An efficient strategy for expression and purification of spider neurotoxic peptide YC1 in E. coli

Figures & data

Figure 1. Construction of pET-GST-YC1 expression vector. (A) The composition of the pET-GST-YC1 vector. (B) The amino acid sequences of ProTX II, GsAF II, and YC1.

Figure 2. Expression of GST-YC1 in SHuffle^TM.

Notes: The protein was isolated by 10% glycine-SDS-PAGE and used for experiments as described in the Materials and Methods. Lane M, marker; lane 1, uninduced bacteria total cells protein using the ZYM-505 medium; lane 2, induced bacterial total cells protein using the ZYM-5051 medium; lane 3, the centrifuged supernatant after sonication (soluble fractions); lane 4, the centrifuged precipitate after sonication (insoluble fractions); lane 5, the flow through from the GST column; lane 6, the flow through from the GST column; lane 7, the fusion protein after ultrafiltration desalination; lane 8, the fusion protein after ULP1 cleavage. The arrow indicates GST-SUMO-YC1 (~49 kDa) and YC1 (~4 kDa).

Figure 3. RP-HPLC chromatography of rYC1. (A) Absorption of the eluted proteins was measured at 280 nm, and fractions corresponding to detected peaks were collected. (B) Absorption of the eluting solution was measured at 215 nm, and fractions corresponding to detected peaks were collected.

Figure 4. Identification of rYC1. (A) The results of the glycine-SDS-PAGE analysis. Lane 1, protein from eluent marked with asterisk in Figure 3; lane 2, the protein after the ultrafiltration purification of eluent marked with asterisk (Figure 3). (B) Mass spectra of the rYC1. The calculated theoretical molecular weight of rYC1 was 3,937.7 Da, and the measured molecular weight was 3,939.9 Da,

Figure 5. Inhibitory effects of rYC1 on the rNa_v 1.3 currents. (A) The inhibitory effects of 1 μM or 10 μM rYC1 on rNa_v1.3 current. (B) The effects of rYC1 on rNa_v1.3 channels expressed in HEK293 cells. The IC₅₀ value of rYC1 for rNa_v1.3 was 2.94 μmol/L.