caa a22 4500 39757505X CHVBK 20180308164853.0 cr unu---uuuuu 161202e199603 xx s 000 0 eng 10.1093/hmg/5.3.331 doi (NATIONALLICENCE)oxford-10.1093/hmg/5.3.331 Chaperonin-mediated Assembly of Wild-Type and Mutant Subunits of Human Propionyl-CoA Carboxylase Expressed in Escherichia Coli [Elektronische Daten] 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 C1283T (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. © 1996 Oxford University Press Kelson Todd L. Department of Pediatrics, Box C-233, University of Colorado School of Medicine, 4200 E. 9th Avenue, Denver, CO 80262, USA Ohura Toshihiro Department of Pediatrics, Tohoku University School of Medicine, Sendai, Japan Kraus Jan P. Department of Pediatrics, Box C-233, University of Colorado School of Medicine, 4200 E. 9th Avenue, Denver, CO 80262, USA Human Molecular Genetics Oxford University Press 5/3(1996-03), 331-337 0964-6906 5:3<331 1996 5 hmg https://doi.org/10.1093/hmg/5.3.331 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/hmg/5.3.331 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kelson Todd L. Department of Pediatrics, Box C-233, University of Colorado School of Medicine, 4200 E. 9th Avenue, Denver, CO 80262, USA NATIONALLICENCE 700 1- Ohura Toshihiro Department of Pediatrics, Tohoku University School of Medicine, Sendai, Japan NATIONALLICENCE 700 1- Kraus Jan P. Department of Pediatrics, Box C-233, University of Colorado School of Medicine, 4200 E. 9th Avenue, Denver, CO 80262, USA NATIONALLICENCE 773 0- Human Molecular Genetics Oxford University Press 5/3(1996-03), 331-337 0964-6906 5:3<331 1996 5 hmg Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford