Chaperonin-mediated assembly of wild-type and mutant subunits of human propionyl-CoA carboxylase expressed in Escherichia coli |
| |
Authors: | Kelson TL; Ohura T; Kraus JP |
| |
Affiliation: | Department of Pediatrics, University of Colorado School of Medicine, Denver 80262, USA. |
| |
Abstract: | We developed a bacterial expression system for the human alpha and beta
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 alpha and beta subunits as indicated
by Western blot analysis and the almost complete biotinylation of the alpha
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 alpha
and beta subunits was demonstrated, thereby confirming the assembly of both
subunits into an active enzyme. 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 (Thr428lle) in beta PCC.
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
alpha PCC and beta PCC subunits. This bacterial expression system can be
useful for analysis of either alpha PCC or beta PCC mutations. Our findings
indicated that the groES and groEL chaperonin proteins were essential for
folding and assembly of the human PCC heteromeric subunits.
|
| |
Keywords: | |
本文献已被 Oxford 等数据库收录! |
|