Synthetic spider dragline silk proteins and their production in Escherichia coli.
Synthetic genes were designed to encode analogs of the two proteins of Nephila clavipes dragline silk, spidroins 1 and 2. The genes were constructed of tandem repeats of relatively long (more than 300 bp) DNA sequences assembled from synthetic oligonucleotides, and encoded proteins of high molecular mass (65-163 kDa). Both analogs were produced efficiently in Escherichia coli. The yield and homogeneity of the products of longer genes were limited by premature termination of synthesis, probably as a result of processivity errors in protein synthesis. Average termination rates were determined to be 1 in 1100 codons to 1 in 300 codons, depending on the length and synonymous codon choices of the gene. Both analog proteins could be induced to form stable aqueous solutions without denaturants. Circular dichroism spectra of the purified proteins in dilute solution resembled spectra of redissolved natural dragline silk in reflecting a largely disordered structure in water and more ordered structures in mixed solvents with methanol and trifluoroethanol.
Fahnestock
S R
SR
Central Science and Engineering Laboratories, E.I. DuPont de Nemours & Co., Wilmington, DE 19880-0328, USA. fahnessr@esvax.dnet.dupont.com
Irwin
S L
SL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005349:Fibroins; D015870:Gene Expression; D005813:Genes, Synthetic; D005822:Genetic Vectors; D019476:Insect Proteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D047011:Silk; D012995:Solubility; D013112:Spiders / Q000737:chemistry* / Q000235:genetics
D019476:Insect Proteins; D011506:Proteins; D011994:Recombinant Proteins; D047011:Silk; C086087:spidroin 1; C076815:spidroin 2; D005349:Fibroins