Total synthesis and expression in Escherichia coli of a gene encoding human 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 constructed for expression in Escherichia coli. To this end, a model of codon adjustment was tested which better suits the polypeptide biosynthetic 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 large synthetic TEL gene was expressed at a high level as the recombinant counterpart of human TEL and as a C-terminal fusion with glutathione S-transferase. This demonstrates that a synthetic approach based upon matching codon usage to that of the host organism can support significant expression of recombinant sequences. The synthetic gene incorporates 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 easily purified and displays properties expected for this protein.
Martin
S L
SL
Department of Biochemistry, University of Sydney, NSW, Australia.
Vrhovski
B
B
Weiss
A S
AS
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon; D004274:DNA, Recombinant / Q000138:chemical synthesis*; D004926:Escherichia coli; D015870:Gene Expression; D005813:Genes, Synthetic; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014333:Tropoelastin / Q000235:genetics*
D003062:Codon; D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D014333:Tropoelastin; D005982:Glutathione Transferase