A large fragment approach to DNA synthesis: total synthesis of a gene for the protease inhibitor eglin c from the leech Hirudo medicinalis and its expression in E. coli.
A DNA containing the coding sequence for the proteinase inhibitor protein, eglin c, from the leech Hirudo medicinalis has been obtained by enzymatic assembly of chemically synthesized DNA fragments. The synthetic gene consists of a 232 base-pair fragment containing initiation and termination codon signals with restriction enzyme recognition sites conveniently placed for cloning into a plasmid vector. Only six oligonucleotides from 34 to 61 bases in length, sharing pairwise stretches of complementary regions at their 3'-termini, were prepared by phosphotriester solid-phase synthesis. The oligomers were annealed pairwise and converted into double stranded DNA fragments by DNA polymerase I mediated repair synthesis. The fragments were assembled by ligation, and the synthetic gene was expressed in high yield in E. coli under the transcriptional control of the E. coli tryptophan promoter. The expression product was purified to homogeneity and was shown to have similar physicochemical and identical biological properties as the authentic protein isolated from the leech.
D000595:Amino Acid Sequence; D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000138:chemical synthesis*; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D007202:Indicators and Reagents; D007865:Leeches / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis*; D009841:Oligonucleotides / Q000138:chemical synthesis*; D010957:Plasmids; D011480:Protease Inhibitors / Q000235:genetics*; D011506:Proteins / Q000235:genetics*; D015843:Serpins
D004274:DNA, Recombinant; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D011480:Protease Inhibitors; D011506:Proteins; D015843:Serpins; C028656:eglin proteinase inhibitors; D004247:DNA