Synthesis of a modified gene encoding human ornithine transcarbamylase for expression in mammalian mitochondrial and universal translation systems: a novel approach towards correction of a genetic defect

• Published in: Gene 1996-03, Vol.169 (2), p.251-255
• Main Authors: Wheeler, Vanessa C., Prodromou, Chrisostomos, Pearl, Laurence H., Williamson, Robert, Coutelle, Charles
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Cell Line; chaperone; Gene Expression; gene transfer; Gene Transfer Techniques; genetic code; Genetic Diseases, Inborn; Humans; Mammals; Mitochondria - metabolism; Molecular Sequence Data; organelle; Ornithine Carbamoyltransferase - genetics; Ornithine Carbamoyltransferase Deficiency Disease; OTC deficiency; Protein Biosynthesis; Recursive PCR; urea cycle
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(95)00812-8

The mitochondrial (mt) genome is a potential means of gene delivery to human cells for therapeutic expression. As a first step towards this, we have synthesised a gene coding for mature human ornithine transcarbamylase (OTC) by recursive PCR using 18 oligodeoxyribonucleotides, each 70–80 nucleotides in length, with codons which should allow translation in accordance with both mammalian mt and universal codon usage. Flanking mt DNA sequences were incorporated which are designed to facilitate site-specific cloning into the mt genome. Expression of this human gene in Escherichia coli leads to an immunoreactive OTC product of the correct size and N-terminal amino-acid sequence, but which forms inclusion bodies and lacks enzymatic activity