Supplementary methods S1. Purification of the native three native

Supplementary methods S1. Purification of the native three native SPNs from the hemolymph of Tenebrio laevae. The purification flow-chart of Tenebrio SPN40, SPN55 and SPN48 from the hemolymph of T. molitor larvae are shown in supplementary methods Fig. 1. The detailed purification methods are described as follows: S2. Preparation of the eluate using a Toyopearl AF-Heparin HC column. The collected Tenebrio hemolymph (11g of protein in 1,000 ml) was treated with diisopropyl fluorophosphate (DFP, 0.5 mM final concentration) for 50 min at 4 °C to inactivate the serine proteases as previously described (1). Then, DFP-treated hemolymph was dialyzed against buffer A (50 mM Tris-HCl containing 3 mM EDTA, pH 6.0) for 12 h at 4 °C, and then applied to a Toyopearl AF-Heparin HC 650 M column (3 cm x 15 cm) equilibrated with the same buffer. After washing the column with buffer A, adsorbed proteins were eluted with a NaCl gradient (0 to 1.0 M NaCl) in 500 ml of the buffer at a flow rate of 2 ml/min (2 g of protein).The eluate was used for the following purification experiments. S3. Preparation of the flow-through and the eluate using Q-Sepharose FF column. The pooled fractions (2g of protein) were dialyzed against 20 mM Tris containing 3 mM EDTA, pH 8.0. And loaded onto a Q-Sepharose FF column (3.5 x 15 cm) equilibrated with the same buffer. The flow-through fractions and washing solution were collected and then this pooled solution used for purification of SPN55 and SPN40. The adsorbed proteins were eluted with a NaCl gradient (0 to 1 M NaCl) in 400 ml of 20 mM Tris containing 3 mM EDTA, pH 8.0 at a flow rate of 4 ml/min. The collected solution (800mg of protein) as an eluate was used for the purification following purification of SPN48. S4. Purification of Tenebrio SPN55 from flow-through solution. Step I: HiTrap Heparin FPLC column chromatography. The concentrated flow-through solution (500mg of protein) was loaded onto a HiTrap Heparin FPLC column equilibrated with 20 mM Tri-HCl buffer containing 3 mM EDTA, pH 8.0. After washing the column with the same buffer, retained proteins were eluted with a NaCl gradient (0 to 1.0 M) in 400 ml of the buffer at a flow rate of 4 ml/min. Each fraction (100 μg protein) was incubated with activated SAE (0.2μg protein) and then the reaction mixture was analyzed by western blot using polyclonal anti-SAE antibody. The fractions forming SDS-stable complex were collected (supplementary methods Fig. 2) and used for further purification.

1

Step2: HiTrap SP High Pressure FPLC column chromatography. The eluted solution from Step 1 was loaded onto a HiTrap SP HP FPLC column (5 ml) equilibrated with 20 mM sodium citrate buffer containing 3 mM EDTA, pH 5.0. After washing the column with the same buffer, retained proteins were eluted with a NaCl gradient (0 to 1.0 M) in 400 ml of the buffer at a flow rate of 4 ml/min. The fractions eluted at 200 mM NaCl was used for the purification of SPN55, but the fractions eluted at 500 mM NaCl concentration were used for the purification of SPN40 as described below. The fractions capable of making a SDS-stable complex with activated SAE were collected and used for purification of the next column. Step3: Mono-S FPLC column chromatography. The concentrate from Step 2 was loaded onto a Mono-S column (HR5/5, Amersham Pharmacia Biotech) equilibrated at a flow rate of 0.5 ml/min with 20 mM sodium citrate buffer containing 3 mM EDTA, pH 6.0. The column was washed with 2.5 ml of the same buffer, followed by elution with a 25 ml gradient from 0.1 to 1.0 M NaCl in the same buffer. Fractions (1 ml) were collected and 2.5 µl of each fraction were used for examination of SDS-stable complex formation with activated SAE. The fractions containing the pure 55 kDa protein were pooled and used as purified Tenebrio SPN55. To determine the partial amino acid sequences of the purified SPN55, the purified protein (25 μg) was reduced, alkylated, and digested with 2 μg of lysylendopeptidase at 37 °C for 13 h. The digested peptides were separated by HPLC on a C18 reverse phase column (Gilson) with a linear gradient between 0.05 % trifluoroacetic acid (TFA) in water and 0.052 % TFA in 80 % acetonitrile. The amino-terminal amino acid sequence of the purified protein and the internal peptides from HPLC were determined on an Applied Biosystem Procise automated gas-phase amino acid sequencer. The obtained amino acid sequences are as follows: QEVPKPLPSVDDQLVRR (N-terminal sequence of mature protein), TMRVINSWVSK, TVLAGPPAPTTASIFTNAVYFK, NFVQTLKSREILE TIAK, FRIERPFVFFIRHEATSA. S5. Purification of Tenebrio SPN40 from the flow-through solution. Step 1: Hydroxylapatite column chromatography. The pooled solution (10 mg of protein) eluted at 500 mM NaCl from Step 2 during purification of SPN55 was loaded onto a hydroxylapatite FPLC column (5 mm x 50 mm, Bio-Rad) equilibrated in buffer B (20 mM sodium phosphate, pH 7.0). The column was washed with 4 ml of the same buffer, and bound material was eluted with a 25 ml gradient from 20 to 500 mM sodium phosphate in the same buffer. Fractions capable of making SDS-stable complex with activated MSP were pooled (2 mg of protein).

2

Step 2: HiTrap Heparin FPLC column chromatography. The concentrated solution was loaded onto a HiTrap Heparin (1ml) FPLC column equilibrated with 20 mM Tri-HCl buffer containing 3 mM EDTA, pH 8.0. After washing the column with the same buffer, retained proteins were eluted with a NaCl gradient (0 to 1.0 M) in 100 ml of the buffer at a flow rate of 1 ml/min. Fractions (1 ml) were collected and 2.5 µl of each fraction were used for examination of SDS-stable complex formation with activated MSP. The fractions containing the pure 40 kDa protein were pooled and used as purified Tenebrio SPN40. To determine the partial amino acid sequences of the purified SPN40, the purified protein (25 μg) was reduced, alkylated, and digested with 2 μg of lysylendopeptidase at 37 °C for 13 h as described above. The obtained N-terminal and internal amino acid sequences are as follows: SDVSLQEFVSS (N-terminal sequence), TTDGNFLVSPF, TVLLNALYFK, LELPFEGGQASMVVVLPNR, ANHPFIFYIK.

S6. Purification of Tenebrio SPN48 from the eluate solution. Step 1: CM-Toyopearl 650 M column chromatography. The eluate from Q-sepharose FF column (800 mg of protein in 32ml) was dialyzed against buffer A. The dialysate was applied to a CM-Toyopearl 650 M column (3 x 15 cm, Tosho), which was developed with a 0–1 M NaCl gradient. The flow-through fractions (200 mg of protein) capable of making SDS-stable complex with SPN48 were collected as shown supplementary methods Fig. 2. Step 2: HiTrap SP High Pressure FPLC column chromatography. The collected solution (200 mg of protein) from Step 1 was applied to a HiTrap SP High Pressure FPLC column (5 ml) equilibrated with buffer A (50 mM Tris-HCl containing 3 mM EDTA, pH 6.0). After washing the column with the same buffer, retained proteins were eluted with a NaCl gradient (0 to 1.0 M) in 400 ml of the buffer at a flow rate of 4 ml/min. Step 3: TSKgel G2000SW size exclusion column chromatography. The eluate containing the SPN48 protein (30 mg of protein) was concentrated and then loaded onto a TSKgel G2000SW column (4.6 mm x 30 cm) and eluted at a flow rate of 0.5 ml/min with 50 mM Tris-HCl containing 3 mM EDTA and 0.2 M NaCl, pH 6.0. The fractions that can make a SDS-stable complex with SPN48 were pooled and concentrated (8 mg of protein). Step 4: Hydroxylapatite column chromatography. The saturated sodium phosphate solution, pH 7.0, was added to the eluate of step 3 to a final concentration of 20 mM and the pool was loaded onto a 1 ml hydroxylapatite FPLC column (5 mm x 50 mm, Bio-Rad) equilibrated in buffer D (20 mM sodium phosphate containing 3 mM EDTA, pH 7.0). The column was washed with 4 ml of the same buffer followed by the elution of the bound

3

material with a 25 ml gradient from 20 to 500 mM sodium phosphate in the same buffer. The SPN48-containing fractions were pooled (1.2mg of protein). The amino-terminal amino acid sequence of the purified SPN48 protein and the internal peptides from HPLC were determined on an Applied Biosystem Procise automated gas-phase amino acid sequencer. The obtained amino acid sequences are as follows: EDATLQEFPNAVNS (Nterminal sequence of mature protein), ANFLVSPFSAATLL, QFIVDHPFIFYIK. S7. cDNA cloning and nucleotide sequencing of the three SPNs. Total RNA was isolated from the fat body of Tenebrio molitor larvae using ISOGEN (WAKO, Osaka, Japan). The cDNA was prepared using SuperScript II reverse transcriptase (Life Technologies, Inc.). A DNA fragments encoding central portion sequence of SPN40, SPN55 and SPN48 were amplified by PCR using degenerated primers (see the supplementary methods Tables 1-3 for used primer sequences). The PCR program was 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 52 °C for 30 s, 72 °C for 1 min and a final extension step of 72 °C for 5 min. The obtained central portion DNA fragments with 195, 474, 990 bps were labeled with [α-32P] dCTP and used as a probe by using Megaprime hybridization (GE Healthcare). The positive plaques were screened and purified from Tenebrio cDNA library and transformed to SOLR cells by in vivo excision. Nucleotide sequences of the positive clones were determined. S8. Expression and purification of the recombinant SPN55, SPN40, SPN48, and SPN1 in E. coli. A DNA fragments that encodes a full-length SPNs were amplified by PCR using as primers (see primers’ sequences in Table 1) with BamH1 and XhoI sites. To make constructs for each SPNs, the PCR product of each SPN was inserted into ProEx vector using EcoR1 and Xha1 restriction sites. The recombinant plasmid pProEX-SPNs construct was transformed into BL21 to produce the SPNs. Protein expression was induced by adding 0.4 mM isopropyl-D-thiogalactoside to the culture at 30 oC. Cells were harvested by centrifugation for 4 h after induction, and the cells were disrupted by sonication in 60 ml of 20 mM Tris (pH 8.0) buffer containing 150 mM NaCl and 2mM β-mercaptoethanol. The soluble lysate was centrifuged at 13,000 rpm for 30 min and the supernatant was mixed with Ni-NTA affinity resin (Qiagen, The Netherlands) that had been pre-incubated with the Tris buffer, and the mixture was stirred for 1 h at 4 oC. After the slurry was loaded into the column, unbound proteins were washed with 400 ml of the Tris buffer supplemented with 20 mM imidazole. Recombinant SPNs with the hexahistidine tag was eluted with the Tris buffer supplemented with 200 mM imidazole. Eluted fractions (total 14 ml) were analyzed by SDS-PAGE. After TEV protease treatment, the solution was purified as follows: Firstly, the expressed SPN was purified using Hitrap Q (1ml, Amersham Pharmacia, U.S.A.) pre-

4

equilibrated with 20 mM Tris (pH 8.0). The protein was eluted from the column using a 01M NaCl linear gradient in 20 mM Tris buffer (pH 8.0). Fractions (10 ml) containing SPNs were pooled and concentrated to 2 ml using Centriprep (Millipore, U.S.A.). Subsequently, the SPNs were purified using HiLoad Superdex 200 (Amersham Pharmacia, U.S.A.) preequilibrated with 20 mM Tris (pH 8.0) containing 150 mM NaCl. Finally, the pool from HiLoad Superdex 200 was concentrated to a volume of 0.5 ml using a Centricon device (Millipore). The concentrated material was applied to a TSKgel G3000SW column (Tosoh) equilibrated in 50 mM Tris-HCl containing 3 mM EDTA, pH 6.0 containing 0.2 M NaCl. Total proteins were analyzed by SDS-PAGE on a 15% gel and stained with Coomassie blue. The purity of the SPN sample was estimated through the protein band intensities on the Coomassie-blue-stained gel. Also, the N-terminal amino acid sequence of purified recombinant SPN was carried out to verify the identities of the purified proteins. The purified protein was stored at 4 oC for use within a week, or stored frozen at -80 oC until use.

Supplementary methods Fig. 1. Flow chart showing the basic scheme for the purifying Tenebrio serpins. The scheme involves fractionations by column chromatographies. FT indicates flow-through. The column names are shown in each purification step.

5

Supplementary methods Fig. 2. Immunoblot analysis for the searching fractions making a complex with activated SAE. The eluate fractions (fraction no. 20 to 30, 50 μg of protein) after Hi-Trap-Heparin HC column were incubated with activated SAE (0.2 μg of protein) for 30 min at 30 oC. The reaction mixtures were separated on SDSPAGE and then transferred to PVDF membrane. The ability of serpin-SP complex formation was examined by the immunoblot analysis using affinity-purified anti-SAE antibody as described previously. References 1. Park, J. W., Je, B. R., Piao, S., Inamura, S., Fujimoto, Y., Fukase, K., Kusumoto, S., Söderhäll, K., Ha, N. C., & Lee, B. L. (2006) J Biol Chem 281, 7747-7755.

6

Supplementary methods Table 1. Primers used to obtain the cDNA of SPN40

7


8


9