Recombinant Spider Silk Genetically Functionalized with Affinity Domains

Abstract

Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule.

Silk‐Based Antimicrobial Polymers as a New Platform to Design Drug‐Free Materials to Impede Microbial Infections

“…The presence of a β‐conformation in the 6mers proteins suggests that two or more disulphide bonds were present in the antimicrobial domain, thus inferring that protein folding was maintained. The same result was reported on previous studies with silk fusion proteins …”

“…The same result was reported on previous studies with silk fusion proteins. [19,39] Also in the 15mer fusion protein (Figure 2E), the presence of a negative elasticity with a minimum of 200 nm suggested a random coil configuration, with a negative shoulder at 220 nm, thus indicating the presence of additional structures like α-helix or β-sheet. [40] However, in the 15mer proteins with the antimicrobial peptides, 15mer-Hep, and 15mer-HNP1, a positive ellipticity with a maximum of 195-200 nm and a negative ellipticity with a minimum of 210 nm was detected ( Figure 2E).…”

Formation of a thin-walled spider silk tube on a micromachined scaffold

“…Silk tube self-assembly Silk encapsulation of the scaffold was achieved using recombinantly produced miniaturized spider silk fusion proteins containing either a domain capable of binding IgG (Z) [8] or an integrin-binding motif from fibronectin (FN) [9] (Z-4RepCT and FN-4RepCT, Spiber Technologies, Sweden). The encapsulation process is shown in detail in the "Silk self-assembly" part of Figure 1.…”

Protein‐based functional nanomaterial design for bioengineering applications

“…Recently, 4RepCT films fused with cell adhesion motifs such as RGD, IKVAV, and YIGSR were shown to promote better cell adhesion than controls especially for Schwann cells on IKVAV functional 4RepCT compared with laminin‐coated control . Furthermore, 4RepCT fusions with different biomolecule‐binding domains such as IgG binding, biotin binding, and albumin binding have also been prepared . Site‐specific immobilization of different biomolecules can be a powerful method to impart extracellular matrix‐like activity to SLP or any other PBP used for cell interaction and tissue engineering.…”