Botanical Studies (2011) 52: 407-416.
MOLECULAR BIOLOGY
Cloning, functional expression and characterization of a phytocystatin gene from jelly fig (Ficus awkeotsang Makino) achenes
Douglas J.H. SHYU1, Yi-Mei YOUNG2, Hsi-Chi LU3, Yueh-Mei CHENG2, Jason T.C. TZEN4, and Wing-Ming CHOU2 *
1Department of Life Sciences, National Pingtung University of Science and Technology, Pingtung 807, Taiwan
2Department of Biotechnology, National Formosa University, Yunlin 632, Taiwan
3Department of Food Science, Tunghai University, Taichung 407, Taiwan
(Received August 25, 2010; Accepted March 30, 2011)
ABSTRACT. A cDNA clone encoding a phytocystatin was isolated and identified from about 300 expressed sequence tag (EST) clones in maturing jelly fig (Ficus awkeotsang Makino) achenes. This clone, named FaCYS, consists of 582 bp encoding 114 amino acids with a putative signal peptide. The predicted mature protein contains no cysteine and has a molecular mass of 10.8 kDa with an isoelectric point (pI) of 9.7. Fa-CYS constructed in nonfusion and fusion vectors were overexpressed in Escherichia coli as nonfusion and his-tagged recombinants, respectively. Both recombinants were found in the soluble fractions of the cell extracts. The purified nonfusion and his-tagged FaCYS exhibited papain inhibitory activity with similar Ki values of 2.7 x 10-7 M and 2.4 x 10-7 M, respectively. In addition, his-tagged recombinant proteins showed inhibitory activity toward human cathepsin B, cathepsin L and ficin with Ki values of 5.6 x 10-7 M, 3.0 x 10-8 M and 2.0 x 10-7 M, but no inhibitory activity against stem bromelain. It was tolerant at a wide range of pH values and thermally stable up to 50°C for 30 min. Furthermore, his-tagged FaCYS could arrest the fungal growth of Glomerella cingulata and Sclerotium rofsii.
Keywords: Cystatin; Cysteine protease inhibitor; Jelly fig achene; Phytocystatin.
INTRODUCTION
Phylogenetic analyses of known phytocystatins show that two groups are mainly present in plant kingdoms, which have the molecular weight of∼11 kDa for group I, and∼23 kDa for group II. Basically, group I shares high homology with the N-terminal region of group II. Group II contains an additional C-terminal extension of∼10 kDa, which may have physiological importance (Martinez et al., 2007). Plant multicystatins, comprised eight tandem cystatin domains and a high molecular weight of∼85 kDa, were also found in several plant species, including potato and tomato (Waldron et al., 1993; Girard et al., 2007). They showed inhibitory activity against papain. Follow­ing trypsin digestion, the obtained intact potato cystatin domains also showed inhibitory activity (Nissen et al.,2009).
Cystatins play important roles in a variety of biologi­cal and pathological processes by regulating endogenous cysteine protease activities. The physiological roles of phytocystatins are diverse and complicated. They have been proposed to intervene in germination, seed matura­tion, storage protein turnover and programmed cell death (Solomon et al., 1999; Corr-Menguy et al., 2002; Martinez et al., 2009). They may protect plants from environmental
Cystatins, the natural inhibitors of papain-like cysteine proteases, have been found extensively throughout the ani­mal and plant kingdoms. They reversibly act as pseudo-substrates to enter the active-site cleft of proteases and inhibit proteolytic activity. Three conservative regions were found in the cystatins of animal and plant origin, a G residue near the N-terminus, a central QXVXG motif, and a W residue near the C-terminus. These three con­served regions could form a tripartite wedge interacting with the cysteine protease to cause the inhibition (Turk et al., 1997; Nagata et al., 2000). Plant cystatins, namely phytocystatins particularly possess a unique conserved se­quence, [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-X-[EDQV]-[HYFQ]-N at the N-terminus of a 1-helix. In addition, the typical structure element of phytocystatins include four (3 sheets linked to an a-helix in the N-terminal region and two hairpin loops (Arai et al., 2002; Fabienne et al., 2002).
*Corresponding author: E-mail: wingming@sunws.nfu.edu. tw; Phone: +886-5-6315499; Fax: +886-5-6315502.
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stimuli in response to biotic and abiotic stresses (Gaddour et al., 2001; Zhang et al., 2008). Some of them act as de­fense proteins when invaded by pathogens whose cysteine proteases play a role in cell replication, or when attacked by pests whose cysteine proteases serve as digestive en­zymes in the gut (Gutierrez-Campos et al., 1999; Arai et al., 2002). Moreover, due to their broad specificity of pro­teases, phytocystatins are targets of interest for biotechno-logical applications, as stabilizers in food processing, for anti-cancer, anti-viral and chemotherapy of leishmaniasis (Aoki et al., 1995; Shyu et al., 2004; Gmnotti et al., 2008; Ordonez-Gutierrez et al., 2009).
Jelly fig (Ficus awkeotsang Makino), a member of the family Moraceae, is a unique woody vine growing in the mountain areas of Taiwan. A water extract of the pericarpi-al portion of jelly fig achenes has been utilized to produce jelly curd, a beverage popular in local markets for around two hundred years. To extend the application of jelly fig achenes, several achene proteins have been identified and characterized (Wang and Tzen, 2005; Chua et al., 2008). In this study, a cDNA clone encoding a putative phytocys-tatin, FaCYS was isolated and identified from in-house generated expressed sequence tag (EST) clones in matur­ing jelly fig (Ficus awkeotsang Makino) achenes. Based on sequence alignment, FaCYS was classified as group I phytocystatin. The recombinant FaCYS was functionally over-expressed in Escherichia coli, purified and charac­terized as a thermal and pH-stable protein. Its antifungal activity was further examined.
using a Miniprep Purification kit (Protech, Taipei, Taiwan) prior to EST sequencing by the dideoxy chain-termination method with a BigDye terminator cycle sequencing kit and an ABI 377 DNA sequencer (Perkin-Elmer, CT, USA). Approximately 200 and 100 EST clones derived from the two cDNA libraries were sequenced and analyzed with the Blast program (http://www.ncbi.nlm.nih.gov/blast/) at the National Center for Biotechnology Information in the USA (Altschul et al., 1997; Zhang and Madden, 1997). A cDNA clone named FaCYS encoding a putative phy-tocystatin was subsequently isolated and identified. The N-terminal signal peptide was predicted using the SignalP program(http://www.cbs.dtu.dk/
services/SignalP/) in the World Wide Web Prediction Server Center for Biological Sequence Analysis (Emanuelsson et al., 2007). Sequence alignment was executed by the CLUSTAL W program at Pole Bioinformatique Lyonnais (PBIL) (Lyons, France) (Thompson et al., 1994).
Bacterial over-expression of recombinant phy-tocystatin
According to the FaCYS open reading frame, two primers (JF-CYS5, 5'-TCCATATGCGTCCTCACTGC-CTCCTCG-3'; and JF-CYS3, 5'-CCGCTCGAGCTAAT-TGACAAGCGTAAA-3') were designed and synthesized. Primer JF-CYS5 had an NdeI recognition sequence; and primer JF-CYS3 had an XhoI recognition sequence and the stop codon. Using two primers, JF-CYS5 and JF-CYS3, the 0.35 kb FaCYS DNA fragment was amplified by PCR reaction (30 cycles at 95oC for 30 s, 50°C for 30 s, and 72°C for 30 s). The amplified DNA fragment was ligated into the pGEM-T easy vector (Promega, Madison, WI, USA) then transformed into E. coli DH5a. The nucleotide sequence of the insert was confirmed by sequencing. The insert DNA fragment was digested with NdeI and XhoI and then constructed into the 6xhis-tag fusion expression vec-tor, pET28a, or the nonfusion expression vector, pET29a (Novagen, Madison, WI, USA). Recombinant protein over-expression in E. coli BL21(DE3) was induced after adding 1 mM isopropyl thio-( -D-galactoside (IPTG) under the T7 RNA polymerase/promoter system. After induction for 4 h at 37°C, the E. coli cells were harvested and lysed by sonication in a 10 mM phosphate buffer (pH 8.0). They were fractionated into soluble and precipitated fractions by centrifugation at 10,000 xg, then subjected to SDS/PAGE and western blot analyses.
MATERIALS AND METHODS
Plant materials
Mature and fresh maturing jelly fig (Ficus awkeotsang Makino) achenes were purchased from local growers. Ma­turing achenes, that had been pollinated approximately 30 days before, were used for mRNA extraction and cDNA library construction.
RNA isolation, cDNA library construction, in-house generated EST sequencing and se­quence analyses
Total RNA was prepared by grinding 2 g maturing achenes in liquid nitrogen using the phenol/SDS method (Wilkins and Smart, 1996). The mRNA was purified from total RNA using oligo-dT magnetic beads (Dylan Bio­tech, Oslo, Norway). cDNA was synthesized with 5 jag of mRNA using a Stratagene ZAP-cDNA Synthesis Kit (Stratagene, La Jolla, CA, USA). It was size fractioned into two parts of 0.5-1.0 kb and 1.0-2.0 kb (Chua et al.,2008). Two ZAP cDNA libraries were subsequently con-structed with the two recovered DNA fractions. Mass excision was performed to convert the phage libraries into bacterial pBluescript libraries.
Bacterial libraries were sprayed and grown on LB plates with antibiotic ampicillin. Colonies were randomly selected and recombinant cDNA plasmids were prepared
Recombinant phytocystatin affinity purification
To enable his-tagged recombinant protein purification, the extract was incubated overnight with TALON res­ins (Clontech, Palo Alto, CA, USA) at 4°C under gentle agitation. The resin was equilibrated and washed with appropriate buffers according to the manufacturer's in-structions. His-tagged FaCYS were eluted with a buffer containing 150 mM imidazole, and underwent dialysis with a 10 mM phosphate buffer (pH 8.0). For nonfusion recombinant phytocystatin purification, papain was used
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as a ligand enzyme to prepare an affinity column as in our previous work (Shyu et al., 2004). Papain was coupled to CNBr-activated Sepharose 4B (GE Healthcare, Uppsala, Sweden) at a concentration of 5 mg/ml of gel according to the manufacturer's instructions. The nonfusion recom-binant FaCYS from the soluble fraction of the E. coli cell lysate was incubated overnight with papain coupled resin at 4°C under gentle agitation. The affiliated proteins were eluted with 50 mM K3PO4 at pH 11.5 containing 0.5 M NaCl and 10% (v/v) glycerol. The eluate was adjusted to pH 7.4 with a 5 M sodium formate buffer at pH 3.0, then underwent dialysis with a 10 mM phosphate buffer (pH 8.0). The eluate was further applied to a cation-exchange column (HiTrap SP FF column, GE Healthcare, Uppsala, Sweden) previously equilibrated with a 50 mM phosphate buffer at pH 7.5. The flowthrough fraction of the HiTrap SP column was collected for SDS/PAGE analyses and the purified nonfusion protein purified to apparent homogene-ity was obtained. The protein concentration was deter-mined using a protein assay kit (Bio-Rad, Richmond, CA, USA) with bovine serum albumin as standard.
for 15 min, and the absorbance of the mixture was mea­sured at 540 nm. The reaction without any protease and FaCYS was used as the zero control. The protease inhibi­tory activity (%) was calculated by the equation, [(A-A*)/A] x 100%, and protease residual activity (%) was calculated by the equation, (A*/A) x 100%, where A and A* represent­ed the OD540 in the absence and presence of FaCYS. The Ki value was determined by plots (1/v versus [I]) (Dixon, 1953), where v represented protease residual activity and [I] was the concentration of FaCYS. Moreover, molar ratio of [FaCYS]/[papain] was determined by [23 x the amount of FaCYS (μg)] /[10 μg x (molecular mass of recombinant FaCYS)] according to 10 μg of papain used in the assay and the molecular mass, ~23 kDa (for papain),∼10.9 kDa (for nonfusion FaCYS) and ~11.7 kDa (for fusion FaCYS).
When using another protease, cathepsin B, cathepsin L or bromelain as target enzyme, Z-Phe-Arg-MCA (Peptide Institute, Osaka, Japan) was used as substrate (Rassam and Laing, 2004). Various amounts of recombinant FaCYS were incubated with 2.6 μg of cathepsin B (200 U/mg) (Calbiochem, Darmstadt, Germany), 20 ng of cathepsin L (6000 U/mg) (BioVision, CA, USa) or 20 μg of bromelain (2.4-3 FIP U/mg) (Acros Organics, NJ, USA) in a 250-μl assay solution containing 0.1 M sodium phosphate at pH 6.1, and 2 mM EDTA, at 37°C for 10 mm. The pro-teolytic reaction was carried out by adding 250 μl of 20 μM Z-Phe-Arg-MCA at 37°C for 10 min, and terminated
after addition of 500 μl of stop buffer, pH 4.3 containing 30 mM sodium acetate and 70 mM acetic acid. The liber­ated product, 7-amino-4-methyl-coumarin, was estimated by a spectrofluorometer (Beckman coulter Inc., Fuller-ton, USA) at an excitation of 360 nm and an emission of 465 nm. Protease residual activity (%) and the Ki value was then determined according to the equation described above.
Antibody preparation and western blot analysis
The recombinant his-tagged phytocystatin over-expressed in E. coli was first purified, resolved by SDS/ PAGE, then eluted from the gel with Electro-Eluter, ac­cording to its instruction manual (Bio-Rad, Hercules, CA, USA). Antibodies against the recombinant his-tag fusion FaCYS were raised in chickens, and chick egg im-munoglobulins were collected for immunoassays (Polson, 1990). Proteins resolved by SDS/PAGE in these immuno-assays were transferred onto a nitrocellulose membrane (Pall, Ann Arbor, MI, USA) in a Trans-Blot system (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. The membrane was subjected to immuno-detection by using secondary antibodies conjugated with horseradish peroxidase (Jackson ImmunoResearch Labo­ratories, West Grove, PA, USA), then incubated for color development with 4-chloro-1-naphthol (Sigma) containing H2O2.
Thermal and pH stability test
To examine its thermal stability, the purified recombi-
nant FaCYS was incubated at 30-80oC for 30 min in a 10 mM phosphate buffer, pH 7.5; while one protein sample was placed on ice as the control. After incubation at room temperature for another 30 min, the recombinant protein was subjected to papain inhibitory activity measurement as described above. Furthermore, the protein was incubated at 50°C and 60°C for 0-30 min prior to determine papain inhibitory activity (%). To examine its pH stability, the recombinant FaCYS was in a 50 mM sodium citrate buffer at pH 2.0-4.0, 50 mM sodium acetate buffer at pH 4.0-6.0, 50 mM sodium phosphate at pH 6.0-8.0 and 50 mM Tris-HCl at pH 8.0-10.0. Papain inhibitory activity was deter-mined after incubation at 37°C for 30 min.
Protease activity assay and protease inhibitory measurement
A protease inhibitory assay was performed using papain or ficin as the target enzyme and N-benzoyl-L-arginine-2-naphthylamide (BANA) (BANA, papain and ficin were purchased from Sigma-Aldrich, MO, USA) as the substrate (Abe et al., 1994). Various amounts of FaCYS were incubated with 10 j g of papain (2.9 U/mg) or 30 j g of ficin (0.21 U/mg) in a 250-jal assay solution containing 0.1 M sodium phosphate at pH 6, 1 mM EDTA, and 2 mM 2-mercaptoethanol at 37°C for 5 min. Subsequently, the proteolytic reaction was initiated by adding 0.1 ml of 1 mM BANA at 37°C for 10 min, and terminated by adding 0.5 ml of 2% HCl/ethanol. The color was developed fol­lowed by the addition of 0.5 ml of 0.06% p-dimethylamin-ocinnamaldehyde (Sigma-Aldrich, MO, USA) in ethanol
Antifungal activity assay
The purified recombinant FaCYS was applied to ex­amine the growth inhibition of phytopathogenic fungi including Alternaria brassicae, Fusarium oxysporum, Glomerella cingulata, Phellinus noxius, Rhizoctonia solani
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and Sclerotium rofsii. These fungi were cultured in potato dextrose agar (PDA) for 1-2 weeks. The sclerotinia of S. rofsii was inoculated in 1/2 PDA plate with or without Fa-CYS at 24°C for 24 h. The hyphal growth inhibition was observed and recorded under a light microscope.
For A. brassicae, F. oxysporum and G. cingulata, the spores of pathogens were suspended in 0.1% (w/v) glu­cose to a concentration of 104 spores/mL. The fungal spore stock (approximately 500 spores) was cultured in the glucose solution with or without FaCYS at 24°C for 24 h. Inhibition of spore germination was examined under a light microscope. For P. noxius and R. solani, mycelium discs of 1 cm in diameter were placed at the center of PDA plates and incubated at 24°C. As the colonies reached 2.5 cm in diameter, 20 j l of PBS buffer, with or without FaCYS, were poured onto the discs (1 cm in diameter and four discs per plate). The plates were incubated at 24°C for 2-3 days. The experiment was carried out three sepa­rate times under the same conditions.
nus, a central QXVXG and a W residue near the C-termi-nus (Figure 1). These three conserved regions could form a tripartite wedge and interact with the cysteine protease to cause inhibition. The conserved glycine residue was for the optimal orientation of its N-terminal domain toward cysteine protease. The signature motif QXVXG and the conserved W residue were localized on the first and sec­ond hairpin loops, which may interact directly with the ac­tive site of target cysteine protease. FaCYS also contains a unique consensus sequence of phytocystatins at its N-terminal proximity, [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-X-[EDQV]-[HYFQ]-N, only at the third position where D replaces [RKE]. Phytocystatins from kiwifruit (KCPI1, AAR9224, and AAR9225) and from grapefruit (CystL-1) showed that Q replaces [RKE] (Rassam and Laing, 2004; Shatters et al., 2004).
Heterologously over-expression and western blot analysis of FaCYS
RESULTS AND DISCUSSION
The cDNA fragments of FaCYS were constructed in nonfusion and his-tagged fusion expression vectors, pE-T28a and pET29a. In comparison with no IPTG induction, his-tagged FaCYS was induced after 4 hours of adding IPTG and was mostly found in E. coli cell lysate soluble fractions (Figure 2A). Nonfusion FaCYS induction was not as clear as for his-tagged FaCYS (Figure 2B). Purified his-tagged FaCYS was obtained by Ni2+-containing affin­ity chromatography in a single step of purification; while nonfusion FaCYS was purified via a papain-coupled af­finity column and a cation-exchange column. Both recom­binant FaCYSs were purified to apparent homogeneity as revealed by SDS/PAGE (Figure 3A and 3B). Western blot analyses indicated that these purified recombinants could be recognized by antibodies raised against his-tagged Fa-CYS (Figure 3A and 3B); therefore, both recombinants were heterologously expressed in E. coli BL21(DE3).
The expression of recombinant his-tagged FaCYS was better than that of recombinant nonfusion FaCYS. The expression of his-tagged FaCYS with 1.2 mg/L yield was not as efficient as the previous work; in contrast, bacterial over-expression of pineapple phytocystatins reached 63.7 mg/L and 3.7 mg/L of yields by using pET28a and pET29a as expression vectors, respectively (Shyu et al., 2004). Moreover, no FaCYS was immunodetected either inside the seeds or in the pericarp of jelly fig achenes (data not shown). It is thus suggested that FaCYS is a minor protein in jelly fig achenes.
Isolation and characterization of a cDNA clone coding for a putative jelly fig phytocystatin
A full-length cDNA fragment (FaCYS, Genbank acces­sion numbers: GU084175) encoding a putative phytocys-tatin was obtained during sequencing and analyses of 300 EST clones in maturing jelly fig achenes. The nucleotide sequence of FaCYS consists of 582 bp including a 69-nucleotide 5'-untranslated region, an open reading frame of 345 nucleotides and a 168-nucleotide 3'-untranslated region. Its deduced protein sequence comprised 114 amino acids with a putative signal peptide (1-19 a.a.). The predicted mature protein contains no cysteine and had a molecular mass of 10.8 kDa with an isoelectric point (pI) of 9.7. According to the NCBI Blast search, FaCYS is closely related to the reported or putative group I phytocystatins from grapefruit (CystL-1, Genbank ac­cession numbers: AAG38521) (Shatters et al., 2004), wine grape (XP_002274494), kiwifrmt (KCPI1, Q6TPK4; AAR92224; AAR92225) (Rassam and Laing, 2004), Chi-nese cabbage (ADK13077), castor bean (XP_002531171), and black cottonwood (XP_002307893). FaCYS showed about 50-60% similarity to these phytocystatins. For ex-ample, it shares 53% identity with grapefruit CystL-1 in 108 a.a., 59% identity with putative phytocystatin from wine grapes (XP_002274494) in 108 a.a, and 56-58% identity with phytocystatin from kiwifruit (KCPI1, Q6TPK4; AAR92224; AAR92225) in 87 a.a.. In addition, FaCYS shares about 30% identity to OC-I and OC-II, also group I rice phytocystatins (AAA33903, CAA40860) (Abe et al., 1987; Kondo et al., 1991). OC-I was the first cloned and well studied phytocystatin to show homology with animal cystatins.
After sequence alignment with the phytocystatin ho-mologs and a chicken egg-white cystatin, FaCYS showed the typical inhibitory motifs, a G residue near the N-termi-
Inhibitory activity of recombinant FaCYS toward papain and other cysteine proteases
The nonfusion and his-tagged FaCYSs were bacterial-expressed, purified and then characterized. The purified recombinants showed inhibitory activity against papain in a dose-dependent manner with Ki value of 2.7 x 10-7 M for nonfusion FaCYS and 2.4 x 10-7 M for his-tagged FaCYS, respectively (Figure 4). FaCYS possessed papain inhibi-tory activity with similar Ki values to those of reported
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Figure 1. Protein sequence alignment of FaCYS (accession number, GU084175) with cystatins from kiwifruit (KCPI1, Q6TPK4; AAR9224; AAR9225), grapefruit (CystL-1, AAG38521), wine grape (XP_002274494), castor bean (XP_002531171), black cot-tonwood (XP—002307893), maize (CCA60610), Chinese cabbage (ADK13077), Job's tear (CysM, AB037156), chestnut (CsC, CAA11899), rice (OC-I, AAA33903; OC-II, CAA40860), and chicken (GgCYS, P01038). Short names of those reported cystatins were indicated before accession number. Signal peptides for the aligned sequences were indicated by under lines. Broken lines in the sequences represent gaps introduced for maximum sequence similarity. Five conserved amino acid residues, including a G residue near the N-terminus, a central [QXVXG] motif, and a W residue near the C-terminus, were marked by asterisks. The unique conserved amino acid sequence, [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-X-[EDQV]-[HYFQ]-N, in the phytocystatin subfamily was marked by the upper broken line on the top of the sequences. Amino acid residues that were identical in at least eleven out of the fifteen sequences were highlighted. The location of secondary structures, including a a helix and four p sheets, were indicated.
group I phytocystatins such as from sugarcane (Ki of Can-eCPI-2, 2 x 10-7M), Job's tears (Ki of CysM, 1.9 x 10-7M) and rice (Ki of OC-II, 8.3 x 10-7 M) (Kondo et al., 1990; Yoza et al., 2002; Gianotti et al., 2006). The Ki value of FaCYS against papain were 1-3 orders magnitude higher than other reported group I phytocystatins including OC-I from rice (Ki of 3.0 x 10-8 M), CsC from chestnut (Ki of
2.9 x 10-8 M), and KCPI1 from kiwifruit (Ki of 1.6 x 10-10 M) (Kondo et al., 1990; Rassam and Laing, 2004). How-ever, it should be noted that these experimental conditions were different.
Papain almost lost its activity when incubated with equimolar FaCYS (Figure 3). Since both recombinants showed similar Ki values, it suggested that the polyhisti-
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Figure 2. SDS/PAGE analysis of fusion and non-fusion FaCYS over-expressed in E. coli. (A) Total proteins were extracted from E. coli cells with or without IPTG induction, and fractionated into supernatant and precipitate; (B) Total proteins were extracted from E. coli cells with or without IPTG induction, and fractionated into supernatant and precipitate.
dine tag did not structurally interfere with FaCYS activ­ity. Besides, FaCYS showed inhibitory activity against human cathepsin B, cathepsin L and ficin with Ki values of 5.6 x 10-7 M, 3.0 x 10-8 M and 2.0 x10-7 M, but showed no inhibitory activity against pineapple stem bromelain (data not shown). Regarding other reported phytocysta-tins, Ki of KCPI1 to human cathepsin B, cathepsin L, ficin and bromelain were 8.0 x 10-9, 1.0 x 10-12, 3.2 x 10-11, and 1.5 x 10-10 M (Rassam and Laing, 2004); while Ki of CsC against human cathepsin B and ficin were 4.7 x 10-7 and 6.5 x 10-8 M (Pernas et al., 1998). A pineapple phy-tocystatin, AcCYS1 had a putative signal peptide and an extended unique AE-rich N-terminal trunk (NTT). NTT-containing AcCYS1 could inhibit bromelain, and was post-translationally removed during ripening (Neuteboom et al., 2009). Without NTT, AcCYS1 exhibited poor inhi-bition activity against bromelain (Neuteboom et al., 2009; Shyu et al., 2004). KCPI1 exhibited good inhibition activ-ity to papain and bromelain; in contrast, FaCYS showing inhibitory activity against papain was unable to inhibit the proteolytic activity by bromelain.
binant proteins were subjected to a heat treatment of 30-80°C for 30 min. FaCYS was shown to be thermally stable up to 50°C (Figure 5A). However, it rapidly lost almost 60% of its inhibitory activity when the treatment temperature was 60°C for 10 min (Figure 5B). To exam pH effect on FaCYS, the purified his-tagged FaCYS was incubated at various pH values (pH=2-10) for 2 h prior to measuring papain inhibitory activity. Different pH en­vironments, including those strongly acidic or basic, had no apparent affect on FaCYS activities (data not shown). Despite the lack of intra-disulfide bonds to sustain pro­tein structure, our results indicated that FaCYS was a pH-and thermo-stable protein. Although phytocystatins were known as a proteins without disulfide bonds, several phy-tocystatins were reportedly thermo-stable proteins, such as pineapple cystatin, stable up to 60°C for 30 min (Shyu et al., 2004), and rice OC-II, up to 65°C for 10 min (Ohtsubo et al., 2007).
Antifungal activity of recombinant FaCYS
Phytocystatins have gained attention for protecting plants from herbivorous insect and fungal attack. The growth inhibition effect of purified recombinant FaCYS was tested on some severe tropical phytopathgenic fungi, including Fusarium oxysporum, Glomerella cingulata,
pH and thermal stability of the recombinant Fa-CYS
To assess the thermal stability of FaCYS, the recom-
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Figure 3. SDS/PAGE and western blotting analysis of the purified fusion and non-fusion FaCYS over-expressed in E. coli. (A) To­tal proteins were extracted from E. coli cells with or without IPTG induction, and fractionated into supernatant and precipitate. The recombinant his-tagged FaCYS was then purified from soluble proteins of the E. coli lysate by affinity column. These proteins were resolved by SDS/PAGE. A duplicate gel was transferred onto a nitrocellulose membrane and subjected to immunoblotting with anti­bodies against his-tagged FaCYS; (B) Total proteins were extracted from E. coli cells with or without IPTG induction, and fractionated into supernatant and precipitate. The nonfusion recombinant was then purified from soluble proteins of the E. coli lysate by papain-coupling affinity column and cation exchange column. These proteins were resolved by SDS/PAGE. A duplicate gel was transferred onto a nitrocellulose membrane and subjected to immunoblotting with antibodies against his-tagged FaCYS.
Phellinus noxius, Rhizoctonia solani and Sclerotium rofsii. The results showed no FaCYS inhibitory effect on F. oxysporum, P. noxius and R. solani, but an FaCYS inhibitory effect was observed on spore germination of G. cingulata and the mycelia growth of S. rofsii (Figure 6). G. cingulata spore germination was inhibited to 27% and the of S. rofsii mycelia growth was almost completely inhib­ited as the FaCYS concentration reached 400 [ig/mL.
Various phytocystatins cause different growth inhibi­tions on fungi, and these inhibitions vary depending on fungal species. Recently, a tarocystatin from Colocasia esculenta, a group II phytocystatin, inhibited the myce-lia growth of S. rofsii (Yang and Yeh, 2005). Similarly, FaCYS showed a toxic effect on the mycelia growth of S. rofsii. Though the mechanism for phytocystatin anti-fungal activity is still unclear, S. rofsii appears sensitive to phytocystatins. It is known that S. rofsii causes great dam­age (Southern Blight) to tomato, peanut and banana; while G. cingulata causes Anthracnose to mango and papaya. The inhibition effects of FaCYS provide potential bio-technological applications on these fruits. In addition, full
Figure 4. Inhibition of papain activity by the recombinant FaCYS. Ten microgram of papain was incubated with various amounts of the purified his-tagged and nonfusion FaCYS. The residual hydrolytic activity was determined.
length, N-terminal and C-terminal extension regions of tarocystatin were produced separately (Wang et al., 2008). Full length tarocystatin possess stronger papain inhibition
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Figure 5. Thermal stability of his-tagged FaCYS. The purified recombinant FaCYSs were subjected to a heat treatment either under different temperatures for 30 min (A) or at 50oC and 60oC for different periods of time (B) prior to measure papain inhibitory activity.
knowledge, it is the first report of phytocystatin from the Ficus genus as well as the Moraceae family. However, the current work revealed that recombinant FaCYS in pET28a and pET29a expression was not efficient. Obtaining enough recombinant FaCYS for further detailed study and application of this unique protein is now our mission.
Acknowledgements. The authors gratefully acknowledge Wei-Chung Chiou, Tzu-Ying Lee and Kai-Ting Hsu for their technical assistance. This work was supported by a grant from National Science Council of Taiwan (NSC 97-2313-B-001-MY3) to W. M. Chou and (NSC 098-2811-B-
150-001) to Y. M. Cheng.
Figure 6. Growth inhibition of phytopathgenic fungi by his-tagged FaCYS. It was observed by microscopy that recombi-nant FaCYS inhibited the spore germination of G. cingulata and the mycelia growth of S. rofsii at a concentration of 400 [ig/mL.
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CONCLUSION
Jelly fig is a native plant in Taiwan and the pericarpial portion of its achenes has been long used to produce jelly curd, a popular local beverage. We recently evaluated jelly fig achene proteins for further applications. FaCYS encod­ing a jelly fig phytocystatin was subsequently isolated and identified. This cDNA clone was bacterial-expressed, and the purified his-tagged recombinant protein was character­ized as a pH and thermo-stable protein. We found that the recombinant protein showed anti-fungal activity, and ex­hibited inhibitory activity toward papain, cathepsin B and ficin, but no inhibitory activity against bromelain. FaCYS presents a minor protein in jelly fig achenes; and to our
SHYU et al. ― Jelly fig phytocystatin
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愛玉瘦果植物性硫氫蛋白?抑制蛋白基因的選殖、功能性表
達與重組蛋白質特性分析
徐志宏1  楊翊梅2  盧錫祺3  鄭月媚2  曾志正4  周文敏2
1國立屏東科技大學生命科學系
2國立虎尾科技大學生物科技系
3東海大學食品科學系
4國立中興大學生物科技學研究所
由成熟愛玉瘦果的cDNA資料庫中分析約300個表現序列標籤(EST clone)。其中一 cDNA選殖株
之DNA序列經比對及演譯分析結果，預測爲植物性硫氫蛋白酶抑制蛋白（phytocystatin)。此選殖株被
命名爲FaCYS，全長含有582 bp ，可轉譯得到114個胺基酸。經預測帶有訊息胜肽'成熟的FaCYS不
含任何cysteine ，分子量爲10.8 kDa， pI値爲9.7 。將FaCYS分別選殖至 非融合與融合(his-tagged)的表
達載體中，將重組質體轉殖於大腸桿菌並表達得到可溶性的非融合與融合FaCYS重組蛋白質。純化後
所得之非融合與his-tagged融合FaCYS重組蛋白皆具有papain之抑制活性,其Ki値極爲接近，分別爲
2.7 × 10-7 M與2.4 × 10-7 M 。進一歩分析發現his-tagged FaCYS對人類cathepsin B、cathepsin L及ficin
亦具有抑制活性'其Ki値分別爲5.6 × 10-7 M、3.0 x10 8 M和2.0 × 10-7 M ；但對bromelain則無抑制活
性。his-tagged FaCYS在不同的pH環境中具有很好的耐受性，亦具有不錯的熱穩定性，於50°C下30
分鐘後仍維持對papain的抑制活性。此外'此重組蛋白可抑制Glomerella cingulata及Sclerotium rofsii
菌絲的生長。
關鍵詞：硫氫蛋白酶抑制蛋白；硫氫蛋白酶；愛玉瘦果；植物性硫氫蛋白酶抑制蛋白；重組蛋白。