2000 Fiscal Year Final Research Report Summary
A systematic analysis of the effects of fine-structures of antibodies on microbial expression and secretion and construction of a general-purpose system for antibody production.
| Project/Area Number |
09556019
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B).
|
| Allocation Type | Single-year Grants |
|---|
| Section | 展開研究 |
|---|
| Research Field |
応用微生物学・応用生物化学
|
|---|
| Research Institution | Tokyo University of Pharmacy and Life Science |
|---|
Principal Investigator |
YAMAGATA Hideo Tokyo University of Pharmacy and Life Science, School of Life Science, Professor, 生命科学部, 教授 (20023468)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
OHTA Toshihiro Tokyo University of Pharmacy and Life Science, School of Life Science, Associate Professor, 生命科学部, 助教授 (10266893)
IWASA Susumu Takeda Chemical Industries, DDS Research Laboratories, Senior Research Head, DDS研究所, 主席研究員
UDAKA Shigezo Tokyo University of Agriculture, School of Agriculture, Professor, 農学部, 教授 (70023463)
KOJIMA Masaki Tokyo University of Pharmacy and Life Science, School of Life Science, Research Associate, 生命科学部, 助手 (90277252)
|
|---|
| Project Period (FY) |
1997 – 2000
|
| Keywords | Bacillus brevis / Fab / Secretion-vector / Cryptic plasmid / Single-chain antibody |
|---|
| Research Abstract |
(1) Two genes coding for single-chain Fabs containing a linker peptide of 73 and 83 amino acid residues, respectively, were constructed and then expressed in Bacillus brevis. Their products were accumulated in the medium at a level of 200 mg/l.
(2) To construct a new expression-secretion vector for production of antibody fragments by B.brevis, functional analysis of ORFs of pWT481, a cryptic plasmid in B.brevis 481, was carried out. Deletion of an ORF (ORF3) was found to decrease the survival rate of the bacterium at the stationary phase of growth.
(3) Various deletions were introduced into the cDNA encoding CH1 domain. The resultant cDNAs were directly fused to the 5' terminus of the Bacillus licheniformis α-amylase (BLA) gene and then expressed in B.brevis. Amounts of the products of the fused genes accumulated in the medium were determined by Western blot analysis by using an antiserum against BLA.The results suggested that the 4 amino acid residues at the N-terminus of CH1 domain are inhibitory for the expression and/or secretion of CH1 domain.
|
