Isolation and purification of dragline silk protein from Crossopriza lyoni web

Abstract

Spider silk is a robust biomaterial due to its desirable tensile strength, biocompatibility and biodegradability. It is composed of spidroins which are recently used in wide technical applications. At present, dragline spidroins from Nephila clavipes are heavily studied thus, neglecting the presence of the protein in other species. The aim of this study is to isolate and purify dragline silk protein from Crossopriza lyoni web. Spider webs were pre-treated with 0.02 M of sodium carbonate and followed by spidroin extraction using 20% (w/v) SDS-ethanol extraction solution. Two-level full factorial design was applied to analyse the significant effect of parameters namely; temperature (°C), agitation speed (rpm) and incubation time (hour) on maximum spidroin recovery. Optimisation study was performed by Response Surface Methodology (RSM) via Box-Behnken Design (BBD). The crude sample was purified by gel filtration chromatography and characterised by one-dimensional SDS-PAGE for dragline spidroin presence. Agitation speed, incubation time and temperature posed significant effect on spidroin extraction with p < 0.0500 in decreasing order, respectively. From the optimisation study, the highest spidroin concentration (1210.78 ± 0.974 μg/ml) was obtained at 90 °C, 102.5 rpm and 4.5 hours with a fitted model at p value of 0.0001. Validation experiment was conducted and the result showed that the model was good to fit to the experimental data with percentage error less than 1 %. Purification of the crude sample resulted in the dragline silk protein putative peaks on the chromatogram at 280 nm with 0.1 ml of column volume. SDS-PAGE analysis further confirmed the presence of the dragline silk protein with a band of molecular weight of more than 250 kDa. Isolation and purification of dragline spidroin in the present study is anticipated to bridge the gap of knowledge in the dragline spidroins from non-araneoid species. This would be an initiative to expand the spider silk technology at national level in the near future.