Expression in Pichia pastoris and characterization of echistatin, an RGD-containing short disintegrin

Yi Chun Chen, Chun Ho Cheng, Jia Hau Shiu, Yao Tsung Chang, Yung Sheng Chang, Chun Hau Huang, Jenq Chang Lee, Woei Jer Chuang

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6 Citations (Scopus)

Abstract

Echistatin (Ech) is a potent inhibitor of integrins and was isolated from snake Echis carinatus. To facilitate the study on the molecular determinants of integrin-ligand interactions, we expressed recombinant Ech and its mutants in the Pichia pastoris (P. pastoris) expression system and purified them to homogeneity with the yields of 2-7 mg/L. Ech produced in P. pastoris inhibited platelet aggregation with the IC50 value of 210.5 nM. The sequential assignment and structure analysis of recombinant Ech were obtained by 2D and 3D 15N-edited NMR spectra. These data suggests that Ech produced in P. pastoris retained its function and native fold. The results presented here provide the evidences that four disulfide-bonded peptide inhibitor of integrin, Ech, can be expressed in P. pastoris with correct fold and high yield. Platelet aggregation analysis of Ech mutants showed that the length of C-terminus and the K45 residue of Ech are important for interacting with integrin αIIbβ3. We also found that recombinant Ech can inhibit the migration of human A375 melanoma cell. These findings may serve as the basis for understanding the activities of Ech.

Original languageEnglish
Pages (from-to)1342-1348
Number of pages7
JournalToxicon
Volume60
Issue number8
DOIs
Publication statusPublished - 2012 Dec 5

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All Science Journal Classification (ASJC) codes

  • Toxicology

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