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High-level expression of Myrothecium verrucaria bilirubin oxidase in Pichia pastoris, and its facile purification and characterization

フォーマット:
論文
責任表示:
片岡, 邦重 ; Kataoka, Kunishige ; Tanaka, Kazuhiro ; Sakai, Yoko ; Sakurai, Takeshi
言語:
英語
出版情報:
Elsevier, 2005-05-01
著者名:
掲載情報:
Protein expression and purification
ISSN:
1046-5928  CiNii Research  Webcat Plus  JAIRO
巻:
41
通号:
1
開始ページ:
77
終了ページ:
83
バージョン:
author
概要:
Bilirubin oxidase (BO) from Myrothecium verrucaria (authentic BO) catalyzing the oxidation of bilirubin to biliverdine was overexpressed in the methylotrophic yeast, Pichia pastoris. The cDNA encoding BO was cloned into the P. pastoris expression vector pPIC9K under the control of the alcohol oxidase 1 promoter and its protein product was secreted using the Saccharomyces cerevisiae α-mating factor signal sequence. The productivity of recombinant BO (rBO) in P. pastoris was approximately 5000 U/L of culture broth, being about 2.5- and 250-fold higher than rBO expressed in Aspergillus oryzae and S. cerevisiae, respectively. The calculated molecular mass of rBO consisting of 538 amino acids was 60,493 kDa, however, that of SDS–PAGE was 66 kDa because of non-native type N-linked sugar chains. The spectroscopic properties of rBO were typical of multicopper oxidase containing four Cu ions per protein molecule. The specific activity to oxidize bilirubin was 57 U/mg, having a value about twice that of authentic BO and rBO expressed in A. oryzae. Moreover, the thermostability of rBO expressed in P. pastoris was significantly high compared to the authentic BO previously reported. Accordingly, a heterologous expression system of rBO to meet clinical and industrial needs was constructed 続きを見る
URL:
http://hdl.handle.net/2297/1732
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