English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Zendy Plus

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Zendy Tools

Zendy Open

We developed a bacterial expression system for the human α and β cDNAs of propionyl-CoA carboxylase (PCC). These cDNAs (less the putative mitochondrial matrix targeting presequences) were co-expressed in  Escherichia coli  on one plasmid vector with each cDNA having its own IPTG-inducible promoter. Only negligible amounts of active PCC were measured despite the presence of both α and β subunits as indicated by Western blot analysis and the almost complete biotinylation of the α subunit. Co-expression of this plasmid with a second plasmid vector over-expressing the  E.coli  chaperonin proteins, groES and groEL, resulted in a several hundred-fold increase in PCC specific activity, to a level comparable with that found in crude human liver extracts. PCC was partially purified on monomeric avidin affinity resin and the presence of both α and β subunits was demonstrated, thereby confirming the assembly of both subunits into an active enzyme. Deficiency of either αPCC or βPCC 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 C 1283 T (Thr428Ile) in βPCC. This mutation, when expressed in  E.coli  under the same conditions as that of wild-type PCC, had null activity, despite the presence of assembled αPCC and βPCC subunits. This bacterial expression system can be useful for analysis of either αPCC or βPCC mutations. Our findings indicated that the groES and groEL chaperonin proteins were essential for folding and assembly of the human PCC heteromeric subunits.

Chaperonin-mediated Assembly of Wild-Type and Mutant Subunits of Human Propionyl-CoA Carboxylase Expressed in Escherichia Coli