TOTAL SYNTHESIS AND EXPRESSION IN ESCHERICHIA-COLI OF A GENE ENCODINGHUMAN TROPOELASTIN

To elucidate the structural features and interactions of tropoelastin (TEL) molecules which assist in giving the elastic fibre its physical properties, a 2210-bp synthetic human TEL-encoding gene (SHEL) was con structed for expression in Escherichia coli. To this end, a model of c odon adjustment was tested which better suits the polypeptide biosynth etic needs of E. coli than the human sequence, where over one-third of this natural sequence contains expression-limiting rare codons and 4 amino acids alone account for 75% of the resulting polypeptide. This l arge synthetic TEL gene was expressed at a high level as the recombina nt counterpart of human TEL and as a C-terminal fusion with glutathion e S-transferase. This demonstrates that a synthetic approach based upo n matching codon usage to that of the host organism can support signif icant expression of recombinant sequences. The synthetic gene incorpor ates the facility for simple cassette replacement in future insertion, deletion and mutagenesis experiments, including the introduction and removal of exon homologues. The resulting soluble polypeptide is easil y purified and displays properties expected for this protein.