Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber

Abstract

Spider dragline silk is a remarkably strong fiber that makes it attractive for numerous applications. Much has thus been done to make similar fibers by biomimic spinning of recombinant dragline silk proteins. However, success is limited in part due to the inability to successfully express native-sized recombinant silk proteins (250-320 kDa). Here we show that a 284.9 kDa recombinant protein of the spider Nephila clavipes is produced and spun into a fiber displaying mechanical properties comparable to those of the native silk. The native-sized protein, predominantly rich in glycine (44.9%), was favorably expressed in metabolically engineered Escherichia coli within which the glycyl-tRNA pool was elevated. We also found that the recombinant proteins of lower molecular weight versions yielded inferior fiber properties. The results provide insight into evolution of silk protein size related to mechanical performance, and also clarify why spinning lower molecular weight proteins does not recapitulate the properties of native fibers. Furthermore, the silk expression, purification, and spinning platform established here should be useful for sustainable production of natural quality dragline silk, potentially enabling broader applications.metabolic engineering | glycyl-tRNA | silk fiber | Nephila clavipes | spinning S pider dragline silk, used by spiders as the safety line and the web frame, is exceptionally strong and elastic; it is five times stronger by weight than steel, three times tougher than the top quality man-made fiber Kevlar (1, 2). The dragline silk is primarily composed of two proteins, the major ampullate spidroins 1 (MaSp1) and 2 (MaSp2) (3, 4). These spidroins are highly modular, each with a long repetitive sequence that is flanked on both sides by nonrepetitive amino-and carboxy-termini of approximately 100 amino acids (5). The repetitive sequence is rich in glycine and alanine, and characterized by stretches of alanine that are interrupted by glycine-rich repeats (4). The poly alanine regions form hydrophobic crystalline domains that are responsible for the high tensile strength, whereas the glycine-rich regions are hydrophilic and responsible for the links between crystalline domains as well as the elasticity of dragline fiber (6).Due to the unique mechanical properties, spider dragline silk has received much attention as a promising material for numerous industrial applications such as parachute cords, protective clothing, and composite materials in aircrafts. Also, many biomedical applications are envisioned due to its biocompatibility and biodegradability. For example, silk-based materials have been developed as sutures for wounds, coatings for biomedical implants, drug carriers for drug delivery, and scaffolds for cell culture and organ replacement (7-9). Unfortunately, natural spider dragline silk cannot be conveniently obtained by farming spiders because they are highly territorial and aggressive. Thus, many attempts have been made to produce recombinant dragline silk proteins (10-13) followed ...

With great structure comes great functionality: Understanding and emulating spider silk

“…52 High molecular weight expression, matching that of native silks, appears critical in obtaining high strength synthetic silk. This was demonstrated by Xia et al 85 who produced a range of molecular weight silks in Escherichia coli (E. coli) and found a clear dependence of mechanical properties on molecular weight (see later). 85 Interestingly, bacteria with enriched tRNA populations express MaSp proteins with high efficiency, resulting in a reasonable yield for proteins up to native size (285 kDa).…”

“…This was demonstrated by Xia et al 85 who produced a range of molecular weight silks in Escherichia coli (E. coli) and found a clear dependence of mechanical properties on molecular weight (see later). 85 Interestingly, bacteria with enriched tRNA populations express MaSp proteins with high efficiency, resulting in a reasonable yield for proteins up to native size (285 kDa). 85 E. coli has been the system of choice for recombinant production of spider silk dope, however, low protein yields and poor solubility are hallmarks of E. coli expression.…”

“…The previously discussed innovations in metabolic engineering of E. coli have allowed for the expression of spider silk proteins with controlled molecular weight from ;55 to ;285 kDa; the upper value is in the range of natural spider silk proteins. 85 The highest molecular weight silks from this study exhibited breaking strain and tenacity comparable to natural N. clavipes dragline silk, and a Young's modulus that exceeded that of the natural product (21 6 4 GPa compared to 11-14 GPa, respectively).…”

“…85 Interestingly, bacteria with enriched tRNA populations express MaSp proteins with high efficiency, resulting in a reasonable yield for proteins up to native size (285 kDa). 85 E. coli has been the system of choice for recombinant production of spider silk dope, however, low protein yields and poor solubility are hallmarks of E. coli expression. 86 Additionally, unwanted recombination events can lead to shortening of the repetitive genes.…”

Production of a novel silk-like protein from sea anemone and fabrication of wet-spun and electrospun marine-derived silk fibers

“…To investigate any enhancement of the proteins' mechanical properties, we also constructed a higher-molecular-weight (B62 kDa) aneroin protein (designated aneroin-60K; Figure 2d) by repeating the aneroin gene twice, based on reports of the relationship between the molecular weights of recombinant spider silk proteins and the mechanical properties of the spun fibers. 22,23 Materials with high-production capabilities and superior mechanical properties enable feasible and practical applications in various biomedical and industrial fields. Because affinity purification was not sufficient to obtain a high yield of recombinant aneroin proteins (B67 mg ml À1 of aneroin-30K and B53 mg ml À1 of aneroin-60K), we devised an alternative method based on temperature-induced aggregation followed by acid extraction to improve purification yield and generate a simple purification procedure for practical applications.…”