Expression in Pichia pastoris and characterization by circular dichroism and NMR of rhodostomin.
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 alpha(IIb)beta3 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% alpha-helix, 48.2-50.5% beta-structures, and 42.3-47% coil. The sequential assignment and structure analysis of Rho were obtained using 2D and 3D 15N-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.
Guo
R T
RT
Department of Biochemistry, National Cheng Kung University Medical College, Tainan, Taiwan.
Chou
L J
LJ
Chen
Y C
YC
Chen
C Y
CY
Pari
K
K
Jen
C J
CJ
Lo
S J
SJ
Huang
S L
SL
Lee
C Y
CY
Chang
T W
TW
Chaung
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D009682:Magnetic Resonance Spectroscopy; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010974:Platelet Aggregation / Q000187:drug effects; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid; D012995:Solubility
D004220:Disulfides; D010455:Peptides; D011993:Recombinant Fusion Proteins; C072026:rhodostomin