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. [2006]) (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
TcTrx cDNA was subcloned into an E. coli and yeast expression vector, respectively. The coding region of the TcTrx cDNA was amplified using gene specific flanking primers. The 5′ upstream primer contains Eco RI recognition site (5′GAA TTC GAT GTT ATC TTC GCT TGC ATC C3′) and the 3′ downstream primer contains Eco RI recognition site (5′GAA TTC GCG AGG CCC TGG ATG AG3′). Using 0.2 μg of TcTrx cDNA as a template, and 10 pmole of each 5′ upstream and 3′ downstream primers, a 405 bp fragment encoding the putative mature TcTrx gene was amplified by PCR. The fragment was ligated into pCRÒ4-TOPOÒ and transformed into E. coli. The recombinant plasmid was isolated and digested with Eco RI. The digestion products were separated on a 1.0% agarose gel. The 405 bp insert DNA was gel purified and subcloned into Eco RI site of pET-20b(+) expression vector (Novagen, Darmstadt, Germany). The recombinant DNA was then transformed into E. coli C43(DE3). However, the recombinant protein was not expressed in the E. coli expression system. We then subcloned the gene into the Eco RI site of the pYEX-S1 expression vector (Clontech, Mountain View, CA, USA) and introduced into Saccharomyces cerevisiae (trp− ura−), The recombinant protein was still not expressed in the Saccharomyces cerevisiae expression system. We decided to optimize the TcTrx DNA sequenced based on the yeast codon usage table (The codons were optimized by using the Codon Optimization Tool provided by the Integrated DNA Technologies (http://sg.idtdna.com/CodonOpt) with codon usage table of Saccharomyces cerevisiae. The GenScript Codon Usage Frequency Table Tool was used as the reference for yeast usage table. The optimized gene was custom synthesized by Genomics company, Taiwan. The optimized sequence is shown in Figure 1 in red. It was subcloned into a pET-20b(+) expression vector and the recombinant DNA transformed into E. coli C43(DE3). The recombinant protein was still not expressed in the E. coli expression system. We then re-amplified the codon-optimized pET-20b(+)-TcTrx DNA using two gene-specific primers: the 5′ upstream primer contained Eco RI recognition site (5′GAA TTC GAT GTT ATC TTC GCT TGC ATC C3′) and the 3′ downstream primer contained a His8-tag and Eco RI recognition site (5′ GAATTC GAG ACG TCA GTG GTG GTG GTG GTG GTG GTG GTG3′). Using the 0.2 μg optimized recombinant DNA of pET-20b(+)-TcTrx as a template, and 10 pmole of each 5′ upstream and 3′ downstream primers, a 0.4 kb fragment was amplified by PCR. The fragment was ligated into pCRÒ4-TOPOÒ and transformed into E. coli. The recombinant plasmid was isolated and digested with Eco RI. The digestion products were separated on 1.0% agarose gel. The 0.4 kb insert DNA was gel purified and subcloned into the Eco RI site of the pYEX-S1 expression vector and introduced into Saccharomyces cerevisiae (trp− ura−). The transformed yeast cells were selected by YNBDT (0.17% yeast nitrogen base, 0.5% ammonium sulfate, and 2% glucose) agar plates containing 20 μg Trp/mL. The presence of TcTrx cDNA in the selected transformants was verified by PCR using gene-specific flanking primers. The recombinant TcTrx protein was expressed in yeast in YPD medium (1% yeast extract, 2% peptone, 2% glucose). Expression of the functional recombinant TcTrx was analyzed by enzyme activity assay.
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. [2005]). 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 [1967]; 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. [2010]). 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.
Kinetic studies
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.
Enzyme characterization
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.