Cloning, expression and mutational studies of a trypsin inhibitor that retains activity even after cyanogen bromide digestion

Bhattacharjee, Nilanjana; Banerjee, Sayanika; Dutta, Sriparna

doi:10.1016/j.pep.2014.01.013

Cited by 8 publications

(6 citation statements)

References 28 publications

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“…Using E. coli BL21 (DE3) cells for expression, the yields of purified proteins recovered exclusively from the intracellular soluble fraction were 256 mg (D67V), 264 mg (R68L), 248 mg (R68L/D67V), and 287 mg (recombinant FtTI) per 1 L of culture. These expression levels are very similar to those reported for other recombinant plant protease inhibitors, such as mustard, mung bean and winged bean TIs, with yields of approximately 180-200 mg per 1 L of culture [4,5,26]. E. coli BL21 (DE3) cells were induced for 5 h with isopropyl β-D-thiogalactoside.…”

Section: Expression and Purification Of Ftti And Ftti Mutantssupporting

confidence: 77%

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Molecular Cloning and Structure–Function Analysis of a Trypsin Inhibitor from Tartary Buckwheat and Its Application in Combating Phytopathogenic Fungi

et al. 2018

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Host plant protease inhibitors offer resistance to proteases from invading pathogens. Trypsin inhibitors (TIs), in particular, serve as protective agents against insect and pathogen attacks. In this study, we designed a pair of degenerate primers based on highly conserved motifs at the Nand C-termini of the TI from tartary buckwheat (Fagopyrum tataricum; Ft) to clone the central portion. Genomic walking was performed to isolate the 5 and 3 flanking regions of FtTI. We demonstrated the successful PCR amplification of a 644 bp portion of FtTI. The full-length DNA of FtTI contains a complete open reading frame of 264 bp, encoding 87 amino acids with a mass of approximately 9.5 kDa. The FtTI protein sequence was 49% identical and 67% similar to potato protease inhibitors. Site-directed mutagenesis identified the residues, Asp 67 and Arg 68 , as crucial for the inhibitory activity of the FtTI. Recombinant and mutant FtTI inhibited both the hyphal growth and spore germination of Alternaria solani. The calculated 50% inhibitory concentrations of FtTI ranged from 5-100 µg mL −1 for spore germination and 1-50 µg mL −1 for fungal growth. Thus, recombinant FtTI may function in host resistance against a variety of fungal plant pathogens.

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Section: Expression and Purification Of Ftti And Ftti Mutantssupporting

confidence: 77%

“…Site-directed mutagenesis and sequence assays were carried out as described previously [26][27][28].…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

Molecular Cloning and Structure–Function Analysis of a Trypsin Inhibitor from Tartary Buckwheat and Its Application in Combating Phytopathogenic Fungi

et al. 2018

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“…5A and D). Occurrence of such double headed Kunitz inhibitors is found to be rare in nature and reported from the following plants earlier: Leucaena leucocephala (Sattar et al, 2004), black soybean (Lin and Ng, 2008), Adenanthera pavonina (Migliolo et al, 2010), tamarind (Patil et al, 2012) and winged bean (Bhattacharjee et al, 2014). However, the exact nature of the number of TI/CI reactive sites on RsBBI and RsKI has to be studied in detail as it could also be due to oligomerisation.…”

Section: Visualization Of Activity Titration and Inhibition Kineticsmentioning

confidence: 99%

Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea

et al. 2019

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Rhynchosia sublobata, a wild relative of pigeonpea, possesses defensive proteinase/protease inhibitors (PIs). Characterization of trypsin specific PIs (RsPI) separated from seeds by column chromatography using 2-D gel electrophoresis and Edman degradation method identified R. sublobata possessed both Bowman-Birk isoinhibitors (RsBBI) and Kunitz isoinhibitors (RsKI). A quick method was developed to separate RsBBI and RsKI from RsPI based on their differential solubility in TCA and acetate buffer. N-terminus sequencing of RsBBI and RsKI by MALDI-ISD ascertained the presence of Bowman Birk and Kunitz type isoinhibitors in R. sublobata. RsBBI (9216 Da) and RsKI (19,412 Da) exhibited self-association pattern as revealed by western blotting with anti-BBI antibody and MALDI-TOF peptide mass fingerprint analysis, respectively. RsBBI and RsKI varied significantly in their biochemical, biophysical and insecticidal properties. RsBBI inhibited the activity of trypsin (Ki = 128.5 ± 4.5 nM) and chymotrypsin (Ki = 807.8 ± 23.7 nM) while RsKI (Ki = 172.0 ± 9.2 nM) inhibited the activity of trypsin alone, by non-competitive mode. The trypsin inhibitor (TI) and chymotrypsin inhibitor (CI) activities of RsBBI were stable up to 100°C. But, RsBBI completely lost its TI and CI activities on reduction with 3 mM DTT. Conversely, RsKI lost its TI activity on heating at 100°C and retained > 60% of its TI activity in presence of 3 mM DTT. CD spectroscopic studies on RsBBI and RsKI showed their secondary structural elements in the following order: random coils > β-sheets/β-turns > α-helix. However, RsKI showed reversible denaturation midpoint (Tm) of 75°C. Further, the significant inhibitory activity of RsBBI (IC 50 = 24 ng) and RsKI (IC 50 = 59 ng) against trypsin-like gut proteases of Achaea janata (AjGPs) and Helicoverpa armigera (HaGPs) suggest them as potential biomolecules in the management of A. janata and H. armigera, respectively. vegetative organs during biotic and abiotic stresses (Jamal et al., 2013; Yamchi et al., 2017). They also act as pseudosubstrates of proteases and stabilize them during desiccation. The PIs are rapidly degraded during seed germination to release essential amino acids and they reappear in cotyledons to protect them from invading pests and pathogens. They also take part in programmed cell death in plants. Bowman-Birk

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“…However, if a high concentration of PIs is ingested, then this adaptation mechanism can only result in a substantial loss of protein through the feces, aggravating the lack of essential amino acids . This particular situation was reported in the case of the ingestion of PIs from flowers of Nicotiana alata (NaPIs) by H. armigera larvae: most of the trypsin activity was inactivated but the chymotrypsin activity increased.…”

Section: Insect Adaptation Mechanisms To Protease Inhibitors and Lectinsmentioning

confidence: 99%

Insect midgut structures and molecules as targets of plant‐derived protease inhibitors and lectins

Napoleão

Albuquerque

Santos

et al. 2018

Pest Management Science

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The midgut of insects is involved in digestion, osmoregulation and immunity. Although several defensive strategies are present in this organ, its organization and function may be disturbed by some insecticidal agents, including bioactive proteins like lectins and protease inhibitors (PIs) from plants. PIs interfere with digestion, leading to poor nutrient absorption and decreasing amino acid bioavailability. Intake of PIs can delay development, cause deformities and reduce fertility. Ingestion of PIs may lead to changes in the set of proteases secreted in the insect gut, but this response is often insufficient and results in aggravation of the malnutrition status. Lectins are proteins that are able to interact with glycoconjugates, including those linked to cell surfaces. Their effects on the midgut include disruption of the peritrophic matrix, brush border and secretory cell layer; induction of apoptosis and oxidative stress; interference with nutrient absorption and transport proteins; and damaging effects on symbionts. In addition, lectins can cross the intestinal barrier and reach the hemolymph. The establishment of resistant insect populations due to selective pressure resulting from massive use of a bioactive protein is an actual possibility, but this can be minimized by the multiple mode-of-action of these proteins, mainly the lectins.

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Cloning, expression and mutational studies of a trypsin inhibitor that retains activity even after cyanogen bromide digestion

Cited by 8 publications

References 28 publications

Molecular Cloning and Structure–Function Analysis of a Trypsin Inhibitor from Tartary Buckwheat and Its Application in Combating Phytopathogenic Fungi

Molecular Cloning and Structure–Function Analysis of a Trypsin Inhibitor from Tartary Buckwheat and Its Application in Combating Phytopathogenic Fungi

Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea

Insect midgut structures and molecules as targets of plant‐derived protease inhibitors and lectins

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