Cloning and expression in Escherichia coli of mature E1β subunit of bovine mitochondrial branched-chain α-keto acid dehydrogenase complex: Mapping of the E1β-binding region on E2

A cDNA encoding the mature E1β subunit of the bovine branched-chain α-keto acid dehydrogenase complex was isolated from a λZAP expression library. The bovine E1β cDNA is 1,393 base pairs in length. It encodes the entire mature E1β subunit consisting of 342 amino acid residues and a partial mitochondrial targeting presequence of 26 residues. The calculated molecular mass of the mature bovine E1β subunit is 37,776 daltons, and the calculated isoelectric point is pI 5.04 The mature bovine E1β subunit was expressed in Escherichia coli via the pKK233-2 vector in the presence of isopropyl β-D-thiogalactopyranoside (IPTG). When expression was induced by IPTG at 37 °C, the soluble recombinant E1β subunit existed as a single high molecular weight form (M_r ≃3.5 x 10⁵), which sedimented during sucrose gradient ultracentrifugation at 2 x 10⁵ x g. However, lowering the induction temperature to 25 °C resulted in the occurrence of both high and low molecular weight forms of the recombinant E1β protein. The low molecular weight form (M_r≃9.1 x 10⁴) remained soluble after sucrose gradient centrifugation and was utilized in binding studies with a series of truncated recombinant E2 proteins. The results showed that the E1β subunit bound to the region between Ala-115 and Lys-150 of the E2 chain, which lay within the putative E3-binding domain. In contrast, the recombinant E1α subunit did not bind the E2 component. The data suggest an apparent binding order of E2-E1β-E1α, which supports and extends the model of E2 inner core deduced previously from the data of scanning transmission electron microscopy (Hackert, M. L., Xu, W.-X., Oliver, R. M., Wall, J. S., Hainfeld, J. F., Mullinax, T. R., and Reed, L. J. (1989) Biochemistry 28, 6816-6821). The relatively inaccessible topology of E1β may explain the lack of antigenicity and resistance to limited proteolysis of this subunit as it exists in the complex.