One-step heating strategy for efficient solubilization of recombinant spider silk protein from inclusion bodies

Abstract Background Spider silk, an ideal biomaterial with remarkable mechanical properties, is built of spider silk proteins (spidroins). Engineering spidroins have been successfully produced in a variety of heterologous hosts and the most widely used expression system is Escherichia coli ( E. coli ). However, recombinant expression of spidroins often results in water insoluble inclusion bodies (IBs). Target protein solubilization from IBs in a traditional way is often solved under extremely harsh conditions, e.g. 8 M urea or 6 M guanidine hydrochloride, which highly risk to poor bioactive protein recovery as well as precipitation during the following dialysis process.Results Here we present a new mild solubilization strategy called one-step heating method to solubilize spidroins from IBs, with combining together spidroins’ high thermal stability with low concentration of urea. We expressed a 430-aa recombinant protein (designated as NM) derived from the minor ampullate spidroin of Araneus ventricosus in E.coli ; the NM proteins are present in the insoluble fraction as IBs. The isolated NM IBs were solubilized parallelly by both traditional urea-denatured method and one-step heating method. The efficiency of solubilization of NM IBs in Tris-HCl (pH 8.0) containing 4 M urea by one-step heating method was comparable to that of 7 M urea by the traditional urea-denatured method. The effects of buffer, pH and temperature conditions on solubilization of NM IBs by one-step heating method were evaluated, and the optimum conditions are following: hearting temperature 70–90°C for 20 min, pH 7.0–10, urea concentration 2–4 M in normal buffers. Prepared via the one-step heating method, the recombinant NM spidroin could self-assembly into nanoparticles.Conclusions The one-step heating method introduced here could be used to efficiently solubilize NM IBs under relatively mild conditions, which might by important for the downstream nanoparticle or fiber formation. The one-step heating method can be applied under broad buffer, pH and temperature conditions, conferring the potential to apply to not only spidroins but also other thermal stable protein IBs' solubilization.