Honeybee Silk Research Articles

Biocompatibility and immunogenic response to recombinant honeybee silk material.

If tolerated in biological environments, recombinant structural proteins offer the advantage that biological cues dictating cell attachment and material degradation can be modified as required for clinical application using genetic engineering. In this study, we investigate the biological response to materials generated from the recombinant honeybee silk protein, AmelF3, a structural protein that can be produced at high levels by fermentation in Escherichia coli. The protein can be readily purified from E. coli host cell proteins after transgenic production and fabricated into various material formats. When implanted subcutaneously according to International Standard ISO 10993 tests, materials generated from the purified recombinant protein were found to be noncytotoxic, inducing a transient weak immunogenic response and a chronic inflammatory response that resolved over time. While preliminary, this study supports the ongoing development of materials generated from this protein for biomedical applications. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1763-1770, 2019.

Confirmation of Bioinformatics Predictions of the Structural Domains in Honeybee Silk.

Honeybee larvae produce a silk made up of proteins in predominantly a coiled coil molecular structure. These proteins can be produced in recombinant systems, making them desirable templates for the design of advanced materials. However, the atomic level structure of these proteins is proving difficult to determine: firstly, because coiled coils are difficult to crystalize; and secondly, fibrous proteins crystalize as fibres rather than as discrete protein units. In this study, we synthesised peptides from the central structural domain, as well as the N- and C-terminal domains, of the honeybee silk. We used circular dichroism spectroscopy, infrared spectroscopy, and molecular dynamics to investigate the folding behaviour of the central domain peptides. We found that they folded as predicted by bioinformatics analysis, giving the protein engineer confidence in bioinformatics predictions to guide the design of new functionality into these protein templates. These results, along with the infrared structural analysis of the N- and C-terminal domain peptides and the comparison of peptide film properties with those of the full-length AmelF3 protein, provided significant insight into the structural elements required for honeybee silk protein to form into stable materials.

Design of silk proteins with increased heme binding capacity and fabrication of silk-heme materials

In our previous studies, heme was bound into honeybee silk to generate materials that could function as nitric oxide sensors or as recoverable heterogeneous biocatalysts. In this study, we sought to increase the heme-binding capacity of the silk protein by firstly redesigning the heme binding site to contain histidine as the coordinating residue and secondly, by adding multiple histidine residues within the core of the coiled coil core region of the modified silk protein. We used detergent and a protein denaturant to confirm the importance of the helical structure of the silk for heme coordination. Aqueous methanol treatment, which was used to stabilize the materials, transformed the low-spin, six-coordinate heme to a five-coordinate high-spin complex, thus providing a vacant site for ligand binding. The optimal aqueous methanol treatment time that simultaneously maintains the helical protein structure and stabilizes the silk material without substantial leaching of heme from the system was determined.

Modification of Honeybee Silk by the Addition of Antimicrobial Agents.

Honeybee silk proteins can be produced at high levels in recombinant systems, fabricated into materials, and are tolerant of amino acid modifications: properties that make them exciting templates for designing new functional materials. Here, we explore the properties of materials either made from silk-antimicrobial peptide (AMP) fusion proteins or silk containing entrapped AMPs or silver nanoparticles. Inclusion of AMP within the silk protein sequence did not affect our ability to express the proteins or process them into films. When AMP-silk proteins and Escherichia coli cells were coincubated in solution, a reduction in cell numbers was observed after degradation of the chimeric protein to release a truncated version of the AMP. In films, the AMP was retained in the silk with leaching rates of <1% per day. Films containing silver nanoparticles were antimicrobial, with the silk preventing aggregation of nanoparticles and slowing the rate of dissolution of the particles.

Applicability of biotechnologically produced insect silks

Silks are structural proteins produced by arthropods. Besides the well-known cocoon silk, which is produced by larvae of the silk moth Bombyx mori to undergo metamorphosis inside their silken shelter (and which is also used for textile production by men since millennia), numerous further less known silk-producing animals exist. The ability to produce silk evolved multiple independent times during evolution, and the fact that silk was subject to convergent evolution gave rise to an abundant natural diversity of silk proteins. Silks are used in air, under water, or like honey bee silk in the hydrophobic, waxen environment of the bee hive. The good mechanical properties of insect silk fibres together with their non-toxic, biocompatible, and biodegradable nature renders these materials appealing for both technical and biomedical applications. Although nature provides a great diversity of material properties, the variation in quality inherent in materials from natural sources together with low availability (except from silkworm silk) impeded the development of applications of silks. To overcome these two drawbacks, in recent years, recombinant silks gained more and more interest, as the biotechnological production of silk proteins allows for a scalable production at constant quality. This review summarises recent developments in recombinant silk production as well as technical procedures to process recombinant silk proteins into fibres, films, and hydrogels.

Bioinspired electrocatalysts for oxygen reduction using recombinant silk films

Hemin-loaded honeybee silk reduces oxygen with low peroxide formation.

Topochemistry, optical anisotropy and FT-IR microspectroscopy of the cocoon of Lithurgus chrysurus (Hymenoptera, Megachilidae)

A previous study has not revealed the participation of a mucous component in the cocoon wall of the solitary bee, Lithurgus chrysurus, differing from the cocoon structure reported for many other bee species. However, uncertainty remains, because only the median and rear zones of this cocoon type have thus far been analyzed. Here, we studied the front zone of this cocoon, searching its components and their organization, to fill this knowledge gap. Topochemical assays, polarization microscopy and Fourier transform-infrared (FT-IR) microspectroscopy were used to study cross sections from L. chrysurus cocoon. Three main layers differing in structural organization were found to compose the cocoon wall. Silk fibroins were assumed to constitute the filamentous threads of the inner and outer layers and the laminar structure of the intermediate layer. Deduced from its topochemical properties and FT-IR spectral signature, a foamy material containing mucin glycoproteins and carboxylated acid glycosaminoglycans was found in the intermediate layer. FT-IR analysis using a Savitzky-Golay 2nd-derivative and absence of linear dichroism and birefringence phenomena suggest that a random-coil secondary structure predominates in the foam component. Co-existence of α-helical and β-sheet conformations is also hypothesized for the fibroin component of this cocoon. It is thus concluded that in addition to fibroin elements, a mucous component, likely contributed by a Malpighian tubule secretion, integrates the composition of the front zone of the cocoon wall of L. chrysurus. In addition, the FT-IR analysis of the inner layer silk of this cocoon suggests significant differences in comparison to the silk fibroins of the silkworm, and some minor spectral differences in comparison to published data on the honeybee silk, with respect to protein secondary structure.

Recombinant honeybee silk proteins and their use in future materials

Recombinant honeybee silk proteins and their use in future materials

Native honeybee silk membrane: a potential matrix for tissue engineering and regenerative medicine

Biomimetic natural origin biomaterials are noteworthy targets for further innovation in biomedical and tissue engineering.

The molecular structure of the silk fibers from Hymenoptera aculeata (bees, wasps, ants).

Silks from the Hymenoptera aculeata (bees, wasps, ants) contain ropes with four α-helical strands, rather than the more usual two strands found, for example, in α-keratin and myosin molecules. Extensive studies of the chemical structure of the silks have shown that each of the four chains in the molecule contains a central coiled-coil rod domain. However, little progress has been made in modeling the three-dimensional structure. X-ray diffraction data on honeybee silk (Apis mellifera), recorded by Rudall and coworkers, has been re-examined in detail and possible structures developed for the various types of filament seen in the silk glands, and for the packing arrangement in the spun fibers. The original X-ray data were re-collected by scanning figures in the original publications, de-screening and averaging perpendicular to the direction of interest, thereby reducing the graininess of the original images. Sufficient numbers of equatorial and meridional reflections were collected to define the axial projection of the base of the unit cell in fibers drawn from the contents of the silk glands, and to suggest that the axial period is different from that suggested by Rudall and coworkers. Models for two types of filament of increasing diameter are developed based on the node-internode packing scheme observed in protein crystals containing four-strand α-helical ropes. The central domains of the four component chains in the molecule are enclosed by N- and C-terminal domains with widely different lengths and compositions. The fibers thus have a composite filament-matrix texture, and possible locations for the matrix are discussed.

Stabilization of viruses by encapsulation in silk proteins.

Viruses are important for a range of modern day applications. However, their utility is limited by their susceptibility to temperature degradation. In this study, we report a simple system to compare the ability of different dried protein films to stabilize viruses against exposure to elevated temperatures. Films from each of three different silks, silkworm, honeybee silk and hornet silk, stabilized entrapped viruses at 37 °C better than films of albumin from bovine serum (BSA) and all four proteins provided substantially more stabilization than no protein controls. A comparison of the molecular structure of the silks and BSA films showed no correlation between the ability of the proteins to stabilize the virus and the secondary structure of the protein in the films. The mechanism of stabilization is discussed and a hypothesis is suggested to explain the superior performance of the silk proteins.

Micromolar biosensing of nitric oxide using myoglobin immobilized in a synthetic silk film

In this work we investigate the use of coiled-coil silk proteins, produced in recombinant Escherichia coli, as a new material for immobilizing biosensors. Myoglobin was embedded in transparent honeybee silk protein films. Immobilized myoglobin maintained a high affinity for nitric oxide (KD NO=52µM) and good sensitivity with a limit of detection of 5µM. The immobilized myoglobin–silk protein film was stable and could be stored as a dry film at room temperature for at least 60 days. The effect of immobilization on the structure of myoglobin was fully investigated using UV/visible, Fourier Transform Infrared and Raman spectroscopy, which indicated a weakening in the strength of the iron–histidine bond. This study demonstrates that recombinant coiled-coil silk proteins provide a safe and environmentally friendly alternative to sol–gels for stabilizing heme proteins for use as optical biosensors.

Correction: Controlling the Molecular Structure and Physical Properties of Artificial Honeybee Silk by Heating or by Immersion in Solvents

Correction: Controlling the Molecular Structure and Physical Properties of Artificial Honeybee Silk by Heating or by Immersion in Solvents

Continuous Production of Flexible Fibers from Transgenically Produced Honeybee Silk Proteins

Flexible and solvent stable fibers are produced after concentrated recombinant honeybee protein solutions are extruded into a methanol bath, dried, drawn in aqueous methanol, then covalently cross-linked using dry heat. Proteins in solution are predominantly coiled coil. Significant levels of non-orientated ß-sheets form during drying or after coagulation in aqueous methanol. Drawing generally aligns the coiled coil component parallel with the fibre axis and ß-sheet component perpendicular to the fiber axis. The fibres are readily handled, stable in the strong protein denaturants, urea and guanidinium, and suitable for a range of applications such as weaving and knitting.

Physical properties of honeybee silk: a review

Honeybee silk is released from secretory cells and polymerises as birefringent tactoids in the lumen while silk is spun by a spinneret at the tip of the labium–hypopharynx and contains ά-helical proteins arranged in a four-strand coiled-coil structure. Wet fibres are only half as stiff as dried ones, but are equal in strength. The fibroin is hygroscopic and lithium thiocyanate and urea eliminate the yield point tested on both dry and wet fibres. The slopes of the solvent-related curves are reduced compared to those tested in water. Silk sheets are independent of temperature when deformed in tension. This fibre is rather crystalline and its hydration sensitivity, expressed as the ratio of the elastic modulus of wet to that of dry fibre, is 0.53. The ά-helical fibroins are predicted to have an antiparallel tetrameric configuration that is shown as a possible structural model. The molecular structure of ά-helical proteins maximizes their robustness with minimal use of building materials. In conclusion, it appears that the composition, molecular topology and amino acid content and sequence are a highly conserved feature in the evolution of silk in Apis species.

Artificial Honeybee Silk: A Recombinant Protein as a Biomimetic Structural Material

Honeybee larvae produce silken cocoons that provide mechanical stability to the hive. The silk proteins are small and non-repetitive and therefore can be produced at large scale by fermentation in E. coli. Recombinant silk proteins which have a coiled coil structure can be fabricated into a range of forms including sponges and fibers. The resultant material is soluble in water and requires a post-production stabilizing treatment. Aqueous methanol treatment induces the formation of a stabilizing s-sheet structure, with the amount of s-sheet being controlled by time or methanol concentration. Dry heat treatment at 190°C also produces a water insoluble material but without significant secondary structural changes. Honeybee silk proteins are particularly high in lysine, serine, threonine, glutamic acid and aspartic acid. The stability of the heat treated material is attributed to the generation of covalent cross-links including lysinoalanine and isopeptide groups. The unique ability to stabilize material by controlling secondary structure rearrangement and covalent cross-linking allows us to design recombinant silk materials with a wide range of properties and potential applications.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Controlling the Molecular Structure and Physical Properties of Artificial Honeybee Silk by Heating or by Immersion in Solvents

Honeybee larvae produce silken cocoons that provide mechanical stability to the hive. The silk proteins are small and non-repetitive and therefore can be produced at large scale by fermentation in E. coli. The recombinant proteins can be fabricated into a range of forms; however the resultant material is soluble in water and requires a post production stabilizing treatment. In this study, we describe the structural and mechanical properties of sponges fabricated from artificial honeybee silk proteins that have been stabilized in aqueous methanol baths or by dry heating. Aqueous methanol treatment induces formation of ß-sheets, with the amount of ß-sheet dictated by methanol concentration. Formation of ß-sheets renders sponges insoluble in water and generates a reversibly compressible material. Dry heat treatments at 190°C produce a water insoluble material, that is stiffer than the methanol treated equivalent but without significant secondary structural changes. Honeybee silk proteins are particularly high in Lys, Ser, Thr, Glu and Asp. The properties of the heat treated material are attributed to generation of lysinoalanine, amide (isopeptide) and/or ester covalent cross-links. The unique ability to stabilize material by controlling secondary structure rearrangement and covalent cross-linking allows us to design recombinant silk materials with a wide range of properties.

Production, structure and in vitro degradation of electrospun honeybee silk nanofibers

Honeybees produce silken cocoons containing four related fibrous proteins. High levels of each of the honeybee silk proteins can be produced recombinantly by fermentation in Escherichia coli. In this study we have used electrospinning to fabricate a single recombinant honeybee silk protein, AmelF3, into nanofibers of around 200 nm diameter. Infrared spectroscopy found that the molecular structure of the nanofibers was predominantly coiled coil, essentially the same as native honeybee silk. Mats of the honeybee nanofibers were treated with methanol or by water annealing, which increased their β-sheet content and rendered them water insensitive. The insoluble mats were degraded by protease on a time scale of hours to days. The protease gradually released proteins from the solid state and these were subsequently rapidly degraded into small peptides without the accumulation of partial degradation products. Cell culture assays demonstrated that the mats allowed survival, attachment and proliferation of fibroblasts. These results indicate that honeybee silk proteins meet many prerequisites for use as a biomaterial.

Single Honeybee Silk Protein Mimics Properties of Multi-Protein Silk

Honeybee silk is composed of four fibrous proteins that, unlike other silks, are readily synthesized at full-length and high yield. The four silk genes have been conserved for over 150 million years in all investigated bee, ant and hornet species, implying a distinct functional role for each protein. However, the amino acid composition and molecular architecture of the proteins are similar, suggesting functional redundancy. In this study we compare materials generated from a single honeybee silk protein to materials containing all four recombinant proteins or to natural honeybee silk. We analyse solution conformation by dynamic light scattering and circular dichroism, solid state structure by Fourier Transform Infrared spectroscopy and Raman spectroscopy, and fiber tensile properties by stress-strain analysis. The results demonstrate that fibers artificially generated from a single recombinant silk protein can reproduce the structural and mechanical properties of the natural silk. The importance of the four protein complex found in natural silk may lie in biological silk storage or hierarchical self-assembly. The finding that the functional properties of the mature material can be achieved with a single protein greatly simplifies the route to production for artificial honeybee silk.

Honeybee silk: Recombinant protein production, assembly and fiber spinning

Transgenic production of silkworm and spider silks as biomaterials has posed intrinsic problems due to the large size and repetitive nature of the silk proteins. In contrast the silk of honeybees ( Apis mellifera) is composed of a family of four small and non-repetitive fibrous proteins. We report recombinant production and purification of the four full-length unmodified honeybee silk proteins in Escherichia coli at substantial yields of 0.2–2.5 g/L. Under the correct conditions the recombinant proteins self-assembled to reproduce the native coiled coil structure. Using a simple biomimetic spinning system we could fabricate recombinant silk fibers that replicated the tensile strength of the native material.