Conferring biological activity to native spider silk: A biofunctionalized protein‐based microfiber

Abstract

Spider silk is an extraordinary material with physical properties comparable to the best scaffolding/structural materials, and as a fiber it can be manipulated with ease into a variety of configurations. Our work here demonstrates that natural spider silk fibers can also be used to organize biological components on and in devices through rapid and simple means. Micron scale spider silk fibers (5-10 μm in diameter) were surface modified with a variety of biological entities engineered with pentaglutamine tags via microbial transglutaminase (mTG). Enzymes, enzyme pathways, antibodies, and fluorescent proteins were all assembled onto spider silk fibers using this biomolecular engineering/biofabrication process. Additionally, arrangement of biofunctionalized fiber should in of itself generate a secondary level of biomolecular organization. Toward this end, as proofs of principle, spatially defined arrangement of biofunctionalized spider silk fiber was shown to generate effects specific to silk position in two cases. In one instance, arrangement perpendicular to a flow produced selective head and neck carcinoma cell capture on silk with antibodies complexed to conjugated protein G. In a second scenario, asymmetric bacterial chemotaxis arose from asymmetric conjugation of enzymes to arranged silk. Overall, the biofabrication processes used here were rapid, required no complex chemistries, were biologically benign, and also the resulting engineered silk microfibers were flexible, readily manipulated and functionally active. Deployed here in microfluidic environments, biofunctional spider silk fiber provides a means to convey complex biological functions over a range of scales, further extending its potential as a biomaterial in biotechnological settings. Biotechnol. Bioeng. 2017;114: 83-95. © 2016 Wiley Periodicals, Inc.

Site-specific immobilization of endoglycosidases for streamlined chemoenzymatic glycan remodeling of antibodies

“…On the other hand, MTG displays broad acyl-acceptor substrate specificity [29], enabling the use of a variety of scaffolds with primary amine groups as solid supports. We have developed several applications of this versatile enzyme including functionalization of many support structures, such as the polysaccharide chitosan [33], proteins spider silk [34], and gelatin [35]. This enables not only immobilization of the enzyme, but also its localization among many different structures, surfaces, and delivery vehicles [34, 36, 37].…”

Incorporating LsrK AI‐2 quorum quenching capability in a functionalized biopolymer capsule

“…Hence, while we have described a means for the assembly of the multicomponent capsules for deployment in culture fluids, their application may be anticipated in other environments, including in human wound dressings. The biofabrication assembly methodologies exploited here and shown previously (Wu et al, 2017) would be suitable for a variety of applications. Functionalizing these biopolymers, and others, with bacterial kinase, LsrK, may provide yet another active barrier in the prevention of infection by the quenching of AI-2 mediated QS activity.…”

Biofabricating Functional Soft Matter Using Protein Engineering to Enable Enzymatic Assembly

“…Figure 1 illustrates two examples of fusion tag proteins: N-terminus Tyr-tagged green fluorescent protein (GFP) that can be readily conjugated to stimuli-responsive aminopolysaccharide chitosan [105][106][107][108][109] through tyrosinase-mediated reaction, and C-terminus Gln-tagged red fluorescent protein (RFP) that can be conjugated to lysine containing biopolymers through mTG-catalyzed reaction. 29,96 The use of fusion tags has been reported to offer three advantages. First, the conjugation efficiency of fusion tagged protein is typically more efficient than conjugation of the native protein, presumably because the tag provides readily accessible residues for conjugation.…”

“…101 Importantly, mTG catalyzed conjugation confers excellent selectivity because the nucleophilic attack by the lysine occurs at the enzyme's active site resulting in the direct transfer of the carbonyl from the thioester intermediate to the Gln residue. 96,[102][103][104]…”