Expression in Pichia pastoris and characterization of APETx2, a specific inhibitor of acid sensing ion channel 3

被引:16
作者
Anangi, Raveendra [1 ]
Chen, Chih-Cheng [2 ]
Lin, Yi-Wen [2 ]
Cheng, Yuan-Ren [2 ,3 ]
Cheng, Chun-Ho [1 ]
Chen, Yi-Chun [1 ]
Chu, Yuan-Ping [1 ]
Chuang, Woei-Jer [1 ]
机构
[1] Natl Cheng Kung Univ, Coll Med, Dept Biochem & Mol Biol, Tainan 701, Taiwan
[2] Acad Sinica, Inst Biomed Sci, Taipei, Taiwan
[3] Natl Taiwan Univ, Inst Zool, Taipei 10764, Taiwan
关键词
ASIC3; APETx2; Disulfide bond; Folding; NMR; Pichia pastoris; GATED NA+ CHANNELS; SENSORY NEURONS; PEPTIDE INHIBITOR; POTASSIUM CHANNEL; ASIC3; PROTEINS; TOXIN; CURRENTS; VENOM; ISCHEMIA;
D O I
10.1016/j.toxicon.2010.08.004
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
Acid sensing ion channels (ASICs) are family of proteins predominantly present in the central and peripheral nervous system. They are known to play important roles in the pathophysiology of pain and ischemic stroke. APETx2 is a potent and selective inhibitor of ASIC3-containing channels and was isolated from sea anemone Anthopleura elegantissima. To facilitate the study on the molecular determinants of ASIC3-ligand interactions, we expressed recombinant APETx2 in the Pichia pastoris (P. pastoris) expression system and purified it to homogeneity. Recombinant APETx2 produced in P pastoris inhibited the acid-evoked ASIC3 current with the IC50 value of 37.3 nM. The potency of recombinant toxin is similar to that of native APETx2. The sequential assignment and structure analysis of APETx2 were obtained by 2D and 3D N-15-edited NMR spectra. Our NMR data suggests that APETx2 produced in P. pastoris retained its native fold. The results presented here provide the first direct evidence that highly disulfide bonded peptide inhibitor of ASIC3, APETx2, can be expressed in P. pastoris with correct fold and high yield. We also showed that the R17A mutant exhibited a decrease in activity, suggesting the feasibility of the use of this expression system to study the interactions between APETx2 and ASIC3. These evidences may serve as the basis for understanding the selectivity and activity of APETx2. (C) 2010 Elsevier Ltd. All rights reserved.
引用
收藏
页码:1388 / 1397
页数:10
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