Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman–Birk type protease inhibitor from rice bean

Katoch, Rajan; Singh, Sunil Kumar; Thakur, Neelam; Dutt, Som; Yadav, Sudesh Kumar; Shukle, R. H.

doi:10.1016/j.gene.2014.05.055

Cited by 20 publications

(5 citation statements)

References 64 publications

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“…Rice bean is the potential source of resistance to both the biotic stresses. A new gene encoding Bowman-Birk type protease inhibitor was also isolated from rice bean (Katoch et al 2014). Besides, rice bean has a high yield potential owing to more pods per plant, pod length, number of seeds per pod, etc.…”

Section: Challenges and Opportunitiesmentioning

confidence: 99%

Rice bean: a lesser known pulse with well-recognized potential

Pattanayak

Roy

Sood

et al. 2019

Planta

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Section: Challenges and Opportunitiesmentioning

confidence: 99%

Rice bean: a lesser known pulse with well-recognized potential

Pattanayak

Roy

Sood

et al. 2019

Planta

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“…cDNA amplification was carried out according to modified protocol of Sambrook et al [28]. Confirmation of cDNA synthesis was done by amplification of rice bean trypsin inhibitor gene (RbTI) [24]. The primer sequences used were (i) RBF1: 5′-ATG GTG GTG CTG AAG GTG -3′ and (i) RBR1: 5′-GAC ATC ATC TTT ATC CCT GGA CTT -3′.…”

Section: Cloning and Sequence Analysis Of Rbl Orfmentioning

confidence: 99%

“…However, the separation among these species is possibly due to mutation causing heterogenicity in amino acid sequence. This is probably the way of adaptation to particular physiological and environmental conditions during their evolution [24].…”

Section: Molecular Cloning and Sequence Analysismentioning

confidence: 99%

“…Rice bean seeds have longer storage life than other legumes and remain unaffected from the attack of storage pest [21,22]. It has been suggested that plant inhibitory proteins such as enzyme inhibitor play key role in imparting resistance against insects in rice bean seeds [23,24]. Transferring insect resistance in susceptible plants is one of the important biotechnological applications of legume lectins.…”

Section: Introductionmentioning

confidence: 99%

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Cloning, Characterization, Expression Analysis, and Agglutination Studies of Novel Gene Encoding β-d-Galactose, N-Acetyl-d-Glucosamine and Lactose-Binding Lectin from Rice Bean (Vigna umbellata)

2021

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Lectins are glycoproteins and known for their peculiar carbohydrate-binding activity and their insect-pest-resistant properties. Earlier we have published our research finding on novel gene encoding Bowman-Birk type protease inhibitor with insecticidal properties from rice bean. This paper presents first report on cloning, sequencing, and expression of RbL ORF of 843 bp encoding 280 amino acids long lectin precursor from rice bean (Vigna umbellata) seeds. Blast analysis revealed more than 90% similarity of RbL protein with Vigna aconitifolia and Vigna angularis lectins. Phylogenetic analysis also revealed a close relationship between RbL and other legume lectins. Sequence analysis of genomic DNA revealed intronless nature of RbL gene (GenBank accession No. MT043160). The isolated RbL ORF was expressed in E. coli BL-21(DE3) cells and maximum expression was recorded with 0.5 mM IPTG after 4 h incubation at 37 °C. Western blotting confirmed RbL protein expression in E. coli. Recombinant protein (His 6 -RbL) of ~ 35 kDa m.wt was purified using Ni-NTA affinity chromatography to the extent of 0.26 mg/ml. In silico analysis characterized RbL protein as acidic, stable, hydrophobic, and secretary protein with one signal peptide cleavage site (A 26 -A 27 ) and four N-glycosylation sites. Template-based 3D model of RbL was structured using MODELLER tool and validated as good quality model. Structural analysis revealed dominance of β-pleated sheets and β-turns in RbL protein structure. β-d-galactose, N-acetyl-d-glucosamine, and lactose were predicted as putative ligands for RbL protein. Hydrogen bonding and hydrophobic forces were the major interactions between the predicted ligands and RbL protein. Agglutination and agglutination inhibition assays confirmed the binding specificity of RbL protein with the trypsinized rabbit erythrocytes and with the predicted ligands, respectively. Gene ontology analysis functionally annotated RbL protein as a plant defense protein. The novel information generated in the study is not mere pre-experimental findings but could also lay foundation for future research on exploring RbL gene and encoding protein for different biomedical and biotechnological applications.

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“…As alternative, heterologous expression of BBI is eco-friendly and economical, and thousand important proteins have been prepared in large scale (Ahmad et al 2014). BBIs from soybean, rice bean, and Rhynchosia sublobata were successfully expressed in Escherichia coli with the yields of 0.5 mg/L, mg/L and 2.5 mg/L, respectively (Flecker 1987;Katoch et al 2014;Mohanraj et al 2018). BBI genes from mustard and Glycine max were expressed and prepared in Pichia pastoris with the yields of 40-160 mg/L and 50-56 mg/L, respectively (Volpicella et al 2000;Yakoby and Raskin 2004).…”

mentioning

confidence: 99%

Heterologous Expression and Biochemical Characterization of a Peanut Bowman-Birk Type Inhibitor

Cheng¹,

Kangming²,

Mchunu³

et al. 2021

Preprint

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Objectives To heterologously express peanut ( Arachis ipaensis ) Bowman-Birk inhibitor (BBI) and characterize its properties. Results A putative BBI gene from peanut was overexpressed in Pichia pastoris with the maximal yield of 11.1 mg/L in a 250-mL shaking flask fermentation. The recombinant peanut BBI (rPBBI) was purified and its molecular weight was estimated to be 9 kDa. Purified rPBBI showed 5223.6 TIA/mg inhibitory activity toward trypsin. It retained more than 95% of its inhibitory activity over wide range of temperatures (40 to 90 o C) and pH (2.0 to 10.0) after incubation for 60 min. When presence of 100 mM dithiothreitol, rPBBI lost more than 80% inhibitory activity in 30 min. Conclusion The current investigation expressed a peanut BBI with stable inhibitory activity against trypsin and exposed the potential of heterologous overexpression approach in large scale production of BBI.

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Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman–Birk type protease inhibitor from rice bean

Cited by 20 publications

References 64 publications

Rice bean: a lesser known pulse with well-recognized potential

Rice bean: a lesser known pulse with well-recognized potential

Cloning, Characterization, Expression Analysis, and Agglutination Studies of Novel Gene Encoding β-d-Galactose, N-Acetyl-d-Glucosamine and Lactose-Binding Lectin from Rice Bean (Vigna umbellata)

Heterologous Expression and Biochemical Characterization of a Peanut Bowman-Birk Type Inhibitor

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