1995 Fiscal Year Final Research Report Summary
Propionic acidemia : bacterial expression system for propionyl CoA carboxylase.
| Project/Area Number |
06670762
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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 |
Pediatrics
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| Research Institution | TOHOKU UNIVERSITY |
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Principal Investigator |
OHURA Toshihiro TOHOKU UNIVERSITY,SCHOOL OF MEDICINE,DEPARTMENT OF PEDIATRICS,ASSISTANT PROFESSOR, 医学部, 講師 (10176828)
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| Co-Investigator(Kenkyū-buntansha) |
KURE Shigeo TOHOKU UNIVERSITY,SCHOOL OF MEDICINE,DEPARTMENT OF BIOCHEMICAL GENETICS,CLINICAL, 医学部, 助手 (70211191)
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| Project Period (FY) |
1994 – 1995
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| Keywords | Propionic acdemia / Propiony1 CoA Carboxylase / Chaperonine |
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| Research Abstract |
We developed a bacterial expression system for the human alpha and beta cDNAs of propionyl-CoA carboxylase (PCC). These cDNAs were co-expressed in E.coli on one plasmid vector with each cDNA having its own IPTG-inducible promoter. Only negligible amounts of active PCC were measured. Co-expression of this plasmid with a second plasmid vector overexpressing the E.coli chaperonin proteins, groES and groEL,resulted in a several hundred-fold increase in PCC Specific activity, to a level comparable to that found in crude human liver extracts. Deficiency of either alpha PCC or beta PCC results in propionic acidemia, an autosomal recessive disorder. We used this expression system to characterize one missense mutation previously described in five Japanese alleles, namely C1283T (Thr428Ile) in betaPCC.This mutant, when expressed in E.coli under the same conditions as that of wild type PCC,had null activity. This bacterial expression system can be useful for analysis of either alphaPCC or betaPCC mutations. Our findings indicated that the groES and groEL chaperonin proteins were essential for folding and assembly of the human PCC heteromeric subunits.
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