Expression and Purification of Recombinant Bowman-Birk Trypsin Inhibitor from Foxtail Millet Bran and Its Anticolorectal Cancer Effect In Vitro and In Vivo.

Trypsin inhibitors derived from plants have various pharmacological activities and promising clinical applications. In our previous study, a Bowman-Birk-type major trypsin inhibitor from foxtail millet bran (FMB-BBTI) was extracted with antiatherosclerotic activity. Currently, we found that FMB-BBTI possesses a prominent anticolorectal cancer (anti-CRC) activity. Further, a recombinant FMB-BBTI (rFMB-BBTI) was successfully expressed in a soluble manner in host strain Escherichia coli. BL21 (DE3) was induced by isopropyl-ß-d-thiogalactoside (0.1 mM) at 37 °C for 3.5 h by the pET28a vector system. Fortunately, a purity greater than 93% of rFMB-BBTI with anti-CRC activity was purified by nickel-nitrilotriacetic acid affinity chromatography. Subsequently, we found that rFMB-BBTI displays a strikingly anti-CRC effect, characterized by the inhibition of cell proliferation and clone formation ability, cell cycle arrest at the G2/M phase, and induction of cell apoptosis. It is interesting that the rFMB-BBTI treatment had no obvious effect on normal colorectal cells in the same concentration range. Importantly, the anti-CRC activity of rFMB-BBTI was further confirmed in the xenografted nude mice model. Taken together, our study highlights the anti-CRC activity of rFMB-BBTI in vitro and in vivo, uncovering the clinical potential of rFMB-BBTI as a targeted agent for CRC in the future.

A Novel Trypsin Kunitz-Type Inhibitor from Cajanus cajan Leaves and Its Inhibitory Activity on New Cancer Serine Proteases and Its Effect on Tumor Cell Growth.

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 µM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and ß-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

Potent Inhibitor of Human Trypsins from the Aeruginosin Family of Natural Products.

Serine proteases regulate many physiological processes and play a key role in a variety of cancers. Aeruginosins are a family of natural products produced by cyanobacteria that exhibit pronounced structural diversity and potent serine protease inhibition. Here, we sequenced the complete genome of Nodularia sphaerocarpa UHCC 0038 and identified the 43.7 kb suomilide biosynthetic gene cluster. Bioinformatic analysis demonstrated that suomilide belongs to the aeruginosin family of natural products. We identified 103 complete aeruginosin biosynthetic gene clusters from 12 cyanobacterial genera and showed that they encode an unexpected chemical diversity. Surprisingly, purified suomilide inhibited human trypsin-2 and -3, with IC50 values of 4.7 and 11.5 nM, respectively, while trypsin-1 was inhibited with an IC50 of 104 nM. Molecular dynamics simulations suggested that suomilide has a long residence time when bound to trypsins. This was confirmed experimentally for trypsin-1 and -3 (residence times of 1.5 and 57 min, respectively). Suomilide also inhibited the invasion of aggressive and metastatic PC-3M prostate cancer cells without affecting cell proliferation. The potent inhibition of trypsin-3, together with a long residence time and the ability to inhibit prostate cancer cell invasion, makes suomilide an attractive drug lead for targeting cancers that overexpress trypsin-3. These results substantially broaden the genetic and chemical diversity of the aeruginosin family and suggest that aeruginosins may be a source of selective inhibitors of human serine proteases.

Chemical Composition and in vitro Anti-inflammatory Activity of Wheat Germ Oil Depending on the Extraction Procedure.

This study aimed to examine the chemical composition of wheat germ oil extracted by three different methods, and to evaluate its inhibitory effect on the cyclooxygenase and proteinase activities. The results showed that the contents of policosanols, tocopherols and phytosterols were affected by the extraction procedure. However, the fatty acid composition of the different oil extracts was nearly the same. Among the tested oils samples, cold pressed oil exhibited the strongest inhibitory activity against proteinase (93.4%, IC50 =195.7 µg/mL) and cyclooxygenase 1 (80.5%, IC50 =58.6 µg/mL). Furthermore, the cold pressed oil had the highest content of octacosanol, ß-sitosterol and α-linolenic acid, suggesting that those bioactive compounds could be essential for the potent ani-cyclooxygenase activity. The present data revealed that wheat germ oil contained cyclooxygenase and trypsin inhibitors, which are the promising therapeutic target for the treatment of various inflammatory diseases. Thus, wheat germ oil might be used to develop functional foods and pharmaceutic products for the human health.

Identification of sustainable trypsin active-site inhibitors from Nigrospora sphaerica strain AVA-1.

Trypsin is a protein-digesting enzyme that is essential for the growth and regeneration of bone, muscle, cartilage, skin, and blood. The trypsin inhibitors have various role in diseases such as inflammation, Alzheimer's disease, pancreatitis, rheumatoid arthritis, cancer prognosis, metastasis and so forth. From 10 endophytic fungi isolated, we were able to screen only one strain with the required activity. The fungus with activity was obtained as an endophyte from Dendrophthoe falcata and was later identified as Nigrospora sphaerica. The activity was checked by enzyme assays using trypsin. The fungus was fermented and the metabolites were extracted and further purified by bioassay-guided chromatographic methods and the compound isolated was identified using gas chromatography-mass spectrometry. The compound was identified as quercetin. Docking studies were employed to study the interaction. The absorption, distribution, metabolism, and excretion analysis showed satisfactory results and the compound has no AMES and hepatotoxicity. This study reveals the ability of N. sphaerica to produce bioactive compound quercetin has been identified as a potential candidate for trypsin inhibition. The present communication describes the first report claiming that N. sphaerica strain AVA-1 can produce quercetin and it can be considered as a sustainable source of trypsin active-site inhibitors.

Identification and Target-Modification of SL-BBI: A Novel Bowman-Birk Type Trypsin Inhibitor from Sylvirana latouchii.

The peptides from the ranacyclin family share similar active disulphide loop with plant-derived Bowman-Birk type inhibitors, some of which have the dual activities of trypsin inhibition and antimicrobial. Herein, a novel Bowman-Birk type trypsin inhibitor of the ranacyclin family was identified from the skin secretion of broad-folded frog (Sylvirana latouchii) by molecular cloning method and named as SL-BBI. After chemical synthesis, it was proved to be a potent inhibitor of trypsin with a Ki value of 230.5 nM and showed weak antimicrobial activity against tested microorganisms. Modified analogue K-SL maintains the original inhibitory activity with a Ki value of 77.27 nM while enhancing the antimicrobial activity. After the substitution of active P1 site to phenylalanine and P2' site to isoleucine, F-SL regenerated its inhibitory activity on chymotrypsin with a Ki value of 309.3 nM and exhibited antiproliferative effects on PC-3, MCF-7 and a series of non-small cell lung cancer cell lines without cell membrane damage. The affinity of F-SL for the ß subunits in the yeast 20S proteasome showed by molecular docking simulations enriched the understanding of the possible action mode of Bowman-Birk type inhibitors. Further mechanistic studies have shown that F-SL can activate caspase 3/7 in H157 cells and induce apoptosis, which means it has the potential to become an anticancer agent.

LUTI: a double-function inhibitor isolated from naked flax seeds.

Acute glucose fluctuation during the postprandial period causes a risk for type 2 diabetes mellitus (T2DM). α-Glucosidase inhibitors have been approved as therapeutic agents for diabetes. In the present study, a protein with α-glucosidase inhibitory activity from Flax (Linum usitatissimum) seeds was isolated using a one-step purification with Q-Sepharose4B column, followed by Sephacryl S-200 size-exclusion chromatography. It was identified as a trypsin inhibitor, named L. usitatissimum trypsin inhibitor (LUTI). The half maximal inhibitory concentration (IC50) of LUTI was 113.92 µM for α-glucosidase and 6.17 µM for trypsin. Lineweaver-Burk kinetic experiment showed that the protein exhibited two distinct inhibitory modes, a competitive inhibitor type for α-glucosidase and a non-competitive type for trypsin. The interaction between LUTI and α-glucosidase was detected through gel filtration chromatography and dynamic light scattering. Increased glucose consumption and lactic acid production were also observed following LUTI treatment in Caco-2 and HepG2 cells. LUTI inhibits not only the activity of trypsin but also the activity of α-glucosidase. It is expected that LUTI will become an oral hypoglycemic polypeptide drug for T2DM.

Isolation and partial structural characterization of new Kunitz-type trypsin inhibitors from the pike cestode Triaenophorus nodulosus.

The inhibitors produced by the parasitic worms successfully protect them from the host's proteases and are supposed to underlie the host-parasite specificity. Our previous study has shown that the extracts from the pike tapeworm Triaenophorus nodulosus inhibit host proteinases and commercial trypsin. We aimed to isolate and identify the components responsible for trypsin inactivation. After a two-step separation the molecular masses were measured by SE-HPLC. The sample proved to contain four fractions represented by polypeptides (1-45 kDa) and low-molecular hydrophobic compounds. According to SDS-PAGE analysis, the major polypeptides in the fractions displaying the highest inhibition had masses of 14.4 kDa. The study culminated in partial N-terminal amino acid sequence analysis with a further search for homology. The research revealed two novel Kunitz-type proteins potentially responsible for the inhibitory capacity of the tapeworms against trypsin. Our findings extend the list of cestodes relying on Kunitz-type proteins in the host-parasite molecular cross-talk.

Discovery and Characterization of Cyclic and Acyclic Trypsin Inhibitors from Momordica dioica.

Momordica trypsin inhibitors (TIs) such as those isolated from the seeds of the gac fruit, Momordica cochinchinensis (MCoTI-I and MCoTI-II), are widely used as scaffolds for drug design studies. To more effectively exploit these molecules in the development of therapeutics, there is a need for wider discovery of the natural sequence diversity among TIs from other species in the Momordica subfamily. Here we report the discovery of the encoding gene and six TIs from the seeds of the spiny gourd, Momordica dioica, four of which possess novel sequences (Modi 1, 3, 5, and 6) and two (Modi 2 and 4) of which are known peptides (TI-14, TI-17) previously identified in Momordica subangulata. Modi 6 is an acyclic peptide featuring a pyrrolidone carboxylic acid modification, whereas the remaining five TIs are cyclic. All Modi peptides display similar overall structures and trypsin inhibitory activities. No toxicity was observed for these peptides when tested against cancer and insect cells. All Modi peptides were exceptionally stable over 24 h in human serum, indicating a dual strategy to stabilize the peptides in nature, either head-to-tail cyclization or N-pyrolation, which suggests these peptides might be excellent candidates as scaffolds for epitope stabilization in drug design studies.

Characterization of a new member of kunitz-type protein family from the venom of Persian false-horned viper, Pseudocerastes persicus.

A new member of kunitz-type protein family, PPTI (PseudocerastesPersicusTrypsin Inhibitor), was isolated from the venom of Persian false horned viper Pseudocerastes persicus and characterized. Mass spectrometry and amino acid sequencing revealed that PPTI is a 68 amino acid protein with molecular weight of about 7.6 kDa. The first amino acid residue of PPTI is N-terminally blocked via a post translational modification to pyroglutamyl. Sequence comparison against UniProtKB shows a high sequence similarity of PPTI with kunitz-type proteins, especially serine protease inhibitors and dendrotoxins (DTXs). The number of cysteines and disulfide bonding pattern of PPTI are the same as kunitz-type proteins. Based on sequence derive information, anti-protease activity of PPTI against trypsin was experimentally examined. The constructed homology models of PPTI confirmed the ability of PPTI to fold similarly to kunitz domain. The presence of characteristic basic-hydrophobic functional dyad of DTXs in PPTI supports its inhibitory potential against potassium channels. In summary, this study hypothesized the dual functionality of PPTI according to its inhibitory effect on trypsin and its potential ability in blocking potassium channel.

CgTI, a novel thermostable Kunitz trypsin-inhibitor purified from Cassia grandis seeds: Purification, characterization and termiticidal activity.

Cassia grandis trypsin inhibitor (CgTI) is a novel plant serine proteinase inhibitor. This study sets out to purify a thermostable inhibitor from the seeds of Cassia grandis and to provide biochemical information about a novel peptide belonging to the Kunitz family. Moreover, toxicity assays against Artemia, Aedes aegypti larvae-L4 and Nasutitermes corniger are evaluated. The purification process was performed using acetone precipitation, Trypsin-Sepharose-CL4B and Superdex-G75. The inhibitor showed an apparent molecular mass of around 19.8 kDa on Superdex-G75 gel filtration, and a mass of around 19.0 kDa visualized by SDS-PAGE under reducing conditions, and it also showed the protein consists of two polypeptide chains. N-terminal sequencing by Edman's degradation of 16 residues revealed a sequence of amino acids SVVLDTSGEPIRNGGG. 2D-electrophoresis identified a pI value of 6.3 and a 1:1 stoichiometric ratio was noted during CgTI-trypsin complex formation. The inhibitor retained the inhibitory activity over a broad range of pH (5-10) and showed thermostable activity at temperatures 30-80 °C. Furthermore, in vivo assays showed no lethality effect against Artemia and Aedes aegypti larvae, but mortality against Nasutitermes corniger with termiticidal activity LC50 of 0.685 mg/mL on workers and 0.765 mg/mL on soldiers. Preliminary investigations of CgTI revealed it to be a promising biotechnological and biomedical candidate.

A Bowman-Birk type chymotrypsin inhibitor peptide from the amphibian, Hylarana erythraea.

The first amphibian skin secretion-derived Bowman-Birk type chymotrypsin inhibitor is described here from the Asian green frog, Hylarana erythraea, and was identified by use of molecular cloning and tandem mass spectrometric amino acid sequencing. It was named Hylarana erythraea chymotrypsin inhibitor (HECI) and in addition to inhibition of chymotrypsin (Ki = 3.92 ± 0.35 µM), the peptide also inhibited the 20 S proteasome (Ki = 8.55 ± 1.84 µM). Additionally, an analogue of HECI, named K9-HECI, in which Phe9 was substituted by Lys9 at the P1 position, was functional as a trypsin inhibitor. Both peptides exhibited anti-proliferation activity against the human cancer cell lines, H157, PC-3 and MCF-7, up to a concentration of 1 mM and possessed a low degree of cytotoxicity on normal cells, HMEC-1. However, HECI exhibited higher anti-proliferative potency against H157. The results indicate that HECI, inhibiting chymotryptic-like activity of proteasome, could provide new insights in treatment of lung cancer.

Biochemical properties of a bacterially-expressed Bowman-Birk inhibitor from Rhynchosia sublobata (Schumach.) Meikle seeds and its activity against gut proteases of Achaea janata.

Crude proteinase inhibitors (CPIs) extracted from the seeds of Rhynchosia sublobata, a wild relative of pigeon pea showed pronounced inhibitory activity on the larval gut trypsin-like proteases of lepidopteran insect pest - Achaea janata. Consequently, a full-length cDNA of Bowman-Birk inhibitor gene (RsBBI1) was cloned from the immature seeds of R. sublobata. It contained an ORF of 360 bp encoding a 119-amino acid polypeptide (13.3 kDa) chain with an N-terminus signal sequence comprising of 22 amino acids. The amino acid sequence and phylogenetic analysis together revealed that RsBBI1 exhibited a close relation with BBIs from soybean and Phaseolus spp. A cDNA sequence corresponding to RsBBI1 mature protein (89 amino acid stretch) was expressed in E. coli. The recombinant rRsBBI1 protein with a molecular mass of 9.97 kDa was purified using trypsin affinity chromatography. The purified rRsBBI1 exhibited non-competitive mode of inhibition of both bovine trypsin (Ki of 358 ±â€¯11 nM) and chymotrypsin (Ki of 446 ±â€¯9 nM). Its inhibitory activity against these proteases was stable at high temperatures (>95 °C) and a wide pH range but sensitive to reduction with dithiothreitol (DTT), indicating the importance of disulphide bridges in exhibiting its activity. Also, rRsBBI1 showed significant inhibitory activity (IC50 = 70 ng) on A. janata larval gut trypsin-like proteases (AjGPs). Conversely, it showed <1% inhibitory activity (IC50 = 8 µg) on H. armigera larval gut trypsin-like proteases (HaGPs) than it has against AjGPs. Besides, in vivo feeding experiments clearly indicated the deleterious effects of rRsBBI1 on larval growth and development in A. janata which suggests it can be further exploited for such properties.

Trypsin Inhibitors from Cajanus cajan and Phaseolus limensis Possess Antioxidant, Anti-Inflammatory, and Antibacterial Activity.

Protease inhibitors are one of the most promising and investigated subjects for their role in pharmacognostic and pharmacological studies. This study aimed to investigate antioxidant, anti-inflammatory, and antimicrobial activities of trypsin inhibitors (TIs) from two plant sources (Cajanus cajan and Phaseolus limensis). TI was purified from C. cajan (PUSA-992) by ammonium sulfate precipitation followed by ion exchange chromatography. TI from Phaseolus limensis (lima bean trypsin inhibitor; LBTI) was procured from Sigma-Aldrich, St. Louis, Missouri, United States. The antioxidant activity was analyzed by ferric ion reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH). The anti-inflammatory property of TIs was determined by inhibition of albumin denaturation assay. Ascorbic acid and aspirin were used as standards for antioxidant and anti-inflammatory assays, respectively. These TIs were tested against various bacterial and fungal strains. The TIs showed DPPH radical-scavenging activity in a concentration-dependent manner with IC50 values comparable to ascorbic acid. The FRAP values were also observed comparable to ascorbic acid and followed the trend of dose-dependent manner. The half maximal inhibitory concentration (IC50) values of CCTI and LBTI in anti-inflammatory test showed that LBTI is more potent than CCTI. The TIs showed potent antibacterial activity, but apparently no action against fungi. This study has reported the biological properties of CCTI and LBTI for the first time. The results show that TIs possess the ability to inhibit diseases caused by oxidative stress, inflammation, and bacterial infestation.

Inga laurina trypsin inhibitor (ILTI) obstructs Spodoptera frugiperda trypsins expressed during adaptive mechanisms against plant protease inhibitors.

Plant protease inhibitors (PIs) are elements of a common plant defense mechanism induced in response to herbivores. The fall armyworm, Spodoptera frugiperda, a highly polyphagous lepidopteran pest, responds to various PIs in its diet by expressing genes encoding trypsins. This raises the question of whether the PI-induced trypsins are also inhibited by other PIs, which we posed as the hypothesis that Inga laurina trypsin inhibitor (ILTI) inhibits PI-induced trypsins in S. frugiperda. In the process of testing our hypothesis, we compared its properties with those of selected PIs, soybean Kunitz trypsin inhibitor (SKTI), Inga vera trypsin inhibitor (IVTI), Adenanthera pavonina trypsin inhibitor (ApTI), and Entada acaciifolia trypsin inhibitor (EATI). We report that ILTI is more effective in inhibiting the induced S. frugiperda trypsins than SKTI and the other PIs, which supports our hypothesis. ILTI may be more appropriate than SKTI for studies regarding adaptive mechanisms to dietary PIs.

Isolation and Characterization of a Phaseolus vulgaris Trypsin Inhibitor with Antiproliferative Activity on Leukemia and Lymphoma Cells.

A 17.5-kDa trypsin inhibitor was purified from Phaseolus vulgaris cv. "gold bean" with an isolation protocol including ion exchange chromatography on DEAE-cellulose (Diethylaminoethyl-cellulose), affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-sepharose (Sulfopropyl-sepharose), and gel filtration by FPLC (Fast protein liquid chromatography) on Superdex 75. It dose-dependently inhibited trypsin with an IC50 value of 0.4 µM, and this activity was reduced in the presence of dithiothreitol in a dose- and time-dependent manner, signifying the importance of the disulfide linkage to the activity. It inhibited [methyl-³H] thymidine incorporation by leukemia L1210 cells and lymphoma MBL2 cells with an IC50 value of 2.3 µM and 2.5 µM, respectively. The inhibitor had no effect on fungal growth and the activities of various viral enzymes when tested up to 100 µM.

Anti-angiogenic potential of trypsin inhibitor purified from Cucumis melo seeds: Homology modeling and molecular docking perspective.

Melons have a good source of protease inhibitors. Its fruit and seeds have been used as a traditional medicine. However, its effects on angiogenesis and mechanism of its action remain elusive. Herein trypsin inhibitor from aqueous extract of C. melo seeds (TICMS) was purified. Its effects on different steps of angiogenesis were evaluated. Also, we examined its effects on migration and angiogenesis of endothelial cells. Three dimensional model of TICMS protein was accurately built in which TICMS docked to αVß3 integrin and VEGFR1. Electrophoresis analysis of the purified protein revealed a single band with a molecular mass of about 3kDa. Treatment with TICMS at six doses resulted in a significant decrease of endothelial cell proliferation with an IC50 value of about 20µg/ml. Tubulogenesis assay revealed that a dose dependent anti-angiogenic activity of TICMS (5-40µg/ml). Also, TICMS had inhibitory effects on VEGF, MMP-2 and MMP-9 secretion. Our docking result speculated that TICMS could bind to the cleft between the αVß3 integrin and it able to decrease the activity of this receptor. The TICMS was also able to interact with VEGFR1 receptor, but with low probability. Based on our study, TICMS could be used as a specific angiogenesis inhibitor.

A Bowman-Birk protease inhibitor purified, cloned, sequenced and characterized from the seeds of Maclura pomifera (Raf.) Schneid.

MAIN CONCLUSION: A new BBI-type protease inhibitor with remarkable structural characteristics was purified, cloned, and sequenced from seeds of Maclura pomifera , a dicotyledonous plant belonging to the Moraceae family. In this work, we report a Bowman-Birk inhibitor (BBI) isolated, purified, cloned, and characterized from Maclura pomifera seeds (MpBBI), the first of this type from a species belonging to Moraceae family. MpBBI was purified to homogeneity by RP-HPLC, total RNA was extracted from seeds of M. pomifera, and the 3'RACE-PCR method was applied to obtain the cDNA, which was cloned and sequenced. Peptide mass fingerprinting (PMF) analysis showed correspondence between the in silico-translated protein and MpBBI, confirming that it corresponds to a new plant protease inhibitor. The obtained cDNA encoded a polypeptide of 65 residues and possesses 10 cysteine residues, with molecular mass of 7379.27, pI 6.10, and extinction molar coefficient of 9105 M-1 cm-1. MpBBI inhibits strongly trypsin with K i in the 10-10 M range and was stable in a wide array of pH and extreme temperatures. MpBBI comparative modeling was applied to gain insight into its 3D structure and highlighted some distinguishing features: (1) two non-identical loops, (2) loop 1 (CEEESRC) is completely different from any known BBI, and (3) the amount of disulphide bonds is also different from any reported BBI from dicot plants.

Psc-AFP from Psoralea corylifolia L. overexpressed in Pichia pastoris increases antimicrobial activity and enhances disease resistance of transgenic tobacco.

Psc-AFP, isolated from the seeds of Psoralea corylifolia L., is an antimicrobial protein with trypsin inhibitor activity. Its encoding gene was cloned by 3'- rapid amplification of cDNA ends (RACE) combined with Y-shaped adaptor-dependent extension (YADE) method. The gene Psc-AFP encodes a protein of 203 amino acids with a deduced signal peptide of 24 residues. The growth inhibition effect exerted by the heterologously expressed Psc-AFP in Pichia pastoris revealed that the recombinant Psc-AFP inhibited mycelium growth of Aspergillus niger, Rhizoctonia solani, and Alternaria brassicae and conidial germination of Alternaria alternata. The recombinant Psc-AFP also showed protease inhibitor activity manifested by the inhibition of trypsin. The transgenic tobacco bioassays confirmed that overexpressing Psc-AFP significantly enhanced the disease resistance of tobacco and that some of the transgenic lines were almost fully tolerant to Ralstonia solanacearum and A. alternata, whereas no apparent alteration in plant growth and development was observed. Collectively, these results indicate that the recombinant Psc-AFP is an active antimicrobial protein, with protease inhibitor activity that can be successfully produced in the yeast and tobacco and, therefore, maybe a potential antimicrobial candidate for practical use.

Allium sativum Protease Inhibitor: A Novel Kunitz Trypsin Inhibitor from Garlic Is a New Comrade of the Serpin Family.

PURPOSE: This study was aimed to purify and characterize the Protease inhibitor (PI) from a plant Allium sativum (garlic) with strong medicinal properties and to explore its phytodrug potentials. METHODS: Allium sativum Protease Inhibitor (ASPI) was purified using ammonium sulphate fractionation and Fast Protein Liquid Chromatography on anion exchanger Hi-Trap DEAE column. The purified protein was analyzed for its purity and molecular weight by SDS PAGE. The confirmation of presence of trypsin inhibiting PI was performed by MALDI TOF-TOF and analyzed by MASCOT database. The ASPI was further investigated for its kinetic properties and stability under extreme conditions of pH, temperature and chemical denaturants. Secondary structure was determined by Circular Dichorism (CD) spectroscopy. RESULTS: ASPI of ~15 kDa inhibited trypsin and matched "truncated kunitz Trypsin Inhibitor (Glycine max)" in MASCOT database. The purified ASPI showed 30376.1371 U/mg specific activity with a fold purity of 159.92 and yield ~93%. ASPI was quite stable in the range of pH 2-12 showing a decline in the activity around pH 4-5 suggesting that the pI value of the protein as ASPI aggregates in this range. ASPI showed stability to a broad range of temperature (10-80°C) but declined beyond 80°C. Further, detergents, oxidizing agents and reducing agents demonstrated change in ASPI activity under varying concentrations. The kinetic analysis revealed sigmoidal relationship of velocity with substrate concentration with Vmax 240.8 (µM/min) and Km value of 0.12 µM. ASPI showed uncompetitive inhibition with a Ki of 0.08±0.01 nM). The Far UV CD depicted 2.0% α -helices and 51% ß -sheets at native pH. CONCLUSIONS: To conclude, purified ~15 kDa ASPI exhibited fair stability in wide range of pH and temperature Overall, there was an increase in purification fold with remarkable yield. Chemical modification studies suggested the presence of lysine and tryptophan residues as lead amino acids present in the reactive sites. Therefore, ASPI with trypsin inhibitory property has the potential to be used as a non-cytotoxic clinical agents.