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trypsin in a 1:1 molar ratio. FPLC binding studies also confirmed that rWbCTI binds the proteases in a 1:1 molar ratio. Key words: chymotrypsin-trypsin inhibitor; ...
Biosci. Biotechnol. Biochem., 73 (12), 2671–2676, 2009

Genomic and cDNA Cloning, Expression, Purification, and Characterization of Chymotrypsin-Trypsin Inhibitor from Winged Bean Seeds Sanhita R OYy and Samir Kumar D UTTA Department of Molecular and Human Genetics, Indian Institute of Chemical Biology, Kolkata 700032, India Received July 17, 2009; Accepted September 18, 2009; Online Publication, December 23, 2009 [doi:10.1271/bbb.90519]

A 516-bp winged bean chymotrypsin-trypsin inhibitor (WbCTI) gene was amplified from genomic DNA and cDNA isolated from winged bean using a pair of degenerate primers designed on the basis of the amino acid sequences of WbCTI. The amplified PCR products were cloned and sequenced to confirm their authenticity. DNA sequence analysis of the genomic and cDNA clones of WbCTI revealed the same nucleotide sequence in the coding region and showed WbCTI to be an intron less gene. WbCTI was subcloned into pTrc99A and expressed in Escherichia coli to yield a recombinant protein (rWbCTI). rWbCTI was purified by a rapid and single step immunoaffinity chromatography method, with an overall yield of 1.1 mg/g of wet cells. The homogeneity of the purified protein was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed the presence of a single protein band. Functionally rWbCTI is indistinguishable from WbCTI, since both inhibit bovine trypsin and chymotrypsin in a 1:1 molar ratio. FPLC binding studies also confirmed that rWbCTI binds the proteases in a 1:1 molar ratio. Key words:

chymotrypsin-trypsin inhibitor; cloning and expression; intronless gene; Kunitz protease inhibitor; winged bean

Proteinase inhibitors are ubiquitous in nature and play an important role in the defense of plants against insect attack.1) Plant protease inhibitors are also involved in the responses to various biotic and abiotic stresses, e.g., pathogen invasion, wounding, and environmental stresses.2,3) Proteinase inhibitors are common in plants and have drawn attention as possible transgenes for insect defense in crops. They are of particular interest because they are generally the product of a single gene and inhibit proteolytic enzymes of animal and fungal origin, but rarely of plant origin, and therefore are thought to act as protective agents.4–7) Hence, proteinase inhibitor genes from various sources have been transferred to several plants of economic interest, resulting in transgenic plants more resistant to insect invasion.8) In addition, inhibitor proteins themselves might also act as storage proteins.9) Besides their natural biological functions, protease inhibitors might be useful in treating inflammation, hemorrhage,10) and y

cancer.11,12) They are present in huge amounts in Leguminosae seeds. Leguminous plant seeds usually contain two major types of protease inhibitors, the Bowman-Birk type (about 8 kDa) and the Kunitz type (about 20 kDa) with two disulfide bridges.13) Winged bean (Psophocarpus tetragonolobus (L.) DC), regarded as the ‘‘soybean of the tropics,’’ has a high protein and oil content in the seeds13) and thus, like soybean, can substitute for animal protein in the diet.14) The tuberous roots of winged bean, in addition to the seeds, also contain proteinase inhibitors, including trypsin and chymotrypsin inhibitors. Winged bean seeds have been reported to contain at least three BowmanBirk-type trypsin inhibitors and nine Kunitz-type serine protease inhibitors.13,15) Four Kunitz chymotrypsin inhibitors16) and two trypsin inhibitors17) have been purified from winged bean seeds. While trypsin inhibitors and chymotrypsin inhibitors have been studied extensively with respect to biochemical, physiological, and structural properties,18–21) information on a dual inhibitor that inhibits both trypsin and chymotrypsin is limited. Winged bean chymotrypsin-trypsin inhibitor (WbCTI), which belongs to the Kunitz family, was purified from winged bean seeds in our lab.18) It inhibits both trypsin and chymotrypsin in a 1:1 molar ratio, but never forms any ternary complex, although it binds both proteases. Amino acid sequencing of WbCTI showed that it was identical to the sequence of WTI-1 reported by Yamamoto et al.17) WbCTI comprises a single polypeptide chain with a molecular mass of 18.6 kDa, and two disulfide bonds. The amino acid sequence of WbCTI has an identical comparative sequence of related Kunitz inhibitors, including trypsin and chymotrypsin inhibitors from Erythrina variegate,22,23) trypsin inhibitor from Erythrina caffra,24) Glycine max,25) and Bauhinia variegate.26) Finally, WbCTI is an effective inhibitor of both chymotrypsin and trypsin, but has no activity towards -amylase, bromelain, or papain. The physiological roles of WbCTI have not yet been fully investigated. There are reports that protease inhibitors present in mature seeds of winged bean are also active inhibitors of midgut proteinases of Helicoverpa armigera, a devastating pest of several economically important crops.15) The present study aimed to clone the WbCTI gene from genomic DNA and cDNA and to express rWbCTI in E. coli, which

To whom correspondence should be addressed. Current address: Department of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA; Tel: +1-216-368-5182; Fax: +1-216-368-0494; E-mail: [email protected] Abbreviations: BSA, bovine serum albumin; bp, base pair; BTEE, N-benzoyl-L-tyrosine ethyl ester; DAB, 3,3-diaminobenzidine tetrahydrochloride; SDS–PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TAME, N--p-tosyl-L-arginine methyl ester; WbCTI, winged bean chymotrypsin-trypsin inhibitor

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exhibits a strong inhibitory activity on both trypsin and chymotrypsin just like WbCTI obtained from the cotyledons.

Materials and Methods Materials. Winged bean (Psophocarpus tetragonolobus (L.) DC) plants were grown in the institute field, Kolkata, India. The flowers of winged bean were tagged as 0 d when they just blossomed, and the cotyledons were collected when the developing pods were at 20 d. Trypsin, chymotrypsin, TAME, and BTEE were from Sigma (St. Louis, MO). cDNA first strand synthesis kit and TRIzol were from Gibco BRL (USA). Restriction enzymes and the other reagents used in molecular biology techniques were from MBI Fermentas (USA). All the other reagents used were of analytical grade. Organisms, plasmids, and media. E. coli strain DH5 was used in cloning experiments and for plasmid propagation. In the expression of active rWbCTI, E. coli strain BL21-DE3 was used as the host. Plasmid pTz57R/T (MBI Fermentas) was used in the cloning and sequencing of PCR fragments, and high expression vector pTrc99A (Invitrogen, USA) in the recombinant expression of rWbCTI. Expression of recombinant WbCTI was done in Luria-Bertani broth. Isopropyl--Dthiogalactopyranoside (IPTG) was added to the media to induce the expression system. Isolation of genomic DNA from winged bean. Dry winged bean seeds were imbibed overnight and germinated on moist filter paper at 25  C under sterile conditions in the presence of antifungal agents.27) The day on which the seed coats cracked was considered the first day. After 12 d, growing shoots were collected and stored in liquid nitrogen for genomic DNA isolation. The genomic DNA was purified from the developing shoots as described by Shure.28) RNA isolation from developing winged bean seeds and cDNA synthesis. Total RNA was extracted from 20 d old winged bean cotyledons using TRIzol according to the manufacturer’s protocol. The total RNA was reverse transcribed using a first-strand cDNA synthesis kit from Gibco BRL, following the manufacturer’s protocol. In brief, 3.0 mg of total RNA was incubated with 0.5 mg of oligo (dT)12{18 primers at 70  C for 10 min and then on ice for 1 min. To this mixture was added 50 mM Tris–Cl, pH 8.3, 75 mM KCl, 2.5 mM MgCl2 , 10 mM DTT, and 1 mM dNTP mix and this was incubated at 42  C for 5 min. Superscript II RT (200 U) was added, and this was incubated at 42  C for 50 min. The reaction was terminated by incubating the mix at 70  C for 15 min, and this was chilled on ice. The reaction mix was incubated with 1 ml of RNase H at 37  C for 20 min to remove the RNA. Isolation of the WbCTI gene. Genomic DNA and cDNA were PCR amplified with degenerate primers having EcoRI and SalI restriction enzyme sites in a thermal cycler (Eppendorf, Germany). The two primers, forward 50 -GGGAATTCGARCCNTTRCTNGAY-30 and reverse 50 -CCTGTCGACTTANGANGAYTCNACYTTY-30 , where R represents A and G; Y represents C and T; and N represents A, C, G and T, were designed on the basis of the amino acid sequence of WTI1.17) The 23 mer forward primer contained an EcoRI site at the N terminal followed by the gene sequence, whereas the reverse primer was of 28 mer and contained a stop codon after the last amino acid, followed by a SalI site. The restriction enzyme sites are underlined and the stop codon is written in bold in the primer sequence. Genomic DNA (300 ng) or cDNA (300 ng) was used for amplification for 35 cycles under the following cycling conditions: 45 s at 94  C, 60 s at 55  C, and 30 s at 72  C. After the completion of 35 cycles, the reaction was continued for another 10 min at 72  C. DNA sequence analysis of WbCTI. The PCR products were detected by 1.5% agarose gel electrophoresis, and the band corresponding to 0.54 kbp was purified, digested with EcoRI and SalI, and ligated to pTz57R/T with a T/A Cloning Kit (MBI Fermentas). Competent E. coli DH5 cells were transformed with this recombinant plasmid, pTz57R/T–WbCTI, by the CaCl2 method,29) and transformants were selected by blue-white selection. To verify the WbCTI gene and its

orientation, the DNA sequence of the insert was determined by sequencing pTz57R/T–WbCTI using an ABI-PRISM automated DNA sequencer. All the inserts were sequenced at least twice on both strands. Expression of WbCTI in E. coli. The expression plasmid was a derivative of pTrc99A (Invitrogen), and was constructed by ligation of the WbCTI gene excised from the sequencing vector using EcoRI and SalI to generate pTrc-WbCTI. The resulting pTrc-WbCTI construct was transformed into E. coli BL21-DE3, grown in LB media, and was selected on X-gal/IPTG containing ampicillin LB plates. The presence of the chimeric plasmids was ascertained by restriction digestion analysis and DNA sequencing. For expression of the recombinant proteins, E. coli containing pTrc-WbCTI was grown overnight 37  C. Overnight grown culture was used to inoculate fresh culture, and was grown at 37  C until O.D600 was about 0.7. IPTG was added to a final concentration of 0.4 mM and grown further at 30  C, and aliquots were taken out hourly until the 5-h. The cells were pelleted down, suspended, and boiled with 1 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) loading buffer (250 mM Tris–HCl, pH 6.8, 10% SDS, and 40% glycerol with 5% bromophenol blue), and 20% -mercaptoethanol, and centrifuged at 12;000  g for 5 min. Both the pellet and the supernatant were used in SDS–PAGE30) and immunoblot analysis.31) The ratio of the O.D. of the cells taken and the gel loading buffer were kept constant in each case. Purification and characterization of rWbCTI. Starting with 3.0 g of optimally induced E. coli cells containing pTrc-WbCTI plasmids, the recombinant protein (rWbCTI) was purified from the soluble portion of the bacterial lysate by single-step immunoaffinity chromatography using a rabbit anti-WbCTI antibody coupled sepharose column. The immunoaffinity column used for purification of the recombinant protein was prepared as described by Roy et al.32) The cells were harvested by centrifugation at 12;000  g for 15 min. The pellet was resuspended in ice-cold lysis buffer (50 mM Tris–Cl, pH 8.0, 1 mM EDTA, 150 mM NaCl, and protease inhibitor cocktail) and sonicated Labsonic 2000 (Germany) at 10 pulses of 30 s each. All subsequent operations were carried at 4  C, unless stated otherwise. The sonicated cell extract was centrifuged for 10 min at 12;000  g. The supernatant representing the soluble fraction of bacterial lysate was loaded onto an anti-WbCTI antibody coupled sepharose column (1  4 cm) equilibrated with PBS. After thorough washing of the column with PBS, rWbCTI was eluted with 0.1 N HCl. The protein-containing fractions were pooled and the pH was adjusted to about 7.5. The purity and the total protein concentration of rWbCTI were evaluated by SDS–PAGE and Bradford assay, using bovine serum albumin (BSA) as the standard,33) respectively. Assay for the chymotrypsin and trypsin inhibitory activity of rWbCTI. Trypsin and chymotrypsin inhibition assays were carried out using N--p-tosyl-L-arginine methyl ester (TAME)34) and N-benzoyl-Ltyrosine ethyl ester (BTEE)35) respectively as substrates. To determine trypsin and chymotrypsin inhibitory activities, varying amounts of the inhibitor were preincubated with fixed amounts of trypsin and of chymotrypsin, and the residual peptidase activity in the mixture was assayed. One inhibitor unit was defined as the amount of protein that inhibits 50% of trypsin or chymotrypsin activity separately under the assay conditions. Binding studies of rWbCTI with chymotrypsin and trypsin. Binding studies of rWbCTI with both chymotrypsin and trypsin were carried out by FPLC gel filtration chromatography. The inhibitor was incubated with trypsin or chymotrypsin at various molar ratios for 5 min in a total incubation volume of 120 ml. After centrifugation at 10;000  g for 15 min, the incubation mixture was loaded onto a Superose 12 HR10/30 column equilibrated with buffer containing 50 mM Tris–Cl, pH 7.5, and 100 mM NaCl. The flow rate of the column was 0.5 ml/min, and the retention times for the different species were noted. The markers used to calibrate the column were bovine serum albumin (67 kDa), ovalbumin (45 kDa), chymotrypsinogen (25 kDa), and cytochrome c (12 kDa). The molecular sizes of the complexes and the free proteins were determined by reference to the calibrated values.

Cloning, Expression, and Characterization of WbCTI

A

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A

B B

Fig. 1. PCR Amplification and Cloning of the WbCTI Gene. A, PCR amplification was done from winged bean cDNA and genomic DNA using the WbCTI degenerate primer pair. The products were analyzed by 1.5% agarose gel electrophoresis and stained with ethidium bromide. Lane 1, 100-bp DNA ladder marker (sizes are given in the left margin); lane 2, PCR product obtained using cDNA as a template; lane 3, PCR product obtained using genomic DNA as template. B, Restriction sites for EcoRI and SalI were used in the cloning of the WbCTI gene. WbCTI was restriction digested from pTz57R/T by EcoRI and SalI and ligated to 4.1 kb pTrc99A vector.

Results and Discussion Amplification and cloning of WbCTI Degenerate primers designed on the basis of the amino acid sequence for WbCTI were used to amplify specific sequence from winged bean genomic DNA and also from cDNA by RT-PCR. Agarose gel electrophoresis (1.5%) of the PCR products showed the presence of a single DNA band of approximately 540 bp (Fig. 1A) in both cases. The size of the amplified DNA as indicated by gel electrophoresis is consistent with the expected size of 534 bp (516 bp of the gene along with 18 nucleotides from the restriction enzyme sites present on the two primers) for the structural gene of WbCTI. The amplified DNA fragment produced by PCR reaction with cDNA and genomic DNA as templates were purified by gel elution, cloned into pTz57R/T, and sequenced. The resulting recombinant plasmid contained the entire nucleotide region encoding native inhibitor WbCTI. The deduced amino acid sequence of WbCTI using the DNA sequence of the cDNA and genomic clones had the same nucleotide sequences in the coding region. This indicates that the genomic DNA is without any intervening sequences and thus that WbCTI is an intron less gene. This is similar to the findings for other Kunitz inhibitors that are devoid of introns. The genomic clone isolated from soybeans encoding Kunitz-type trypsin inhibitors was also reported to be an intronless gene.36) Another Kunitz-type

Fig. 2. Expression of rWbCTI in E. coli Harboring pTrc-WbCTI. A, SDS–PAGE analysis (15%). Lane 1, Pellet of E. coli–pTrc– WbCTI culture induced with 0.4 mM IPTG for 5 h; lane 2, supernatant of E. coli–pTrc99A culture induced as in lane 1; lane 3, supernatant of uninduced E. coli–pTrc-WbCTI culture; lanes 4–8, supernatant of E. coli–pTrc–WbCTI culture, induced with 0.4 mM IPTG for 1–5 h; lane 9, standard molecular weight marker proteins. B, Western blot analysis of rWbCTI expressed in E. coli. Bacterial lysates were resolved by 15% SDS–PAGE, followed by transfer to PVDF membrane. Immunodecoration was carried out using rabbit anti-WbCTI immune sera, followed by incubation with proteinAperoxidase and final detection using DAB (3,3-diaminobenzidine tetrahydrochloride). Lane 1, Pellet of E. coli–pTrc–WbCTI culture induced with 0.4 mM IPTG for 5 h; lane 2, supernatant of E. coli– pTrc99A culture induced as in lane 1; lane 3, supernatant of uninduced E. coli–pTrc-WbCTI culture; lanes 4–8, Supernatant of E. coli–pTrc–WbCTI culture, induced with 0.4 mM IPTG for 1–5 h; lane 9, prestained molecular weight marker proteins.

trypsin inhibitor gene isolated from Delonix regia (DrTI) was also found to be intronless.37) The amino acid sequence deduced from the genomic and cDNA clones agreed with the amino acid sequence of WTI-1B.17) The GenBank accession numbers of the nucleotide sequences of the WbCTI gene from cDNA and the genomic DNA are AY997024 and GQ166187 respectively. Expression and purification of the recombinant WbCTI gene For expression of recombinant WbCTI in E. coli, the WbCTI gene was subcloned into high expression vector pTrc99A at the EcoRI and SalI sites to generate the recombinant plasmid (Fig. 1B). The expression vector with the insert was confirmed by nucleotide sequencing. To express the recombinant protein, rWbCTI, E. coli containing pTrc-WbCTI was grown under IPTG induction as mentioned above. After induction, the bacterial culture was centrifuged at 12;000  g for 15 min. The supernatant was discarded and the pellet was lysed in lysis buffer by sonication. The bacterial lysate was centrifuged at 12;000  g for 20 min, and the supernatant represented the soluble fraction of bacterial lysate. The bacterial pellet and the soluble portion were

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analyzed by 15% SDS–PAGE and western blot analysis to check the expression of rWbCTI. There was no detectable expression of rWbCTI found in the bacterial pellet induced for 5 h with 0.4 mM IPTG (Fig. 2A, lane 1). The soluble part of the bacterial lysate contained the recombinant protein, where expression of rWbCTI increased with time after IPTG induction, and maximum expression was obtained at 4 h (lanes 4–8). There was no detectable expression in E. coli containing only pTrc99A induced by IPTG for 4 h (lane 2) or in uninduced E. coli containing pTrc-WbCTI (lane 3). The results of Western blot analysis detected with rabbit anti-WbCTI immune sera also indicate that rWbCTI was not expressed in the pellets (Fig. 2B, lane 1). As with SDS–PAGE, there was no detectable expression of rWbCTI in E. coli harboring pTrc99A induced by IPTG (lane 2), but a gradual increase in the expression of rWbCTI with time in the E. coli containing pTrcWbCTI was clearly detected (lanes 4–8). The recombinant protein was purified from the soluble fraction of the bacterial cell lysate by singlestep immunoaffinity chromatography using a rabbit antiWbCTI antibody-sepharose column. This single-step purification of the expressed protein resulted in an overall yield of 6.40 mg of purified rWbCTI per l induced E. coli at 30  C (Table 1). The homogeneity of the purified protein was analyzed by SDS–PAGE (Fig. 3). SDS–PAGE analysis of the purified rWbCTI (lane 2) indicated the presence of a single protein band of about 20 kDa, similar to the expected molecular mass of WbCTI (lane 3).

Inhibitory properties of rWbCTI Bovine chymotrypsin and trypsin were tested for the inhibitory activity of rWbCTI by enzymatic assay. The purified rWbCTI and native WbCTI exhibited identical inhibitory properties against both chymotrypsin and trypsin. Both of the proteases (2 mg) were first titrated against various concentrations of rWbCTI to obtain maximum inhibition. Complete inhibition of chymotrypsin was obtained with the purified rWbCTI at a 1:1 molar ratio, similarly to the WbCTI obtained from winged bean seeds (Fig. 4A). Similarly, rWbCTI also completely inhibited trypsin at a 1:1 molar ratio, as with native WbCTI (Fig. 4B). Dixon plot analysis was employed to determine the Ki values of WbCTI and rWbCTI for trypsin and chymotrypsin.38) The Ki values of rWbCTI and WbCTI for trypsin were 2:50  109 M and 2:56  109 M respectively, and that for chymotrypsin was 1:88  109 M, same for rWbCTI and WbCTI. In this respect, the two proteins were indistinguishable. Although there are reports of inhibitory properties against cysteine proteases in trypsin inhibitors,39) WbCTI and rWbCTI had no inhibitory activities against cysteine proteases or amylases (data not shown). Inhibition of the two proteinases by WbCTI might occur due to the overlapping binding sites for the two enzymes. A

1

2

3

Table 1. Purification of rWbCTI from induced E. coli Cells

Steps Bacterial lysate Anti-WbCTI Column eluate

Total protein (mg)a

rWbCTI (mg)b

Specific protein content (mg/mg)c

Yield (%)

80 3.50

3.50 3.20

0.04375 0.914

100 91.40

a



Fig. 3. Homogeneity of rWbCTI Purified from Immunoaffinity Column. SDS–PAGE (12%) analysis of the purified proteins (5 mg). Lane 1, Molecular weight marker proteins; lane 2, purified rWbCTI; lane 3, purified WbCTI from winged bean seeds.

b

Starting with 3.0 g of E. coli; Protein assayed by Bradford; Protein determined by A280 ; c Specific protein content was defined as rWbCTI/total protein.

A

B

Fig. 4. Inhibition of Chymotrypsin and Trypsin by Purified Inhibitors. A chymotrypsin inhibition study was carried out using WbCTI and rWbCTI (panel A). Similarly, trypsin inhibition was studied with WbCTI and rWbCTI (panel B). Increasing amounts of the inhibitors were mixed with a fixed amount of chymotrypsin (2 mg) or trypsin (2 mg), and the residual protease activities were measured using BTEE and TAME as substrates for chymotrypsin and trypsin respectively. The experiments were been done in triplicate, and error bars represent standard deviation.

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Fig. 5. FPLC Binding Study of rWbCTI with Chymotrypsin and Trypsin. rWbCTI was mixed with chymotrypsin and trypsin in various molar ratios and this was incubated for 15 min. The mixture (100 ml) was injected into a Superose 12 HR10/30 column equilibrated with 50 mM Tris–HCl, pH 8.0, containing 100 mM NaCl, and the protein was monitored at 280 nm. The retention times of the eluted protein peaks were recorded. A, rWbCTI 26.14 min; B, rWbCTI:Chymotrypsin (1:1) 22.60 min; C, rWbCTI:Chymotrypsin (1:3) 22.40 min, 26.30 min; D, rWbCTI:Chymotrypsin (3:1) 22.40 min, 26.18 min; E, rWbCTI:Trypsin (1:1) 24.78 min; F, rWbCTI:Trypsin (1:3) 24.82 min; 28.47 min; G, rWbCTI:Trypsin (3:1) 24.76 min; 26.08 min. Each small divison of the graph along the x-axis corresponds to 5 min. Table 2. Retention Times of Complexes Formed by rWbCTI and Chymotrypsin and/or Trypsin rWbCTI was mixed with various amounts of chymotrypsin and/or trypsin in various molar ratios as described in the table, and injected into an FPLC gel filtration column (Superose 12 HR 10/30), and the retention time of each peak was measured as described in ‘‘Materials and Methods.’’ Protein samples Trypsin Chymotrypsin rWbCTI rWbCTI:Trypsin

rWbCTI:Chymotrypsin

Molar Retention time Retention time ratio of 1st peak (min) of 2nd peak (min) — — — 1:1 1:3 3:1 1:1 1:3 3:1

28.78 26.80 26.14 24.78 24.82 24.76 22.60 22.40 22.40

— — — — 28.47 26.08 — 26.30 26.18

comparison of the reactive site loop (RSL) of various trypsin inhibitors confirmed the presence of arginine at the P1 site and the presence of an apolar, aromatic amino acid residue at the same site for chymotrypsin inhibitors. WbCTI contains Arg (64) at the P1 site, similarly to other trypsin inhibitors such as WbTI40) and the Erythrina trypsin inhibitor, ETI.22) The P2 site of WbCTI contains Leu (63), similarly to WbCI and WCI2, and also to overlapping reactive-site amino acids at P1 and P2 for BSZx, a major barley serpin.41) Although the P1 residue of the inhibitor is the most important site of proteinase-PI contact,42) extended binding interactions between the inhibitor and protease might also contribute to the stability of the association.43) The mutant proteins can be utilized by site-directed mutagenesis to investigate the contribution of Leu (63) and Arg (64) in chymotrypsin and trypsin inhibition of WbCTI respectively.

Binding study of rWbCTI with chymotrypsin and trypsin by FPLC The binding of rWbCTI with chymotrypsin and trypsin was studied by FPLC gel filtration using a Superose 12 HR 10/30 column. The retention times of the complexes formed by rWbCTI with chymotrypsin and or trypsin in presence of excess amounts of the inhibitor and of the protease, indicated the formation of only one type of complex, having a 1:1 molar ratio (Fig. 5). The retention times of the complexes and free proteins are shown in Table 2. Based on the retention times and elution profiles, the molecular masses of the complexes rWbCTI-chymotrypsin and rWbCTI-trypsin were calculated to be 43 kDa and 40 kDa respectively. This is suggestive of a 1:1 molar ratio of the complexes formed. The formation of a ternary complex was not detected even when all the three proteins were mixed together. General discussion There are several reports on cloning of the chymotrypsin inhibitor gene from winged bean,13,16,21,44) and also of a synthetic gene coding for a winged bean trypsin inhibitor,45) but to date there are no reports on the cloning of the chymotrypsin-trypsin dual inhibitor gene from winged bean. The WbCTI gene was cloned by PCR amplification of genomic DNA from developing shoots and from cDNA isolated from cotyledons of winged bean, and was expressed in E. coli. The expressed recombinant protein inhibited chymotrypsin and trypsin in a fashion similar to that of WbCTI obtained from seeds. One of the main goals of this study of proteinase inhibitors was the identification of the corresponding genes, which would allow their use in obtaining transgenic plants with higher resistance to

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insect attack. Transgenic rice lines expressing a synthetic trypsin inhibitor from winged bean has been found to reduce the larval growth of rice stem borers.45) Chymotrypsin inhibitors from winged bean such as WCI2 and WCI5, have been found to have an inhibitory effect on the larval growth of H. armigera.44) Thus WbCTI, being a dual inhibitor of both chymotrypsin and trypsin is a promising candidate to study for its insecticidal properties against H. armigera and other insect pests.

Acknowledgments

17) 18) 19) 20) 21) 22) 23) 24)

This work was supported by the Department of Science and Technology of India. S.R. gratefully acknowledges the financial support of the Council of Scientific and Industrial Research (CSIR) of India. The authors are thankful to the Director of IICB, India. The help of N. Bhattacharjee is also acknowledged.

25) 26) 27) 28) 29)

References 1) 2) 3) 4) 5) 6) 7) 8) 9) 10)

11) 12) 13) 14) 15)

16)

Ryan CA, Annu. Rev. Phytopathol., 28, 425–449 (1990). Ussuf KK, Laxmi NH, and Mitra R, Curr. Sci., 80, 847–853 (2001). Huang Y, Xiao B, and Xiong L, Planta, 226, 73–85 (2007). Baldwin I and Schultz J, Science, 22, 277–279 (1983). Brattsten LB, Arch. Insect Biochem. Physiol., 17, 253–267 (1991). Green TR and Ryan CA, Science, 175, 776–777 (1972). Hilder VA, Gatehouse MR, and Boulter D, Transgenic Plants, 1, 317–338 (1993). Boulter D, Phytochemistry, 34, 1453–1466 (1993). Richardson M, ‘‘Methods in Plant Biochemistry,’’ ed. Rogers L, Academic Press, New York, pp. 259–305 (1991). Oliva MLV, Souza-Pinto JC, Batista IFC, Araujo MS, Silvera VF, Auerswald EA, Mentele R, Eckerskorn C, Sampaio MU, and Sampio CAM, Biochim. Biophys. Acta, 1477, 54–74 (2000). Banerji A, Fernandes A, Bane S, and Ahire S, Cancer Lett., 129, 15–20 (1998). Kennedy AR, Am. J. Clin. Nutr., 68, 1406S–1412S (1998). Peyachoknagul S, Matsui T, Shibata H, Hara S, Ikenaka T, Okada Y, and Ohno T, Plant Mol. Biol., 12, 51–58 (1989). Gillespie JM and Blagrove RJ, Aust. J. Plant Physiol., 5, 357– 369 (1978). Giri AP, Harsulkar AM, Ku MSB, Gupta VS, Deshpande VV, Rajnekar PK, and Franceschi VR, Phytochemistry, 63, 523–532 (2003). Habu Y, Peyachoknagul S, Umemoto K, Sakata Y, and Ohno T, J. Biochem., 111, 249–258 (1992).

30) 31) 32) 33) 34) 35) 36) 37)

38) 39) 40) 41) 42) 43) 44) 45)

Yamamoto M, Hara S, and Ikenaka T, J. Biochem., 94, 849–863 (1983). Datta K, Usha R, Dutta SK, and Singh M, Plant Physiol. Biochem., 39, 949–959 (2001). Dattagupta JK, Chakrabarti C, Podder A, Dutta SK, and Singh M, J. Mol. Biol., 216, 229–231 (1990). Dattagupta JK, Podder A, Chakrabarti C, Sen U, Dutta SK, and Singh M, Acta Cryst., D52, 521–528 (1996). Ghosh S and Singh M, Protein Expr. Purif., 10, 100–106 (1997). Kouzuma Y, Suetake M, Kimura M, and Yamasaki N, Biosci. Biotechnol. Biochem., 56, 1819–1824 (1992). Kimura M, Kouzuma Y, and Yamasaki N, Biosci. Biotechnol. Biochem., 57, 102–106 (1993). Onesti S, Brick P, and Blow DM, J. Mol. Biol., 217, 153–176 (1991). Koide T and Ikenaka T, Eur. J. Biochem., 32, 417–431 (1973). De Souza AF, Torquato RJS, Tanaka AS, and Sampaio CAM, Biol. Chem., 366, 1185–1189 (2005). Punjabi B and Basu RN, Indian J. Plant Physiol., 25, 289–295 (1982). Shure M, Wessler S, and Fedoroff N, Cell, 35, 225–233 (1983). Sambrook J and Russel DW, ‘‘Molecular Cloning, a Laboratory Manual’’ 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 7.4–7.8 (2001). Laemmli UK, Nature, 227, 680–685 (1970). Towbin H, Staehlin T, and Gordon J, PNAS, 76, 4350–4354 (1979). Roy S and Dutta SK, Am. J. Biochem. Biotech., 5, 142–146 (2009). Bradford MM, Anal. Biochem., 72, 248–252 (1976). Fenteany G and Schreiber SL, Chem. Biol., 3, 905–912 (1996). Bode W and Huber R, Eur. J. Biochem., 204, 433–451 (1992). Song SI, Kim CH, Baek SJ, and Choi YD, Plant Physiol., 101, 1401–1402 (1993). Hung CH, Peng PH, Huang CC, Wang HL, Chen YJ, Cheng YL, and Chi LM, Biosci. Biotechnol. Biochem., 71, 98–103 (2007). Dixon M, Biochem. J., 55, 170–171 (1953). Franco OL, de Sa MFG, Sales MP, Melo LV, Oliveira AS, and Rigden DJ, Proteins: Struct. Funct. Genet., 49, 335–341 (2002). Kortt AA, Biochem. Biophys. Acta, 577, 371–382 (1979). Dahl SW, Rasmussen SK, and Hejgaard J, J. Biol. Chem., 271, 25083–25088 (1996). Khamrui S, Dasgupta J, Dattagupta JK, and Sen U, Biochim. Biophys. Acta, 1752, 65–72 (2005). Srinivasan A, Giri AP, and Gupta VS, Cell. Mol. Biol. Lett., 11, 139–161 (2006). Telang MA, Giri AP, Pyati PS, Gupta VS, Tegeder M, and Franceschi VR, Gene, 431, 80–85 (2009). Mochizuki A, Nishizawa Y, Onodera H, Tabei Y, Toki S, Habu Y, Ugaki M, and Ohashi Y, Entomol. Exp. Appl., 93, 173–178 (1999).