© 2005 The Japanese Biochemical Society
Regular Paper |
Characterization of Recombinant Amyloidogenic Chicken Cystatin Mutant I66Q Expressed in Yeast
Department of Biological Chemistry, Yamaguchi University, Yamaguchi 753-8515
To whom correspondence should be addressed: Department of Biological Chemistry, Yamaguchi University, Yamaguchi 753-8515. Tel: +81-83-933-5854, Fax: +81-83-933-5820, E-mail: akiokato{at}yamaguchi-u.ac.jp
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.
* Present address: Laboratory of Biochemistry and Genetics, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, USA.