A Leishmania secretion system for the expression of major ampullate spidroin mimics

Abstract

Spider major ampullate silk fibers have been shown to display a unique combination of relatively high fracture strength and toughness compared to other fibers and show potential for tissue engineering scaffolds. While it is not possible to mass produce native spider silks, the potential ability to produce fibers from recombinant spider silk fibers could allow for an increased innovation rate within tissue engineering and regenerative medicine. In this pilot study, we improved upon a prior fabrication route by both changing the expression host and additives to the fiber pulling precursor solution to improve the performance of fibers. The new expression host for producing spidroin protein mimics, protozoan parasite Leishmania tarentolae, has numerous advantages including a relatively low cost of culture, rapid growth rate and a tractable secretion pathway. Tensile testing of hand pulled fibers produced from these spidroin-like proteins demonstrated that additives could significantly modify the fiber’s mechanical and/or antimicrobial properties. Cross-linking the proteins with glutaraldehyde before fiber pulling resulted in a relative increase in tensile strength and decrease in ductility. The addition of ampicillin into the spinning solution resulted in the fibers being able to inhibit bacterial growth.

Advances in Plant-Derived Scaffold Proteins

“…Non-plant hosts that have been used to produce major ampullate spidroins include E. coli and Salmonella (Lewis et al, 1996;Arcidiacono et al, 1998;Widmaier et al, 2009;Xia et al, 2010;Edlund et al, 2018), yeast Pichia (Fahnestock and Bedzyk, 1997;Gaines and Marcotte, 2011;Liu et al, 2018), protozoa Leishmania (Lyda et al, 2017), mammalian cell lines (Lazaris et al, 2002), silkworm transformed with fusion protein or CRISPR/Cas9 site specific exchange (Miao et al, 2006;Teulé et al, 2011;Zhang et al, 2019), and mice or goats (Service, 2002;Xu et al, 2007). Here we focus on the efforts and strategies of recombinant spidroin production from plants.…”

Bioengineering of spider silks for the production of biomedical materials

“…Finally, a report on the expression and secretion of major ampullate spidroin mimics (MaSp1 and MaSp2) in Leishmania was reported by Lyda et al (2017) . Leishmania was chosen because it offers several advantages over other systems, such as rapid growth, high optical densities, and simple media formulations.…”

Host Systems for the Production of Recombinant Spider Silk

“…In a pilot study, Lyda and co-workers utilised the eukaryotic protozoan parasite Leishmania tarentolae as a silk production host [60]. L. tarentolae are single-celled insect vector parasites that naturally secrete proteins of high molecular weight.…”

“…Recombinant spidroins modelled on N. clavipes MaSp1/MaSp2 and containing eight repeat units (73-81 kDa) were successfully produced, but adequate protein yield remained a concern (exact data was not reported). The authors suggest the use of bioreactors to enable controlled cellular proliferation as a means of increasing yield [60].…”