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.
Highlights
Orb weaving spiders, such as Nephila clavipes, produce up to seven different types of silk [1,2]. One of these spider silk proteins, the Major Ampullate Spidroin 1 (MaSp1), is a 275 kDa protein of known sequence that provides strength to parts of the web [3]. Spiders use this type of silk, which is known as dragline silk, as a frame and safety thread
Precipitation behavior was first investigated at the small scale6.ofA1s5 observed in Figure 4A, the t protein (TP) remains soluble at acetone concentrations up to 50% (v/v)
Because the ELP-tag present at the C-terminus of our TP is based on the elastin sequence, we investigated whether the tropoelastin purification protocol could be adapted for the ELPylated MaSp1 analog
Summary
Orb weaving spiders, such as Nephila clavipes, produce up to seven different types of silk [1,2] One of these spider silk proteins, the Major Ampullate Spidroin 1 (MaSp1), is a 275 kDa protein of known sequence that provides strength to parts of the web [3]. Spiders use this type of silk, which is known as dragline silk, as a frame and safety thread. The structural motifs in dragline silk responsible for its strength are blocks of antiparallel-oriented beta-sheets consisting of poly-alanine and/or poly-glycine blocks. The reasons for researchers desire to uncover the secrets of dragline silk are obvious: dragline silk is: (1) a natural product; (2) produced under ambient conditions; and (3) possesses a strength on the same order of magnitude as the strongest man-made fiber (Kevlar®; DuPont de Nemours, Neu-Isenburg, Germany) [7]
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