Spider Dragline Silk Molecular Properties and Recombinant Expression

Rising, A. 2007. Spider dragline silk – molecular properties and recombinant production. Doctor’s dissertation. ISSN: 1652-6880, ISBN: 978-91-576-7337-4 Spider dragline silk possesses several desirable features of a biomaterial; it has extraordinary mechanical properties, is biocompatible and biodegradable. It consists of large proteins, major ampullate spidroins (MaSp:s), that contain alternating polyalanineand glycine-rich blocks between non-repetitive Nand C-terminal domains. No full length MaSp gene has been cloned, hence the knowledge of their constitution is limited. The spider stores the silk in a liquid form, which is converted into a fibre by a poorly understood mechanism. Even truncated spidroins are difficult to produce recombinantly in soluble form. Most previous attempts to produce artificial spider silk fibres have included solubilization steps in non-physiological solvents and the use of spinning devises for fibre formation. This thesis presents a novel method for production of macroscopic fibres under physiological conditions, without using denaturing agents. A miniature spidroin is identified that can be produced recombinantly in E. coli when fused to a soluble fusion tag. Upon enzymatic release of the fusion tag, the miniature spidroins spontaneously form macroscopic fibres in physiological solution. These fibres resemble native silk and their strength equals that of fibres spun from regenerated silk. Initial studies suggest that the fibres are biocompatible. This represents a major breakthrough for future biomaterial development. Molecular studies of cDNA and genetic sequences encoding the dragline silk revealed an unexpectedly high level of heterogeneity and the presence of at least two MaSp1 genes. Furthermore, the E. australis MaSp2 was characterised for the first time, as well as a new MaSp-like spidroin. Sequence analysis of previously published spidroin N-terminal domains compared with that of E. australis MaSp1, enabled identification of signal peptides and a 130 residue nonrepetitive domain common to dragline, flagelliform and cylindriform spidroins. Moreover, this highly conserved N-terminal domain was concluded to consist of five positionally conserved -helices. Structural studies using circular dichroism spectroscopy on recombinantly produced MaSp1 Nand C-terminal domains showed that these are folded, stable and soluble, and that salts or pH has no major effect on their secondary structures.