The molecular biological and biochemical studies on nitrite reductase and blue copper protein of Achromobacter cycloclastes
| Autor: | Chen Jang-Yi, 陳正繹 |
|---|---|
| Rok vydání: | 1996 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 84 The blue copper protein ( BCP ) gene has been cloned from Achromobacter cycloclastes and characterized. BCP gene encodes a protein of 148 residues with a 24-residue signal peptide. The DNA-derived amino acid sequence of BCP is consistent with its partial N-terminal amino acid sequence. BCP genecontains its own FNR box in the 5'' upstream region and a Pribnow box (TA- rich region ) which could be the transcription start site. Both BCP and NIR ( also cloned from the same species and characterized in our lab. )could be expressed in E. coli. Both of the expressed proteins could be recognized by their respective antisera and the recombinant NIR demonstrated full enzyme activity. The tris- tricine SDS-PAGE has been used to analyze NIR protein. NIR migrated as trimer and monomer in this system while it behaved as dimer in HPLC-gel filtration or tris- gly SDS-PAGE. The copper depleted NIR showed the same pattern with native NIR in HPLC-gel filtration and tricine SDS-PAGE, which means that copper is not necessary in maintaining the quaternary structure of NIR. The dissociation of trimer to monomer is effected by heating, suggesting that the molecular interaction between monomers is heat-labile. The C-terminal truncated NIR genes were constructed and expressed in E.coli M15. The mutated RNIR which have different C-terminaldeletions were analyzed by HPLC-gel filtration and tricine SDS-PAGE. The results show that the deletion in C-terminal would favor the formation NIRmonomer and reduce the absorption spectrum and the enzyme activity. All these phenomena suggest that C-terminal tail from residues 324 to 340 is very important for maintaining the trimer structure and the enzyme acitvity. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
| Externí odkaz: |
|