Ppsq 21

The fifth instar larvae of S. marmorata were collected in the middle reaches of the Chikuma River in Nagano Prefecture, Japan. The silk glands were dissected from the larvae; the major silk proteins (P3′ fraction) were extracted from the silk glands as previously reported [5, 6] . The proteins were separated by SDS-PAGE and blotted onto a PVDF membrane. The individual bands of Smsp-2, Smsp-3, and Smsp-4 were cut out from the membrane; the N-terminal amino acid sequencing was performed on an automated protein sequencer PPSQ-21 (Shimadzu).

The coding region of mature Contractin A was amplified by PCR using two primers (forward: 5′-AAGGAGATATACATATGTCAGTTATCAATTTTGGCTG-3′ and reverse: 5′-GTTAGCAGCCGGATCCGTGAAGATAACTCGGATTGC-3′), and inserted into a pET-3a vector at NdeI and BamHI restriction sites using the In-Fusion HD Cloning Kit. The plasmid was amplified in E. coli JM109 cells, and the protein was expressed in E. coli BL21(DE3)pLysS cells. Recombinant Contractin A expression was induced with 0.4 mM isopropylthiogalactoside, and the cells were incubated for an additional 18 h at 37°C. Because the recombinant proteins were obtained as inclusion bodies after the induction and disruption of cells, they were solubilized in solubilization buffer (50 mM Tris-HCl pH 8.0, 0.2 M NaCl, 1 mM ethylenediamine tetraacetate [EDTA], 6 M guanidine hydrochloride), and the protein was refolded in the refolding buffer (0.1 M Tris-HCl pH 8.0, 0.8 M L-arginine, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione). Next, the refolded protein was dialyzed against Tris-buffered saline (TBS; 10 mM Tris-HCl pH 7.5, 0.15 M NaCl). Protein concentrations were determined from the molar absorption coefficients at 280 nm calculated from the amino acid compositions of the proteins. The N-terminal amino acid sequence of the expressed protein was determined using a protein sequencer, PPSQ-21 (Shimadzu, Kyoto, Japan).