Abstract
Defects in mtDNA replication are the principle cause of severe, heritable metabolic disorders classified as mitochondrial diseases. In vitro analysis of the biochemical mechanisms of mtDNA replication has proven to be a powerful tool for understanding the origins of mitochondrial disease. Mitochondrial single-stranded DNA-binding protein (mtSSB) is an essential component of the mtDNA replication machinery. To facilitate ongoing biochemical studies, a recombinant source of mtSSB is needed to avoid the time and expense of human tissue culture. This chapter focuses on the subcloning, purification, and initial functional validation of the recombinant human mitochondrial single-stranded DNA-binding protein. The cDNA encoding the mature form of the human mtSSB protein was amplified from a HeLa cDNA library, and recombinant human mtSSB was overproduced in Escherichia coli. A procedure was developed to rapidly purify milligram quantities of homogenous, nuclease-free mtSSB that avoids DNA-cellulose chromatography. We show that, similar to E. coli SSB, human mtSSB assembles into a tetramer and binds single-stranded oligonucleotides in a 4-to-1 protein:oligonucleotide molar ratio.