title: Expression in Pichia pastoris and characterization of APETx2, a specific inhibitor of acid sensing ion channel 3.
abstract: 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 IC(50) 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 (15)N-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.
authors.affiliation: Department of Biochemistry and Molecular Biology, National Cheng Kung University, College of Medicine, Tainan 701, Taiwan.
authors.forename: Raveendra
authors.initials: R
authors.lastname: Anangi
authors.forename: Chih-Cheng
authors.initials: CC
authors.lastname: Chen
authors.forename: Yi-Wen
authors.initials: YW
authors.lastname: Lin
authors.forename: Yuan-Ren
authors.initials: YR
authors.lastname: Cheng
authors.forename: Chun-Ho
authors.initials: CH
authors.lastname: Cheng
authors.forename: Yi-Chun
authors.initials: YC
authors.lastname: Chen
authors.forename: Yuan-Ping
authors.initials: YP
authors.lastname: Chu
authors.forename: Woei-Jer
authors.initials: WJ
authors.lastname: Chuang
mesh_terms: D062565:Acid Sensing Ion Channels; D000818:Animals; D003064:Cnidarian Venoms / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors*; D019906:Nuclear Magnetic Resonance, Biomolecular; D018408:Patch-Clamp Techniques; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020539:Sequence Analysis, Protein; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism
chemical_list: C501880:APETx2 protein, Anthopleura elegantissima; C508378:ASIC3 protein, mouse; D062565:Acid Sensing Ion Channels; D003064:Cnidarian Venoms; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels