Propionic acidemia : bacterial expression system for propionyl CoA carboxylase.

我们开发了一个细菌表达系统的人α和β的丙酰辅酶A羧化酶（PCC）的cDNAs。这些cDNA在大肠杆菌中在一个质粒载体上共表达，每个cDNA具有其自身的IPTG诱导型启动子。仅测量到可忽略量的活性PCC。该质粒与过表达大肠杆菌伴侣蛋白groES和groEL的第二质粒载体的共表达导致PCC比活性增加几百倍，达到与在粗制人肝提取物中发现的水平相当的水平。α PCC或β PCC的缺乏导致丙酸血症，这是一种常染色体隐性遗传疾病。我们使用该表达系统来表征先前在五个日本等位基因中描述的错义突变，即β PCC中的C1283T（Thr428Ile）。该细菌表达系统可用于分析alphaPCC或betaPCC突变。我们的研究结果表明，groES和groEL伴侣蛋白的折叠和组装的人PCC异聚体亚基是必不可少的。

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.