Solution Behavior Of Recombinant Spider Dragline Silk Proteins In Biomimetic Conditions

Due to the unique mechanical properties,good biocompatibility and biodegradability,spider dragline silk has received much attention.Unfortunately,natural spider dragline silk cannot be conveniently obtained by farming spiders because they are highly territorial and aggressive.Previous studies confirmed that transforming spiders' gene into other hosts and then expressing the spider silk protein is feasible.However,the fabricated fiber in vitro were not comparable with native silks.Besides,the residual organic solvents also limit the application of artificial spider silks.Therefore,many researchers paid their attention to mimicking the native spinning conditions to fabricate silk fiber recently.Natural spinning process is achieved in a certain gland,accompanied by the change of physicochemical factors,such as p H,ions and shear force,which resulted in the transition from soluble silk protein solution to solid fiber.Spider dragline silk is mainly composed of 90% protein,and the protein contains C-terminal domain(CTD),N-terminal domain(NTD)and Repetitive domain(Rep).Though many researches have been done to explore the response of terminus of spider silk protein on the physicochemical factors,the experimental conditions designed have not taken the change of those factors into full consideration.In addition,their researches mainly focused on recombinant spider silk proteins(spidroins)with one of the terminus or small spidroinscontaining both terminus,which cannot reveal the actual role of terminus.Herein,we comprehensively investigated factors of p H,ions and shear force and explored the solution behavior of recombinant spidroins with CTD,NTD or both domains.Besides,we also preliminarily explored the biomimetic spinning process.Solubility test indicated that the terminus is important for the storage of highly concentrated protein solution.Turbidity and ANS fluorescence test revealed that spidroins with CTD are prone to aggregate in response to the spinning solution,which indicate that CTD can promote the exposure of hydrophobic patches of spidroins.Tryptophan fluorescence test indicated the formation of NTD dimer,which may be important for the alignment of spidroins.SEM and FTIR tests shown the formation of well-alignedb-sheet-rich fibrils,suggesting the combined role of spinning solution and shear force on fiber formation.Finally,we successfully fabricated artificial fibers by using the Spinning solution.Overall,those findings established here enable the biomimetic spinning process,which will lay the foundation for the application of silk fibers in many fields,especially in medicine.