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

Research output: Contribution to journalArticle

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

Fingerprint

Disintegrins
Pichia
Integrins
Platelets
Platelet Aggregation
Agglomeration
echistatin
Snakes
Disulfides
Inhibitory Concentration 50
Melanoma
Nuclear magnetic resonance
Ligands

All Science Journal Classification (ASJC) codes

  • Toxicology

Cite this

Chen, Yi Chun ; Cheng, Chun Ho ; Shiu, Jia Hau ; Chang, Yao Tsung ; Chang, Yung Sheng ; Huang, Chun Hau ; Lee, Jenq Chang ; Chuang, Woei Jer. / Expression in Pichia pastoris and characterization of echistatin, an RGD-containing short disintegrin. In: Toxicon. 2012 ; Vol. 60, No. 8. pp. 1342-1348.
@article{4b695b7eda3f4ae7b9ef3bc5c3e93560,
title = "Expression in Pichia pastoris and characterization of echistatin, an RGD-containing short disintegrin",
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.",
author = "Chen, {Yi Chun} and Cheng, {Chun Ho} and Shiu, {Jia Hau} and Chang, {Yao Tsung} and Chang, {Yung Sheng} and Huang, {Chun Hau} and Lee, {Jenq Chang} and Chuang, {Woei Jer}",
year = "2012",
month = "12",
day = "5",
doi = "10.1016/j.toxicon.2012.08.009",
language = "English",
volume = "60",
pages = "1342--1348",
journal = "Toxicon",
issn = "0041-0101",
publisher = "Elsevier Limited",
number = "8",

}

Expression in Pichia pastoris and characterization of echistatin, an RGD-containing short disintegrin. / Chen, Yi Chun; Cheng, Chun Ho; Shiu, Jia Hau; Chang, Yao Tsung; Chang, Yung Sheng; Huang, Chun Hau; Lee, Jenq Chang; Chuang, Woei Jer.

In: Toxicon, Vol. 60, No. 8, 05.12.2012, p. 1342-1348.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Expression in Pichia pastoris and characterization of echistatin, an RGD-containing short disintegrin

AU - Chen, Yi Chun

AU - Cheng, Chun Ho

AU - Shiu, Jia Hau

AU - Chang, Yao Tsung

AU - Chang, Yung Sheng

AU - Huang, Chun Hau

AU - Lee, Jenq Chang

AU - Chuang, Woei Jer

PY - 2012/12/5

Y1 - 2012/12/5

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84867307396&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84867307396&partnerID=8YFLogxK

U2 - 10.1016/j.toxicon.2012.08.009

DO - 10.1016/j.toxicon.2012.08.009

M3 - Article

C2 - 22982571

AN - SCOPUS:84867307396

VL - 60

SP - 1342

EP - 1348

JO - Toxicon

JF - Toxicon

SN - 0041-0101

IS - 8

ER -