Biotechnology Letters

, Volume 37, Issue 4, pp 863–869 | Cite as

Isolation, structure modeling and function characterization of a trypsin inhibitor from Cassia obtusifolia

  • Zubi Liu
  • Qiankun Zhu
  • Juanjuan Li
  • Gan Zhang
  • Aerguli Jiamahate
  • Jiayu ZhouEmail author
  • Hai LiaoEmail author
Original Research Paper


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.


Cassia 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.

Supporting information

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.

Supplementary material

10529_2014_1744_MOESM1_ESM.doc (1.1 mb)
Supplementary material 1 (DOC 1076 kb)


  1. Bhattacharjee N, Banerjee S, Dutta SK (2014) Cloning, expression and mutational studies of a trypsin inhibitor that retains activity even after cyanogen bromide digestion. Protein Expr Purif 96:26–31CrossRefPubMedGoogle Scholar
  2. Callis J (1995) Regulation of protein degradation. Plant Cell 7:845–857CrossRefPubMedCentralPubMedGoogle Scholar
  3. De Meester P, Brick P, Lloyd LF, Blow DM, Onesti S (1998) Structure of the Kunitz-type soybean trypsin inhibitor (STI): implication for the interactions between members of the STI family and tissue-plasminogen activator. Acta Crystallogr D Biol Crystallogr 54:589–597CrossRefPubMedGoogle Scholar
  4. Dreon MS, Ituarte S, Heras H (2010) The role of the proteinase inhibitor ovorubin in apple snail eggs resembles plant embryo defense against predation. PLoS One 5:e15059CrossRefPubMedCentralPubMedGoogle Scholar
  5. Erlanger BF, Kokowsky N, Cohen W (1961) The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 95:271–280CrossRefPubMedGoogle Scholar
  6. Kim SJ, Kim KW, Kim DS, Kim MC, Jeon YD, Kim SG, Jung HJ, Jang HJ, Lee BC, Chung WS, Hong SH, Chung SH, Um JY (2011) The protective effect of Cassia obtusifolia on DSS-induced colitis. Am J Chin Med 39:565–577CrossRefPubMedGoogle Scholar
  7. Liao H, Ren W, Kang Z, Jiang JH, Zhao XJ, Du LF (2007) A trypsin inhibitor from Cassia obtusifolia seeds: isolation, characterization and activity against Pieris rapae. Biotechnol Lett 29:653–658CrossRefPubMedGoogle Scholar
  8. Liu Z, Song T, Zhu Q, Wang W, Zhou J, Liao H (2014) De novo assembly and analysis of Cassia obtusifolia seed transcriptome to identify genes involved in the biosynthesis of active metabolites. Biosci Biotechnol Biochem 78:791–799CrossRefPubMedGoogle Scholar
  9. Nixon A, Wood CR (2006) Engineered protein inhibitors of proteases. Curr Opin Drug Discov Dev 9:261–268Google Scholar
  10. 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
  11. Rawlings ND, Tolle DP, Barrett AJ (2004) Evolutionary families of peptidase inhibitors. Biochem J 378:705–716CrossRefPubMedCentralPubMedGoogle Scholar
  12. Sali A (1995) Comparative protein modeling by satisfaction of spatial restraints. Mol Med Today 1:270–277CrossRefPubMedGoogle Scholar
  13. Sob SVT, Wabo HK, Tchinda AT, Tane P, Ngadjui BT, Ye Y (2010) Anthraquinones, sterols, triterpenoids and xanthones from Cassia obtusifolia. Biochem Syst Ecol 38:342–345CrossRefGoogle Scholar
  14. Srinivasan T, Kumar KR, Kirti PB (2009) Constitutive expression of a trypsin protease inhibitor confers multiple stress tolerance in transgenic tobacco. Plant Cell Physiol 50:541–553CrossRefPubMedGoogle Scholar
  15. Zhou D, Lobo YA, Batista IF, Marques-Porto R, Gustchina A, Oliva ML, Wlodawer A (2013) Crystal structures of a plant trypsin inhibitor from Enterolobium contortisiliquum (EcTI) and of its complex with bovine trypsin. PLoS One 8:e62252CrossRefPubMedCentralPubMedGoogle Scholar
  16. Zhu Q, Zou J, Zhu M, Liu Z, Feng P, Fan G, Wang W, Liao H (2014) In silico analysis on structure and DNA binding mode of AtNAC1, a NAC transcription factor from Arabidopsis thaliana. J Mol Model 20:2117CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  1. 1.School of Life Science and EngineeringSouthwest Jiaotong UniversityChengduChina
  2. 2.College of Life SciencesZhejiang UniversityHangzhouChina

Personalised recommendations