| Project/Area Number |
11694189
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B).
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Applied molecular and cellular biology
|
|---|
| Research Institution | HOKKAIDO UNIVERSITY |
|---|
Principal Investigator |
SHIMAZAKI Kei-ichi Hokkaido Univ.Grad.School of Agr., Prof., 大学院・農学研究科, 教授 (10091547)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
TANAKA Tetsuya Hokkaido Univ.Grad.School of Agr., Res.Inst., 大学院・農学研究科, 助手 (00322842)
KUMURA Haruto Hokkaido Univ.Grad.School of Agr., Asso.Prof., 大学院・農学研究科, 助教授 (00241365)
NAKAMURA Shingo Hirosaki Univ.Faculty of Agr.& Life Sci., Prof., 農学生命科学部, 教授 (50003570)
|
|---|
| Project Period (FY) |
1999 – 2000
|
| Project Status |
Completed (Fiscal Year 2000)
|
| Budget Amount *help |
¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 2000: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1999: ¥2,700,000 (Direct Cost: ¥2,700,000)
|
|---|
| Keywords | lactoperoxidase / heme-binding / anti-microbial activity / recombinant protein / milk protein / secondary structure / 二次講造 |
|---|
| Research Abstract |
The cDNA encoding bovine lactoperoxidase has been expressed in CHO cells. The recombinant lactoperoxidase was secreted as an enzymatically active single chain molecule presenting two immunoreactive forms of 88 kDa and 82 kDa, differing by their glycosylation. recombinant lactoperoxidase exhibited the characteristic absorbance spectrum with a Soret peak at 413 nm. Biochemical properties of bovine lactoperoxidase isolated from milk and recombinant bovine lactoperoxidase expressed by CHO cells were compared. The natural and recombinant lactoperoxidases showed the same conformational features as determined by CD measurements. The α-helix, β-structure and unordered structure content was found to be 17.8%, 54.2% and 28.0% for the natural lactoperoxidase and 18.6%, 50.1% and 31.3% for the recombinant lactoperoxidase, respectively. The microenvironments of aromatic amino acid residues in both lactoperoxidases seemed to be the same, although the CD spectral band due to the Soret band differed slightly. A difference in the pH-dependent spectral changes of absorbance at 413 nm was observed. From a pepsin hydrolysate of lactoperoxidase, a heme-binding peptide was isolated by reverse-phase HPLC and its amino acid sequence was examined. Engineering of recombinant lactoperoxidase into a myeloperoxidase-like molecule was attempted by substituting Gln-376 by Met, a residue known to achieve covalent binding with the heme in myeloperoxidase. However, the resulting bovine lactoperoxidase mutant failed to acquire the peculiar absorbance spectrum and the chlorinating activity of myeloperoxidase, underlining the complex nature of interactions in the heme vicinity.
|
