CHAPERONIN-MEDIATED ASSEMBLY OF WILD-TYPE AND MUTANT SUBUNITS OF HUMAN PROPIONYL-COA CARBOXYLASE EXPRESSED IN ESCHERICHIA-COLI

We developed a bacterial expression system for the human alpha and bet a cDNAs of propionyl-eoA carboxylase (PCC), These cDNAs (less the puta tive 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 me asured despite the presence of both alpha and beta subunits as indicat ed by Western blot analysis and the almost complete biotinylation of t he cc subunit, Co-expression of this plasmid with a second plasmid vec tor 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, P GC was partially purified on monomeric avidin affinity resin and the p resence of both alpha and beta subunits was demonstrated, thereby conf irming the assembly of both subunits into an active enzyme, Deficiency of either alpha PCC or beta PCC results in propionic acidemia, an aut osomal recessive disorder, We used this expression system to character ize one missense mutation previously described in five Japanese allele s, namely C1283T (Thr42811e) in beta PCC, This mutation, when expresse d in E. coli under the same conditions as that of wild-type PCC, had n ull 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 tha t the groES and groEL chaperonin proteins were essential for folding a nd assembly of the human PCC heteromeric subunits.