Silky smooth solution

Strong, biocompatible and ideal for healing wounds; silk is Nature’s wonder material – and spider silk perhaps holds the most potential to be exploited. However, given the arachnid tendency for cannibalism, spiders are notoriously difficult to farm. The solution says Dr David Harvey, is to simply do away with them by the use of some clever chemistry and a microbiological host...     

Silk is an extraordinary, protein based, natural material that is produced by a variety of organisms including flies, mites and scorpions¹. However the most well-known and prolific silk producers are silk worms (namely Bombyx mori) and spiders; some of which can produce up to seven different types of silk (orb weaving spiders).

Tensile strength is the most obvious utilisable property, and dragline silk has been used in the Indo-Pacific to construct fishing nets for small fish, however this is not its only desirable property

Of the seven different types of spider silk, dragline silk is the strongest and is comprised of two different protein building blocks – major ampullate spidroin 1 and major ampullate spidroin 2 (or MaSp1 and 2)². Spiders use dragline silk to construct the spokes of their webs and as life lines when evading predators. Tensile strength is the most obvious utilisable property, and dragline silk has been used in the Indo-Pacific to construct fishing nets for small fish, however this is not its only desirable property. As far back as ancient Greece, spider silks have been used to dress wounds, suggesting that the silks may somehow aid in wound healing³. Recent research has shown that dragline silks are in fact biocompatible (meaning that it does not trigger immune or rejection responses in mammals) and capable of supporting the growth of human cells?. In addition, intrinsic antimicrobial activity has also been described?. Therefore it would seem that the ancient Greeks were ahead of their time in identifying suitable advanced materials for wound dressings.

Luckily due to the advances made over the last three decades we don’t actually need spiders to make the silk proteins; we just need the genes encoding the spidroins and a host that can express them

Unfortunately spiders, unlike silk worms, cannot be farmed for their silk owing to their highly territorial and sometimes cannibalistic behaviour. This has meant that obtaining large quantities of spider silk has historically been very challenging, and is presumably the reason why spider silk has rarely been used in large scale textile manufacturing. Luckily due to the advances made over the last three decades we don’t actually need spiders to make the silk proteins; we just need the genes encoding the spidroins and a host that can express them.

Several research groups have employed this recombinant approach – taking spidroin genes from a range of different spiders and introducing them into various expression hosts such as tobacco plants, mammalian cells and even transgenic goats?-?. Most often though the expression host of choice is E. coli bacteria; owing to its fast growth, reproduction rate, industrial scalability, sustainability, and the ability to manipulate the proteins at a genetic level with relative ease?. This approach is well on the way to offering a solution to the spider farming problem.

However because natural spidroins are very large in size (up to several hundred kilo Daltons) and are highly repetitive at a genetic level, spiders have developed highly specialised systems in order to produce these challenging proteins, such as increased transfer RNA supplies to cope with translating the large codon repeats in the spidroin gene¹?. Since the expression hosts listed above lack these specialised systems, true full size spidroins have not yet been produced. Instead miniaturised versions of the spidroins have been expressed; however the majority of these spidroins were expressed as insoluble proteins that are difficult to process further. However a 2007 study reported a major breakthrough with the production of a soluble miniature MaSp1 spidroin (called 4RepCT) in E. coli.

In this study the spidroin was N-terminally fused to another protein, thioredoxin that served to solubilise it¹¹. Most notably however this miniature spidroin could, after enzymatic removal of thioredoxin, self-assemble into stable macroscopic fibres. Continued research from this group has led to the very recent production of another soluble miniature MaSp1 spidroin in E. coli (NT2RepCT)¹². This new spidroin does not require thioredoxin to be soluble, and fibre formation closely resembles the behaviour of natural full-size spidroins in response to lowered pH and sheer force. Furthermore, because both 4RepCT and NT2RepCT are expressed in E. coli, production has the potential to be scaled up to industrial proportions with relatively few complications.

Building on the 2007 study, our research utilised E. coli to express the plasmid encoded miniature spidroin 4RepCT. However our primary goal was to produce a variant of 4RepCT that could be easily and reproducibly chemically functionalised with a wide range of functional ligands. Furthermore, we wanted to retain the self-assembling properties of 4RepCT, this meant that any changes made to 4RepCT would have to be small, minimising the risk of disrupting self-assembling.

We decided that the best way of achieving this would be to replace a natural amino acid (methionine) found in the primary sequence of 4RepCT with a manmade amino acid. Importantly, this manmade amino acid should harbour a uniquely reactive chemical handle which could be exploited later to chemically attach functional ligands to the silk protein with high efficiency and specificity. To this end, the methionine substitute L-azidohomoalanine (Figure 1) was used to replace the three methionine residues ordinarily found in 4RepCT (creating 4RepCT3Aha).

[caption id="attachment_58292" align="alignnone" width="380"] Figure 1. The manmade amino acid L-azidohomoalaine with an azide functional group (circled).[/caption]

This was accomplished by expressing 4RepCT in a strain of E. coli that could not synthesise its own methionine (a methionine ‘auxotroph’) grown in media supplemented with the man-made amino acid L-azidohomoalanine; a technique developed by Professor David Tirrell at Caltech. L-azidohomoalanine contains an azide terminated sidechain which allows the functionalisation of 4RepCT3Aha with ligands bearing an alkyne functional group in the presence of a copper catalyst in a so called ‘click’ reaction (Figure 2). This chemical reaction, coined by Nobel Laureate Professor Barry Sharpless of The Scripps Research Institute, is very selective, quick and does not produce unwanted side products. It allows a diverse range of functional ligands to be conjugated to 4RepCT3Aha provided that they have an alkyne group.

[caption id="attachment_58293" align="alignnone" width="620"] Figure 2. A click reaction between an azide and alkyne group in the presence of a copper (I) catalyst to give a 1-4 disubstituted 1, 2, 3-triazole.[/caption]

Following the expression of the thioredoxin- 4RepCT3Aha fusion protein in E. coli, standard metal affinity chromatography techniques were used to purify the protein and soluble 4RepCT3Aha could then be processed into fibres through the enzymatic removal of thioredoxin (as described in the 2007 study). This suggested that the incorporation of L-azidohomoalanine had not abolished the self-assembly properties of 4RepCT, therefore the next step was to try and functionalise 4RepCT3Aha with an alkyne bearing ligand. Fluorophores were chosen as a proof of principle because they could be used to easily report successful functionalisation without the need for further assays.

To check the incorporation of L-azidohomoalanines fluorophores were ‘clicked’ onto premade 4RepCT3Aha fibres. These fibres were incubated in a reaction mixture that contained the alkyne bearing fluorophore and the copper catalyst. After a short time the fibres were removed from the reaction mixture and washed to remove excess reactants. After several washing steps the 4RepCT3Aha fibres retained fluorescence whilst control fibres (which did not contain L-azidohomoalanine) rapidly lost fluorescence and returned to their original appearance. This indicated that successful click reactions between fibre and fluorophore had taken place, and that the fluorophore was now covalently conjugated to the silk fibres.

Moving forwards, attempts to create functionalised fibres from fluorophore functionalised soluble 4RepCT3Aha were carried out. Using the same process to form fibres, permanently fluorescent macroscopic fibres were produced from fluorophore labelled soluble 4RepCT3Aha. This important result demonstrated that self-assembly was not only unaffected by the incorporation of L-azidohomoalanine, but also unaffected by functionalisation. In addition, functionalised soluble 4RepCT3Aha could be mixed in defined ratios and later processed generating a fibre that contained more than one functional ligand. We demonstrated this by incorporating two different fluorophores into the same fibre (Figure 3).

[caption id="attachment_58294" align="alignnone" width="620"] Figure 3. A scheme showing the production of dual fluorophore labelled 4RepCT3Aha fibre.[/caption]

However fluorophores, whilst useful in detecting successful click reactions aren’t particularly functional in biological systems other than allowing us to monitor the degradation of fibres. Therefore building upon the previously reported antimicrobial activity of natural spider silk we decided to functionalise 4RepCT3Aha fibres with an antibiotic. Premade fibres were functionalised with the broad spectrum antibiotic levofloxacin in combination with a pH sensitive linker to mediate release of the antibiotic. To be functionally useful, levofloxacin needed to be released from the fibres in order to disrupt bacterial growth through its inhibition of DNA gyrase and topoisomerase IV inside the bacterial cells.

When tested against a reference strain of E. coli, the levofloxacin conjugated fibres were able to prevent the growth of the bacteria over the 5 days tested whereas controls did not

As bacteria grow the pH of their local environment drops and they also secrete enzymes such as esterases into their immediate vicinity which can cleave the glycerol ester bond. The linker was therefore chosen to break down in the presence of growing bacteria, therefore releasing levofloxacin from the fibre. When tested against a reference strain of E. coli, the levofloxacin conjugated fibres were able to prevent the growth of the bacteria over the 5 days tested whereas controls did not. The short lived antimicrobial activity observed for one of the controls, indicated that some of the antibiotic may have become absorbed to the surface of the functionalised silk fibres, contributing to an initial ‘burst release’ of antibiotic. In the conjugated fibres this meant that there were two phases of antibiotic release; an initial burst followed by a sustained release of antibiotic which could prove useful when treating infected wounds, for example.

Therefore taking inspiration from the ancient Greeks, there is potential for functionalised 4RepCT3Aha to promote wound healing, particularly in prevention of infection during healing. In addition to the creation of functionalised 4RepCT3Aha fibres, other morphologies of the protein will be explored such as hydrogels and films; widening the scope of wound types that can be treated with functionalised 4RepCT3Aha. This has particular promise in producing customised dressings that would topically release antibiotics reducing the need for systemic administration. Finally, the various 4RepCT3Aha based materials could be functionalised with cocktails of synergistic antibiotics (or other drug molecules) in order to combat antibiotic resistant bacteria, a growing problem that will see the return of deaths to otherwise treatable conditions if not actively researched.

Author: Dr David Harvey is based at The University of Nottingham as a BBSRC funded postdoctoral research associate, he is furthering the work on the use of recombinant silk protein materials in tissue engineering and drug delivery.

References

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