Davie J R, Wynn R M, Cox R P, Chuang D T
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9038.
J Biol Chem. 1992 Aug 15;267(23):16601-6.
We have expressed an active recombinant E1 decarboxylase component of the mammalian branched-chain alpha-ketoacid dehydrogenase complex in Escherichia coli by subcloning mature E1 alpha and E1 beta subunit cDNA sequences into a bacterial expression vector. To permit affinity purification under native conditions, the mature E1 alpha subunit was fused with the affinity ligand E. coli maltose-binding protein (MBP) through an endoprotease Factor Xa-specific linker peptide. When co-expressed, the MBP-E1 alpha fusion and E1 beta subunits were shown to co-purify as a MBP-E1 component that exhibited both E1 activity and binding competence for recombinant branched-chain E2 component. In contrast, in vitro mixing of individually expressed MBP-E1 alpha and E1 beta did not result in assembly or produce E1 activity. Following proteolytic removal of the affinity ligand and linker peptide with Factor Xa, a recombinant E1 species was eluted from a Sephacryl S-300HR sizing column as an enzymatically active 160-kDa species. The latter showed 1:1 subunit stoichiometry, which was consistent with an alpha 2 beta 2 structure. The recovery of this 160-kDa recombinant E1 species (estimated at 0.07% of total lysate protein) was low, with the majority of the recombinant protein lost as insoluble aggregates. Our findings suggest that the concurrent expression of both E1 alpha and E1 beta subunits in the same cellular compartment is important for assembly of both subunits into a functional E1 alpha 2 beta 2 heterotetramer. By using this co-expression system, we also find that the E1 alpha missense mutation (Tyr-393----Asn) characterized in Mennonites with maple syrup urine disease prevents the assembly of soluble E1 heterotetramers.
我们通过将成熟的E1α和E1β亚基cDNA序列亚克隆到细菌表达载体中，在大肠杆菌中表达了哺乳动物支链α-酮酸脱氢酶复合体的活性重组E1脱羧酶组分。为了在天然条件下进行亲和纯化，成熟的E1α亚基通过一种内蛋白酶因子Xa特异性接头肽与亲和配体大肠杆菌麦芽糖结合蛋白（MBP）融合。当共表达时，MBP-E1α融合体和E1β亚基被证明可作为一个MBP-E1组分共同纯化，该组分既表现出E1活性，又对重组支链E2组分具有结合能力。相比之下，单独表达的MBP-E1α和E1β在体外混合不会导致组装，也不会产生E1活性。用因子Xa进行蛋白水解去除亲和配体和接头肽后，一种重组E1形式从Sephacryl S-300HR分子筛柱上洗脱下来，成为一种具有酶活性的160 kDa形式。后者显示出1:1的亚基化学计量比，这与α2β2结构一致。这种160 kDa重组E1形式的回收率很低（估计占总裂解物蛋白的0.07%），大多数重组蛋白以不溶性聚集体的形式丢失。我们的研究结果表明，在同一细胞区室中同时表达E1α和E1β亚基对于将两个亚基组装成功能性的E1α2β2异源四聚体很重要。通过使用这种共表达系统，我们还发现，在患有枫糖尿症的门诺派中发现的E1α错义突变（Tyr-393→Asn）会阻止可溶性E1异源四聚体的组装。
