Biofabricating a Silk Scaffold as a Functional Microbial Trap

Abstract

Silk fibroin produced from silkworms has been intensively utilized as a scaffold material for a variety of biotechnological applications owing to its remarkable mechanical strength, extensibility, biocompatibility, and ease of biofunctionalization. In this research, we engineered silk as a novel trap platform capable of capturing microorganisms. Specifically, we first fabricated the silk material into a silk sponge by lyophilization, yielding a 3D scaffold with porous microstructures. The sponge stability in water was significantly improved by ethanol treatment with elevated β-sheet content and crystallinity of silk. Next, we biofunctionalized the silk sponge with a poly-specific microbial targeting molecule, ApoH (apolipoprotein H), to enable a novel silk-based microbial trap. The recombinant ApoH engineered with an additional penta-tyrosine was assembled onto the silk sponge through the horseradish peroxidase (HRP) mediated dityrosine cross-linking. Last, the ApoH-decorated silk sponge was demonstrated to be functional in capturing our model microorganism targets, E. coli and norovirus-like particles. We envision that this biofabricated silk platform, capable of trapping a variety of microbial entities, could serve as a versatile scaffold for rapid isolation and enrichment of microbial samples toward future diagnostics and therapeutics. This strategy, in turn, can expedite advancing future biodevices with functionality and sustainability.

Silk Bioconjugates: From Chemistry and Concept to Application

“…An interesting concept is the scaffold-based traps for in vivo use or sample cleanup . In the latter case, regenerated B. mori liquid silk was lyophilized and scaffolds were functionalized with covalently binding capture tags .…”

“…Conjugated apolipoprotein H showed improved activity, probably due to the optimized spatial arrangement via N-terminal immobilization and the microbial capture motif at the C-terminal domain. The resulting apolipoprotein silk scaffold was able to trap microbial model compounds, including bacteria and viruses, from laboratory suspension samples . Pushing these systems up to the Technology Readiness Level will be an important task for laying the groundwork for similar platforms.…”

“…An interesting concept is the scaffold-based traps for in vivo use 88 or sample cleanup. 89 In the latter case, regenerated B. mori liquid silk was lyophilized and scaffolds were functionalized with covalently binding capture tags. 89 The resulting sponge was treated with ethanol to induce beta sheets and a surface decorated with the poly-specific microbial targeting molecule apolipoprotein H. Apolipoprotein H is a glycoprotein that circulates in the blood and displays the characteristic cationic amino acid sequence KNKEKK toward the C-terminus.…”

“… 89 In the latter case, regenerated B. mori liquid silk was lyophilized and scaffolds were functionalized with covalently binding capture tags. 89 The resulting sponge was treated with ethanol to induce beta sheets and a surface decorated with the poly-specific microbial targeting molecule apolipoprotein H. Apolipoprotein H is a glycoprotein that circulates in the blood and displays the characteristic cationic amino acid sequence KNKEKK toward the C-terminus. Apolipoprotein H is an ideal capture protein because it strongly interacts with negatively charged phospholipids (e.g., lipopolysaccharides, endotoxins, and membrane proteins of microorganisms).…”

A Redox-Based Autoinduction Strategy to Facilitate Expression of 5xCys-Tagged Proteins for Electrobiofabrication

“…Because protein engineering is now fairly routine, the incorporation of "designer" tags has emerged enabling a variety of new functions, among those including protein attachment onto both biotic and abiotic materials. For example, a pentatyrosine protag was shown to allow tyrosinase-mediated covalent coupling of an IgG-binding protein G or a human glycoprotein, ApoH, with both polysaccharides (Wu et al, 2009) and silk fibroin from Bombyx mori (Wu et al, 2020); a polyglutamine tag facilitated the assembly of proteins onto both gelatin (Liu et al, 2015) and spider silk (Wu et al, 2017); and a polylysine tag was added to enzymes for covalent tethering onto engineered tobacco mosaic virus-derived virus like particles (Bhokisham et al, 2020). Other peptide tags of varied amino acid composition enable binding onto solid materials such as gold (Tamerler et al, 2006;Adams et al, 2015;Terrell et al, 2021), silver (Sedlak et al, 2012), silicon (Zhou et al, 2015), as well as various hydrophobic surfaces (Tanaka et al, 2006) through non-covalent interactions.…”