Gene cloning, expression, and purification of mitochondrial transcription factor TFB2M in escherichia coli

Abstract

Characterization of the basic transcription machinery of human mitochondrial DNA is of fundamental biological interest and may also lead to therapeutic interventions for human diseases associated with mitochondrial dysfunction. Recently it was reported that two forms of a transcription factor (TFB1M, TFB2M) have been identified as the missing components of the transcription initiation machinery for human mitochondrial DNA. We cloned the genes encoding TFB1M and TFB2M from a human cDNA library by polymerase chain reactions (PCR) and subcloned them into an expression vector. The genes were expressed in Escherichia coli (E.coli) and the proteins were purified by affinity chromatography. These, together with the transcription factor A (TFAM) and mitochondrial RNA polymerase (POLRMT), constitute the complete transcription initiation machinery in human mitochondria. The reconstituted system will be used to study the control of mitochondrial transcription and DNA replication.