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

Mohanraj, S S and Tetali, S D and Mallikarjuna, N and Dutta-Gupta, A and Padmasree, K (2018) Biochemical properties of a bacterially-expressed Bowman-Birk inhibitor from Rhynchosia sublobata (Schumach.) Meikle seeds and its activity against gut proteases of Achaea janata. Phytochemistry (TSI), 151. pp. 78-90. ISSN 00319422

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Abstract

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 ± 11 nM) and chymotrypsin (Ki of 446 ± 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.

Item Type: Article
Divisions: Research Program : Genetic Gains
CRP: UNSPECIFIED
Uncontrolled Keywords: Achaea janata; Bowman-Birk inhibitor; E. coli; Helicoverpa armigera; Trypsin-like gut proteases
Subjects: Others > Helicoverpa
Depositing User: Mr Ramesh K
Date Deposited: 25 Jun 2018 10:28
Last Modified: 24 Oct 2018 10:10
URI: http://oar.icrisat.org/id/eprint/10771
Official URL: http://dx.doi.org/10.1016/j.phytochem.2018.02.009
Projects: UNSPECIFIED
Funders: UNSPECIFIED
Acknowledgement: This work was supported by grants from the Department of Biotechnology (DBT), New Delhi, India (Ref. BT/PR 13261/AGR/05/ 489/2009/(letter I) dt. 21/12/2010) to KPS (PI) and ADG (Co-PI). SSM is thankful to UoH and DBT for JRF and SRF. We also thank Ms. Monika Kannan and Proteomics facility, SLS, UoH for help in MALDI-TOF experiments. The authors declare no conflict of interests.
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