徳島大学 教育・研究者情報データベース(EDB)

1. 青山学院大学.理工学部
2. 東京都立大学
3. 昭和大学

1. (英) Kaji Hiroyuki

   役割	任意	共著
   貢献度	任意
   学籍番号	推奨

2. (英) Yada-Wakatabe Rumi

   役割	任意	共著
   貢献度	任意
   学籍番号	推奨

3. (英) Uehira Takashi

   役割	任意	共著
   貢献度	任意
   学籍番号	推奨

4. (英) Terai Mayumi

   役割	任意	共著
   貢献度	任意
   学籍番号	推奨

5. (英) Takeda Atsushi

   役割	任意	共著
   貢献度	任意
   学籍番号	推奨

6. 佐藤 高則

   役割	任意	共著
   貢献度	任意
   学籍番号	推奨

7. (英) Samejima Tatsuya

   役割	任意	共著
   貢献度	任意
   学籍番号	推奨

(英) Molecular cloning, Enhancement of Expression Efficiency and Site-Directed Mutagenesis of Rat Epidermal Cystatin A.

(英) A rat cystatin A cDNA clone was isolated from a lambda ZAP library representing newborn rat skin mRNA by screening with a synthetic oligonucleotide designed from amino acid sequence 15-23 of the cysteine proteinase inhibitor. The obtained clone contained a partial coding region of the inhibitor, lacking the 5'-untranslated region and coding sequence for the NH(2)-terminal 13 residues. The amino acid sequence deduced from the base sequence, Glu14-Phe103, coincided with that determined at the amino acid level. To obtain the recombinant cystatin A protein, the DNA was fused with a synthetic linker encoding its missing N-terminal 17 residues and introduced into an expression vector, pMK2. In Escherichia coli, however, the expression level of the semi-synthetic gene was low, 0. 5 mg of the purified recombinant protein per 1 liter culture being produced. Changing of the codon usage of the N-terminal region in a pET-15b expression system led to an increase in the yield depending on the instability of the putative secondary structure around an initiation codon of the mRNA. The expressed cystatin A showed identical characteristics with the authentic form except for the absence of the N-terminal acetyl blocking group. Using the expression system, two kinds of point mutation, the conservative Val54 in the first loop QxVxG region being changed to Lys and Glu, were introduced, but there was almost no effect on the inhibitory activity toward papain. This suggests that the conserved Val in the reactive site is not restricted and that the hydrophobicity of the position is not essential for the activity of rat cystatin A.

(日) Rat由来システインプロテアーゼ・インヒビター(Cyatatin α) cDNAを調製し，大腸菌における発現を試みた．その結果発現量が少量であったため，遺伝子上流に存在するmRNAの二次構造を推定し，この二次構造を不安定化させる変異を導入した．その結果，大腸菌による大量発現が可能となった．さらに，ファミリーにおいて保存されているVal54について，阻害活性への関与を検討した．

1. (英) Amino Acid Sequence
2. (英) Animals
3. (英) Animals, Newborn
4. (英) Base Sequence
5. (英) Binding Sites
6. (英) Cloning, Molecular
7. (英) Cystatins
8. (英) DNA Primers
9. (英) DNA, Complementary
10. 大腸菌(Escherichia coli)
11. 女性(female)
12. 遺伝子発現(gene expression)
13. (英) Molecular Sequence Data
14. (英) Mutagenesis, Site-Directed
15. (英) Nucleic Acid Conformation
16. (英) RNA, Messenger
17. (英) Rats
18. (英) Rats, Sprague-Dawley
19. (英) Recombinant Proteins
20. 皮膚(skin)

1. (英) Article.PublicationTypeList.PublicationType: Journal Article

2. (英) Article.PublicationTypeList.PublicationType: Research Support, Non-U.S. Gov't