Gene cloning, expression and purification of human mitochondrial transcription factor TFB1M

Abstract

Transcription from human mitochondrial DNA promoter has been recently reconstituted in vitro with three recombinant proteins: mitochondrial transcription factor A (TFAM), mitochondrial transcription factor B1, B2 (TFB1M, TFB2M) and the mitochondrial RNA polymerase (POLRMT). TFB1M is an essential component of the mitochondrial promoter dependent transcription process. Here we cloned the gene encoding TFB1M from human cDNA libraries and ligated it into the expression vector. The recombinant TFB1M was overexpressed in Escherichia coli and purified to near homogeneity, using metal chelate affinity chromatography on Ni-NTA affinity resin, followed by heparin-agarose chromatography. Expression of POLRMT alone in E.coli was not successful, because most of the protein was insoluble. We tried to stabilize POLRMT by coexpression POLRMT with TFB1M. The recombinant TFAM was purified by Ni-NTA affinity chromatography and phosphocellulose chromatography. Then the basic components of the mitochondrial transcription machinery in human now seem to be in our hands.