cDNA Cloning of Human Mitochondrial Transcription Factor 1 with Polymerase Chain Reaction and Overexpression of the Factor in Escherichia coli

概要

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A human cDNA encoding mitochondrial transcription factor 1 (mtTF1) was isolated by screening a HeLa cell cDNA library by using polymerase chain reaction. In the present experiment, mtTF1 cDNA clone could be obtained more rapidly, easily and at a low cost as compared with conventional cloning techniques. The size of the cDNA fragent was approximately 2 kb, indicating that the cDNA is almost full length. For the mass production of mtTF1 as a fused protein, the cDNA fragment was inserted into an expression vector pUC19. The recombinant plasmid encodes the mature mtTF1 protein and extra 20 amino acids at its amino terminus derived from Lac Z' in the vector and signal peptide of mtTF1. The fused protein of 26 kDa was overproduced in Escherichia coli, and had the same characteristics of DNA binding as natural mtTF1.