Total RNA preparation from lemon and cDNA synthesis
A fresh lemon (Citrus limonum) was obtained from a local market. The lemon including the skin (4 g) was frozen in liquid nitrogen and ground to powder in a ceramic mortar. PolyA mRNA (35 μg) was prepared using Straight A’s mRNA Isolation System (Novagen, USA). Four μg of the mRNA was used in the 5′-RACE-Ready cDNA and 3′-RACE-Ready cDNA synthesis using Clontech’s SMART RACE cDNA Amplification Kit.
Isolation of ClPDI cDNA
Using the 5′-RACE-Ready cDNA of lemon as a template and two degenerate primers (5′ AGY CAA GGT GCH TTC CAG 3′ and ACY TTM ACW GGC TCR TTG TT), a 0.2 kb fragment was amplified by PCR. The degenerate primers were designed based on the conserved sequences of PDI from AtPDI (Arabidopsis thaliana, AY063059), HsPDI (Homo sapiens, 3F8U_A), MmPDI (Mus musculus, 2DJ2_A), HiPDI (Humicola insolens, 2DJJ_A). The 0.2 kb fragment was subcloned and sequenced. On the basis of this DNA sequence, two primers near both ends, a ClPDI-6R primer (5′ ACY TTM ACW GGC TCR TTG TT 3′) and a ClPDI-7 F primer (5′ GCA CCT TGG GTG AAG GAA TAC 3′) were synthesized. The primers allowed sequence extension from both ends of the 0.2 kb fragment when used with the UPM primer (universal primer A mix, purchased from BD biosciences). Two PCRs were carried out each using 0.1 μg of the 5′-RACE-Ready cDNA or 3′-RACE-Ready cDNA as a template. The primer pairs in each reaction were UPM and ClPDI-6R primers, and UPM and ClPDI-7 F primers. A 1.3 kb fragment (5′-RACE; 5′-DNA end) and a 1.0 kb DNA (3′-RACE; 3′-DNA end) were amplified by PCR. Both DNA fragments were subcloned into pCR4 vector and transformed into E. coli TOPO10. The nucleotide sequences of these inserts were determined in both strands. Sequence analysis revealed that the combined sequences covered an open reading frame of a putative ClPDI cDNA (1828 bp, EMBL no. HM641784). The identity of this ClPDI clone was assigned by comparing the inferred amino acid sequence in various data banks using the basic local alignment search tool (BLAST).
Bioinformatics analysis of ClPDI
The BLASTP 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 3-D structural model of PDI was created by SWISS-MODEL (http://swissmodel.expasy.org/) based on the known crystal structure of Homo sapiens PDI (PDB code 3F8U_A) (Dong et al. 2009). The modeling data was then superimposed with that of PDB ID: 3F8U_A by DeepView Swiss-PdbViewer v4.1 (http://spdbv.vital-it.ch/).
Subcloning of ClPDI cDNA into an expression vector
The coding region of the ClPDI cDNA was amplified using gene specific flanking primers. The 5′ upstream primer contains Eco RI recognition site (5′ CGT CTC GAA TTC GAT GGC CAG TCG ATC GAT 3′) and the 3′ downstream primer contains Not I recognition site (5′ GCG GCC GCG AGC TCA TCT TTT CCA GA 3′). Using 0.2 μg of ClPDI cDNA as a template, and 10 pmole of each 5′ upstream and 3′ downstream primer, a 1.5 kb fragment was amplified by PCR. The fragment was ligated into pCR4 and transformed into E. coli. The recombinant plasmid was isolated and double digested with Eco RI and Not I. The digestion products were separated on a 1.0% agarose gel. The 1.5 kb fragment was gel purified and subcloned into the Eco RI and Not I site of pET-20b(+) expression vector (Novagen). The recombinant DNA was then transformed into E. coli Rosetta (DE3)pLysS and protein expressed by isopropyl β-D-thiogalactopyranoside (IPTG) induction. Expression of functional recombinant protein was demonstrated by enzyme activity assay as described below.
Expression and purification of the recombinant ClPDI
The transformed E. coli containing the ClPDI gene was grown at 37°C in 200 mL of Luria-Bertani containing 50 μg/mL ampicillin until A
600
reached 0.6. Protein expression was induced by the addition of IPTG to a final concentration of 50 μM. The culture was incubated at 80 rpm for an additional 2 h at 32°C. The cells were harvested and soluble proteins extracted in PBS with glass beads as described before (Ken et al. 2005). The recombinant ClPDI was purified by Ni-NTA affinity chromatography (elution buffer: 30% PBS containing 20–250 mM imidazole) as per the manufacture’s instruction (Qiagen). The purified protein was checked by a 10% SDS-PAGE. Protein bands on gel were visualized by staining with Coomassie Brilliant Blue R-250. Protein concentration was determined by a Bio-Rad Protein Assay Kit (Richmond, CA) using bovine serum albumin as a reference standard.
Molecular mass analysis via electrospray ionization quadrupole-time-of-flight (ESI Q-TOF)
The purified recombinant ClPDI (0.21 mg/mL) was prepared in 0.1 mM Tris–HCl containing 0.05 mM NaCl, 0.5 mM imidazole and 0.03% glycerol. The sample (5 μL) was used for molecular mass determination using an ESI Q-TOF mass spectrometer (Micromass, Manchester, England).
ClPDI activity assay
PDI activity was assayed by the method of Ibbetson and Freedman (1976) using scrambled ribonuclease (sRNase A) as a substrate. In this method, PDI was used to activate sRNase A then the ribonuclease activity was monitored spectrophotometriclly as described below. Each 5 μL ClPDI sample (0.2 μg/μL stock solution) was first pre-incubated with 10 μL of 100 μM DTT (dithiothreitol) for 5 min at 25°C. Next, a 0–60 μL portion of the sRNase A (0.036 μM) was added, followed by addition of 10 μL of 0.5 M Tris/HCl, pH 7.5 and appropriate amount of H2O to 90 μL. The mixture was incubated for 20 min at 25°C to allow conversion of sRNase A to active RNase by PDI. The RNase activity was then measured by its ability to degrade RNA. Ten μL of RNA (2 μg/μL) was added to each assay mixture (Total volume was 100 μL). The samples were incubated at 37°C for another 5 min. Three hundred μL of 95% ethanol was added to each assay mixture to precipitate the residual RNA. Ribonuclease activity was monitored by observing the decrease in A
260
of the residual RNA.
Kinetic studies
The kinetic properties of the ClPDI (1.0 μg) was determined by varying the concentrations of sRNase A (3.6 to 21.6 nM) with fixed amount of 20 μg RNA (2 μg/μL). The change in absorbance at 260 nm was recorded between 1 and 20 min. The KM, Vmax and kcat were calculated from Lineweaver-Burk plots.
Enzyme characterization
The ClPDI enzyme was tested for stability in terms of its activity under various conditions. Aliquots of the CIPDI sample (1.0 μg) were treated as follows: (1) Thermal effect. Each enzyme sample (1.0 μg) was heated at 37, 50, 60, 70, or 80°C for 20 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 2.5, or 4.0), 0.2 M phosphate buffer (pH 6.0, 7.0 or 8.0) or 0.2 M CAPS buffer (pH 10.0, or 11.0). Each sample was incubated at 37°C for 1 h. (3) Imidazole effect. During protein purification, the CIPDI enzyme was eluted with imidazole, therefore, its effect on activity was examined. Imidazole was added to each enzyme sample to the final levels of 0.2, 0.4, 0.8 or 1.0 M and incubated at 37°C for 1 h. (4) DTT effect. DTT was added to each enzyme sample to the final levels of 10, 30, 70, 100 or 200 μM and incubated at 37°C for 5 min. At the end of each treatment, samples were checked for CIPDI activity.
