Recombinant Production and Determinants for Fiber Formation

Spider dragline silk is Nature’s high-performance fiber that outperforms the best man-made materials by displaying extraordinary mechanical properties. In addition, spider silk is biocompatible and biodegradable, which makes it suitable as a model for biomaterial production. Dragline silk consists of large structural proteins (spidroins) comprising an extensive region of poly-alanine/glycine-rich tandem repeats, located in between two non-repetitive and folded terminal domains. The spidroins are stored at high concentration in liquid form and are converted to a solid fiber through a poorly understood spinning process. In order to artificially replicate the dragline properties, the protein constituents must be characterized and the silk production pathway elucidated. The large, repetitive sequences of the genes and corresponding proteins have made spidroin analogs difficult to produce in recombinant expression systems. Genetic instability, prematurely terminated synthesis and poor solubility of produced proteins are often observed. This thesis presents a novel method for the efficient recombinant production of a soluble miniaturized spidroin under non-denaturing conditions. The mini-spidroin can be processed under physiological conditions to form fibers with favorable mechanical and cell-compatibility properties, without the use of denaturing spinning procedures or coagulation treatments. The fibers structure and macroscopic appearance resemble native spider silk and the strength equals that of regenerated silk and mammalian tendons. In addition, for the first time, the production of recombinant silk with enhanced mechanical properties was accomplished through introduction of mutations, enabling covalent intermolecular cross-linking of proteins constituting the fiber. Moreover, the effect on structure, stability and fiber forming propensities for all representative parts of MaSp1, due to changes in temperature, pH and salt concentrations was investigated.