title: Construction, cloning, and expression of synthetic genes encoding spider dragline silk.
abstract: Synthetic genes encoding recombinant spider silk proteins have been constructed, cloned, and expressed. Protein sequences were derived from Nephila clavipes dragline silk proteins and reverse-translated to the corresponding DNA sequences. Codon selection was chosen to maximize expression levels in Escherichia coli. DNA "monomer" sequences were multimerized to encode high molecular weight synthetic spider silks using a "head-to-tail" construction strategy. Multimers were cloned into a prokaryotic expression vector and the encoded silk proteins were expressed in E. coli upon induction with IPTG. Four multimer, ranging in size from 14.7 to 41.3 kDa, were chosen for detailed analysis. These proteins were isolated by immobilized metal affinity chromatography and purified using reverse-phase HPLC. The composition and identity of the purified proteins were confirmed by amino acid composition analysis, N-terminal sequencing, laser desorption mass spectroscopy, and Western analysis using antibodies reactive to native spider dragline silk. Circular dichroism measurements indicate that the synthetic spider silks have substantial beta-sheet structure.
authors.affiliation: Biotechnology Division, U.S. Army Natick Research, Development, & Engineering Center, Natick, Massachusetts 01760-5020, USA.
authors.forename: J T
authors.initials: JT
authors.lastname: Prince
authors.forename: K P
authors.initials: KP
authors.lastname: McGrath
authors.forename: C M
authors.initials: CM
authors.lastname: DiGirolamo
authors.forename: D L
authors.initials: DL
authors.lastname: Kaplan
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D016384:Consensus Sequence / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D019476:Insect Proteins; D007544:Isopropyl Thiogalactoside / Q000235:genetics / Q000494:pharmacology; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics*; D011994:Recombinant Proteins; D012091:Repetitive Sequences, Nucleic Acid; D047011:Silk; D013112:Spiders / Q000235:genetics*
chemical_list: D000596:Amino Acids; D019476:Insect Proteins; D011506:Proteins; D011994:Recombinant Proteins; D047011:Silk; D007544:Isopropyl Thiogalactoside