Isolation and characterization of recombinant Drosophila Copia aspartic proteinase

フォーマット:

論文

責任表示:

Athauda, Senarath B. P. ; Yoshioka, Katsuji ; Shiba, Tadayoshi ; Takahashi, Kenji

言語:

英語

出版情報:

Portland Press, 2006-11-01

著者名:

• Athauda, Senarath B. P.
• Yoshioka, Katsuji
• Shiba, Tadayoshi
• Takahashi, Kenji

掲載情報:

Biochemical Journal

ISSN:

0264-6021      

巻:

399

通号:

3

開始ページ:

535

終了ページ:

542

バージョン:

author

概要:

金沢大学がん研究所がん分子細胞制御<br />The wild type Copia Gag precursor protein of Drosophila melanogaster expressed in Escherichia coli was shown to be processed autocatalytically to generate two daughter proteins with molecular masses of 33 and 23 … kDa on SDS/PAGE. The active-site motif of aspartic proteinases, Asp-Ser-Gly, was present in the 23 kDa protein corresponding to the C-terminal half of the precursor protein. The coding region of this daughter protein (152 residues) in the copia gag gene was expressed in E. coli to produce the recombinant enzyme protein as inclusion bodies, which was then purified and refolded to create the active enzyme. Using the peptide substrate His-Gly-Ile-Ala-Phe-Met-Val-Lys-Glu-Val-Asn (cleavage site: Phe–Met) designed on the basis of the sequence of the cleavage-site region of the precursor protein, the enzymatic properties of the proteinase were investigated. The optimum pH and temperature of the proteinase toward the synthetic peptide were 4.0 and 70 °C respectively. The proteolytic activity was increased with increasing NaCl concentration in the reaction mixture, the optimum concentration being 2 M. Pepstatin A strongly inhibited the enzyme, with a Ki value of 15 nM at pH 4.0. On the other hand, the active-site residue mutant, in which the putative catalytic aspartic acid residue was mutated to an alanine residue, had no activity. These results show that the Copia proteinase belongs to the family of aspartic proteinases including HIV proteinase. The B-chain of oxidized bovine insulin was hydrolysed at the Leu15-–Tyr16 bond fairly selectively. Thus the recombinant Copia proteinase partially resembles HIV proteinase, but is significantly different from it in certain aspects. 続きを見る

URL:

http://hdl.handle.net/2297/3237