Expression, purification, and in vitro characterization of the human outermitochondrial membrane receptor human translocase of the outer mitochondrial membrane 20

In order to investigate the biochemical properties of the mitochondrial out er membrane receptor, hTom20, involved in protein recognition, the cytosoli c domain of this receptor was overexpressed and purified to homogeneity. A four-step purification including the purification of thrombin is described as well as an analysis of the function of the highly purified hTom20 protei n. The receptor was concentrated and the subsequent aggregation behavior wa s investigated in order to understand the function of the single cysteine i n the cytosolic domain as well as the function of the proposed "glutamine f ace" for the structure of the protein. It was found that specific dimerizat ion of the cytosolic domain of hTom20 is necessary in order to prevent aggr egation of the protein. In addition, the cysteine and the glutamine face ar e important for the stability of the protein. We propose that the function of the cysteine is to promote dimerization as found in the absence of dithi othreitol. (C) 1999 Academic Press.