxin based on its TcTrx cDNA sequence
Expression and purification of the recombinant optimized TcTrx
Kinetic studies of the purified TcTrx
Kinetic characterization of TcTrx and that from other sources
3.7 × 10−2
3.1 × 101
E. coli Trx
1.1 × 10−2
6.3 × 10−3
5.7 × 10−3
1.9 × 102
Properties of the purified TcTrx
A three dimensional structural model of T. camphorata Trx based on its TcTrx cDNA sequence. This study reported the first cloning and expression of an important reduction enzyme, TcTrx, from T. camphorata. The active form of the TcTrx has been successfully expressed in yeast. The enzyme possesses Trx activity and is capable of reduction of disulfide bonds during the formation of newly synthesized proteins.
The TcTrx has a higher KM value (Table 1) therefore can work under higher substrate concentration. Its kcat value is compatible to that of Trx from another source. It is likely that the Trx is one of the enzymes responsible for reducing protein disulfide targets in T. camphorata.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Phosphate buffer saline
This work was supported by the National Science Council of the Republic of China, Taiwan under grant MOST 103-2313-B-019 -006 to C-T. Lin.
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