Propionic acidemia : bacterial expression system for propionyl CoA carboxylase.

我们建立了一个人丙酰辅酶A羧基酶(PCC)的α和β基因的细菌表达系统。这些cDNAs在同一个载体上的大肠杆菌中共表达，每个cDNAs都有自己的IPTG诱导启动子。只测量了可以忽略不计的活跃PCC。将该质粒与第二个过表达E.Coli伴侣蛋白GroES和GroEL的质粒载体共表达，导致PCC比活性增加数百倍，达到与人肝粗提物中发现的水平相当的水平。缺乏α-PCC或β-PCC都会导致丙酸血症，这是一种常染色体隐性遗传性疾病。我们使用这个表达系统来鉴定之前在5个日本等位基因中描述的一个错义突变，即BetaPC中的C1283T(Thr428Ile)。该突变在大肠杆菌中表达时，在与野生型PCC相同的条件下，没有活性。该细菌表达系统可用于α-PCC或β-PCC突变的分析。我们的发现表明，Groes和GroEL伴侣蛋白对于人PCC异构体亚基的折叠和组装是必不可少的。

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.