The mammalian muskelin/RanBP9/CTLH complex and the S. cerevisiae GID complex are large, multi-protein complexes that each contain a RING E3 ubiquitin ligase. The yeast GID complex acts to degrade a key enzyme of gluconeogenesis, fructose 1,6-bisphosphatase, under conditions of abundant fermentable carbon sources. However, the assembly and functions of the mammalian complex remain poorly understood. A striking feature of these complexes is the presence of multiple proteins that contain contiguous lissencephaly-1 homology (LisH), C-terminal to LisH (CTLH) and C-terminal CT11-RanBP9 (CRA) domains. TWA1/Gid8, the smallest constituent protein of these complexes, consists only of LisH, CTLH and CRA domains and is highly conserved in eukaryotes. Towards better knowledge of the role of TWA1 in these multi-protein complexes, we established a method for bacterial expression and purification of mouse TWA1 that yields tag-free, recombinant TWA1 in quantities suitable for biophysical and biochemical studies. Circular dichroism spectroscopy of recombinant TWA1 indicated a predominantly a-helical protein. Gel filtration chromatography, size exclusion chromatography with multi-angle light scattering and native polyacrylamide gel electrophoresis demonstrated a propensity of untagged TWA1 to form stable dimers and, to a lesser extent, higher order oligomers. TWA1 has a single cysteine residue, C139, yet the dimeric form was preserved when TWA1 was purified in the presence of the reducing agent tris(2-carboxyethyl)phosphine. These findings have implications for understanding the molecular role of TWA1 in the yeast GID complex and related multi-protein E3 ubiquitin ligases identified in other eukaryotes.
- protein purification
- ©2016 The Author(s)
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