Characteristics of antibodies recognizing denatured proteins and application to separation
| Project/Area Number |
06650912
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
生物・生体工学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
KATOH Shigeo Kyoto Univ., Grad.Eng., Associ.Prof., 工学研究科, 助教授 (20026272)
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| Co-Investigator(Kenkyū-buntansha) |
TERASHIMA Masaaki Kyoto Univ., Grad.Eng., Instructor, 工学研究科, 助手 (30172092)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1994: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Anti-peptide antibody / Denatured protein / Immunoaffinity chromatography / Separation of protein / Refolding |
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| Research Abstract |
To purify weakly denatured proteins by immunoaffinity chromatography, the adsorption characteristics of antibodies against peptides consisting a part of target protein were studied. Anti-peptide antibodies are considered to have higher affinity to proteins with high mobility and to be suitable for purification of denatured proteins. The results obtained in this work are as follows ;
1. Anti-peptide antibodies against the C-terminal peptides of myoglobin, insulin and rice alpha-amylase were prepared by immunizing the C-terminal peptides to rabbits, and adsorption characteristics of these antibodies against native and denatured proteins, as well as the peptides, were measured. While native myoglobin was slightly adsorbed by the antibody against the C-terminal peptide of myoglobin, denatured myoglobin in 3M urea was adsorbed with high affinity. On the other hand, native insulin and alpha-amylase, as well as rheir denatured states, were adsorbed by the anti-peptide antibodies against the C-terminal peptides of these proteins. These results show that anti-peptide antibodies can be used for immunoaffinity purification of denatured proteins in weak denaturants such as 3M urea.
2. Refolding conditions and the performance of a continuous refolding apparatus were studied by use of lysozyme. Loose folding of completely denatured lysozyme under 3M urea gave high refolding yields. Continuous refolding was performed by the apparatus.
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Report
(3 results)
Research Products
(3 results)
