Isolation, structure modeling and function characterization of a trypsin inhibitor from Cassia obtusifolia
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A trypsin inhibitor gene (CoTI1) from Cassia obtusifolia was isolated and the deduced amino acid sequence was attributed to the Kunitz-type trypsin inhibitor. The recombined CoTI1, expressed in E. coli, exhibited strong inhibitory effect on bovine trypsin and trypsin-like proteases from Helicoverpa armigera, Spodoptera exigua, and Spodoptera litura. CoTI1 thus presents insecticidal properties that may be useful for the genetic engineering of plants. Leu84, Arg86 and Thr88 were predicted as three key residues by molecular modeling in which Arg86, inserted into the substrate pocket of trypsin, interacted directly with residue Asp189 of trypsin causing the specific inhibition against trypsin. The predicted results were confirmed by site-directed mutagenesis with L84A, R86A and T88A, respectively. The substantial changing expression level of CoTI1 under salt, drought and abscisic acid treatment suggested that CoTI1 might play important role in the resistance against abiotic stress.
KeywordsCassia obtusifolia Insectcidal trypsin inhibitor Lepidopterous pests Molecular cloning Molecular modeling Trypsin inhibitor Trypsin-like proteases
This work was supported by the Grant (no. 31371232) from National Natural Science Foundation of China and Grant (no. SWJTU11CX114) from Fundamental Research Funds for the Central Universities of China.
Supplementary Table 1 Primers used in this paper.
Supplementary Figure 1 Alignment of genomic DNA, cDNA and the translated polypeptide of CoTI1. (A) Alignment of genomic DNA and cDNA of COTI1. (B) Translated polypeptide from cDNA.
Supplementary Figure 2 The expression and purification of recombinant CoTI1. “M” indicated protein maker. Lanes 1–3 indicated collection solution of lysate, penetration and rinse, respectively. Lanes 4–6 indicated the collection solution eluted with 60, 100 and 300 mM imidazole by turns.
Supplementary Figure 3 Sequencing results of CoTI1 and mutants CoTI1R86A, CoTI1L84A and CoTI1T88A.
- Nixon A, Wood CR (2006) Engineered protein inhibitors of proteases. Curr Opin Drug Discov Dev 9:261–268Google Scholar
- Oliveira AS, Migliolo L, Aquino RO, Ribeiro JK, Macedo LL, Bemquerer MP, Santos EA, Kiyota S, de Sales MP (2009) Two Kunitz-type inhibitors with activity against trypsin and papain from Pithecellobium dumosum seeds: purification, characterization, and activity towards pest insect digestive enzyme. Protein Pept Lett 16:1526–1532CrossRefPubMedGoogle Scholar