1998 Fiscal Year Final Research Report Summary
Purification of bioactive proteins by specific elution with antigen peptides
| Project/Area Number |
09650873
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
生物・生体工学
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| Research Institution | KOBE UNIVERSITY |
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Principal Investigator |
KATOH Shigeo Kobe University, Graduate School of Science and Technology, Professor, 自然科学研究科, 教授 (20026272)
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| Project Period (FY) |
1997 – 1998
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| Keywords | Immunoaffinity chromatography / Anti-peptide antibody / Peptide antigen / Specific elution / Inactivation of protein |
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| Research Abstract |
For purification of bioactive proteins produced by use of genetically engineered microorganisms or cells, immunoaffinity chromatography is very effective, However, in immunoaffinity purification, elution of the target proteins from antibody ligands sometimes requires extreme conditions which may cause their denaturation, because of the high affinity. To solve the problem, we studied utilization of anti-peptide antibodies, which were obtained without use of a target protein as antigen. We also tried specific-elution method using an antigen peptide as an eluent from anti -peptide antibodies to avoid denaturation of a target protein during the elution step.
In this work, the feasibility of anti-peptide antibodies as ligands for immunoaffinity purification and the specific elution method were studied by use of anti-peptide antibodies against the C-terminal and N-terminal regions of several bioactive proteins. Anti-peptide antibodies against the C-terminal and N-terminal regions of chimeric alpha-amylase, recombinant CD2 and insulin B-chain were obtained by using peptides corresponding to the C- and N-terminal regions as immunogens. These anti-peptide antibodies adsorbed the native proteins, as well as the antigen peptides. The characteristics of antigen recognition of these anti-peptide antibodies were clarified by use of peptides having replaced amino acids. The proteins were purified from fermentation broth to high purity by use of the anti-peptide antibodies as affinity ligands. These ligands could discriminate the target proteins having different C-terminal regions. The adsorbed proteins were specifically eluted by the eluents containing the antigen peptides under mild pH and/or ionic conditions without denaturation of bioactive proteins. Low concentrations of antigen peptides could effectively elute the proteins.
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