Characterization of recombinant amyloidogenic chicken cystatin mutant I66Q expressed in yeast.
Amyloidogenic chicken cystatin mutant I66Q (cC I66Q) was successfully secreted by yeasts Pichia pastoris and Saccharomyces cerevisiae. The soluble monomer and dimer forms of amyloidogenic cC I66Q were found in the culture medium, while large amounts of insoluble aggregate and polymeric form cC I66Q besides the monomer and dimer forms were secreted into the culture medium. The amyloidogenic cC I66Q showed a comparable circular dichroism spectrum to that of the wild cystatin, and the monomer form exhibited a similar level of inhibitory activity toward papain, but the dimmer form did not. During storage of amyloidogenic cC I66Q under physiological and acidic conditions, typical binding with Congo red and thioflavin T, and the formation of amyloid fibrils were observed, whereas the characteristic of similar amyloidosis was hardly detected for the wild recombinant cystatin.
He
Jianwei
J
Department of Biological Chemistry, Yamaguchi University, Yamaguchi 753-8515.
Song
Youtao
Y
Ueyama
Nobuhiro
N
Harada
Akihito
A
Azakami
Hiroyuki
H
Kato
Akio
A
D000682:Amyloid / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D002645:Chickens; D003224:Congo Red; D015891:Cystatins / Q000096:biosynthesis* / Q000235:genetics*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D009154:Mutation; D010206:Papain / Q000037:antagonists & inhibitors; D010843:Pichia / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism
D000682:Amyloid; D015891:Cystatins; D011994:Recombinant Proteins; C100158:cystatin, egg-white; D003224:Congo Red; D010206:Papain