Premium
Expression in Pichia pastoris and characterization by circular dichroism and NMR of rhodostomin
Author(s) -
Guo ReyTing,
Chou LiJer,
Chen YenChin,
Chen ChiuYueh,
Pari Koteppa,
Jen Chauying J.,
Lo Szecheng J.,
Huang ShouLing,
Lee ChunYann,
Chang TsaiWang,
Chaung WoeiJer
Publication year - 2001
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.1061
Subject(s) - disintegrin , pichia pastoris , circular dichroism , chemistry , recombinant dna , stereochemistry , folding (dsp implementation) , protein secondary structure , biochemistry , biophysics , biology , metalloproteinase , enzyme , gene , engineering , electrical engineering
Rhodostomin (Rho) is a snake venom protein isolated from Calloselasma rhodostoma . Rho is a disintegrin that inhibits platelet aggregation by blocking the binding of fibrinogen to the integrin α IIb β 3 of platelets. Rho produced in Escherichia coli inhibited platelet aggregation with a K I value of 263 nM. Although functional, Rho produced in E. coli is misfolded based on our 2D and 3D NMR studies. In order to correct the folding problem, Rho was expressed in Pichia pastoris . The recombinant Rho expressed in P. pastoris inhibited platelet aggregation with a resulting K I value of 70 nM. This is the same potency as that of native Rho. CD analysis showed that the secondary structures of Rho are pH‐independent and contain 3.5–7.9% α‐helix, 48.2–50.5% β‐structures, and 42.3–47% coil. The sequential assignment and structure analysis of Rho were obtained using 2D and 3D 15 N‐edited NMR spectra. These results provide the first direct evidence that highly disulfide‐bonded disintegrin can be expressed in P. pastoris with the correct fold. This evidence may serve as the basis for exploring the structure and function relationships as well as the dynamics of disintegrin and its variants. Proteins 2001;43:499–508. © 2001 Wiley‐Liss, Inc.