Low-Tech, Pilot Scale Purification of a Recombinant Spider Silk Protein Analog from Tobacco Leaves

Abstract

Spider dragline is used by many members of the Araneae family not only as a proteinogenic safety thread but also for web construction. Spider dragline has been shown to possess high tensile strength in combination with elastic behavior. This high tensile strength can be attributed to the presence of antiparallel β-sheets within the thread; these antiparallel β-sheets are why the protein is classified as a silk. Due to the properties of spider silk and its technical and medical uses, including its use as a suture material and as a scaffold for tissue regeneration, spider dragline is a focus of the biotechnology industry. The production of sufficient amounts of spider silk is challenging, as it is difficult to produce large quantities of fibers because of the cannibalistic behavior of spiders and their large spatial requirements. In recent years, the heterologous expression of genes coding for spider silk analogs in various hosts, including plants such as Nicotiana tabacum, has been established. We developed a simple and scalable method for the purification of a recombinant spider silk protein elastin-like peptide fusion protein (Q-/K-MaSp1-100× ELP) after heterologous production in tobacco leaves involving heat and acetone precipitation. Further purification was performed using centrifugal Inverse Transition Cycling (cITC). Up to 400 mg of highly pure spider silk protein derivatives can be isolated from six kilograms of tobacco leaves, which is the highest amount of silk protein derivatives purified from plants thus far.

Xanthurenic Acid Is the Main Pigment of Trichonephila clavata Gold Dragline Silk

“…Spider silk is utilized for a variety of purposes, including to catch prey, protect offspring, and act as a lifeline; it exhibits remarkable mechanical toughness, which is a combination of high tensile strength and elasticity [ 1 ]. Owing to its outstanding properties and biocompatible and biodegradable proteinaceous nature, spider silk has attracted attention as a potential tool in the commercial production of synthetic threads for medical and industrial applications [ 2 , 3 , 4 , 5 ], and various hosts are being explored for its synthetic production [ 6 , 7 , 8 , 9 , 10 , 11 ]. Protein composition [ 12 , 13 ] and the variability of the physical properties of spider silk have been extensively studied using the amino acid sequences encoded by spidroin genes as well as spider spinning processes [ 14 , 15 , 16 ].…”

Advances in Plant-Derived Scaffold Proteins

“…The yield based on current methods ranges from microgram to about 200 milligram of recombinant protein per kilogram of leaf or seed tissues. The pilot scale purification of MaSp1-ELP fusion proteins from stable transformed tobacco leaves using heat and acetone precipitation followed by centrifugal inverse transition cycling, achieved 80 mg MaSp1-ELP per kg of leaves (Heppner et al, 2016). This yield still does not meet the commercially acceptable level (1-5 g recombinant protein/kg of plant tissue) (Zhang et al, 2009c).…”

Recombinant Spider Silk: Promises and Bottlenecks

“…Oligomeric repeats of ELP were fused to the spider silk sequence and after expression and purification, up to 400 mg spider silk proteins could be isolated from 6 kg of tobacco leaves. This report is the highest amount of spider silk proteins purified from plants (Scheller et al, 2004;Hauptmann et al, 2013b;Heppner et al, 2016).…”