Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
  • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders

Definitions

• This invention relates to spider silk composites; their methods of manufacture and applications thereof.
• Spiders can produce up to seven different types of silk, each intended for a specific purpose.
• Dragline silk is the strongest type of silk and is used by the spider to create the structural 'spokes' of a web, and as a life line to evade predators.
• Dragline silk also exhibits intrinsic biodegradability, biocompatibility and minimal bacterial adherence (Leal-Egana, A. and Scheibel, T. (2010), Silk-based materials for biomedical applications. Biotechnology and Applied Biochemistry, 55 : 155-167. doi: 10.1042/BA20090229).
• RGD functionalised variants of dragline silk have been created by genetic fusion or chemical modification of cysteine residues, improving fibroblast adhesion and proliferation to match that of fibronectin coated plates acting as positive controls.
• using these techniques to create functionalised silk has inherent problems. Genetic manipulation of silk sequences is difficult because of the highly repetitive nature and high GC content of the gene. Furthermore, altering the protein primary sequence may cause mis-folding of the entire protein, resulting in reduced protein yield. Furthermore genetic manipulation can only integrate functional peptide motif sequences, resulting in a protein that contains a linear functional peptide which may not be accessible to impart function if the functionalised region is hidden upon polymerisation or fabrication into macroscopic structures for example.
• cysteine residues offer and alternative route to modify the silk protein with peptides or other functional molecules with good selectivity and reasonable efficiency. Unfortunately, modifying cysteine residues is not appropriate for functionalising silk proteins that self-assemble via C-terminal domain dimerization, since the formation of an essential disulphide bridge would be prevented, thus destroying the self-assembly process.
• An aim of the present invention is to provide improved functionalised spider silk material and improved methods of functionalising spider silk material.
• a modified spidroin comprising spidroin modified to comprise azide, alkyne, allyl or amine moieties.
• a spider silk material comprising:
• -a modified spidroin comprising spidroin modified to comprise one or more azide, alkyne, allyl or amine moieties.
• the invention advantageously provides a biomedically applicable functionalised silk biomaterial by a simple and benign chemical method that preserves self-assembly properties. Incorporation of the non-natural amino acid L-azidohomoalanine (L-Aha) into the methionine sites of a self-assembling miniature dragline silk spidroin, 4RepCT, to give 4RepCT AHA .
• L-Aha non-natural amino acid L-azidohomoalanine
• 4RepCT self-assembling miniature dragline silk spidroin
• These unique azide functional groups allow highly specific and efficient conjugation to peptides or molecules bearing a chemical group that is reactive to azide, such as a terminal alkyne moiety.
• the reaction may be via a Copper (I) (Cu(I)) catalysed Huisgen 1-3, dipolar cycloaddition, or 'click reaction' to give a 1-4 triazole product, which is a technique previously not used to modify silks.
• the 1-4 triazole formed in the Huisgen cycloaddition is a good bioisostere of an amide and is chemically more robust than the reaction between a cysteine thiol and a maleimide.
• Modifying spider silk using click chemistry offers a novel and versatile way to produce functionalised silk protein structures; custom designed to best fit an intended application.
• the spider silk material or conjugate is biocompatible.
• biocompatible is understood to include non-toxic to the human or animal body.
• the spider silk material may not cause an immune response.
• the spider silk material or conjugate may be biodegradeable.
• biodegradeable is understood to include the ability to breakdown over time in the tissue or body of a human or animal, and/or in the environment.
• the "spidroin” may be "major ampullate spidroin” and encompass all known major ampullate spidroin proteins, typically abbreviated "MaSp", or "ADF” in the case of Araneus diadematus.
• the spidroin may comprise dragline silk spidroin.
• the spidroin may comprise 4repCT spidroin.
• the modified spidroin may comprise the sequence of SEQ ID NO: 5.
• the modified spidroin may comprise the sequence of SEQ ID NO: 5, or a variant thereof having at least 50% sequence similarity to SEQ ID NO: 5.
• the variant may have at least 80% similarity to SEQ ID NO: 5.
• the variant may have at least 90% similarity to SEQ ID NO: 5.
• the variant may have at least 95% similarity to SEQ ID NO: 5.
• the variant may have at least 98% similarity to SEQ ID NO: 5.
• the variant may have at least 99% similarity to SEQ ID NO: 5.
• the variant may comprise one or more non-natural azido amino acid residues in place of one or more methionine residues of an equivalent non-modified or wild-type sequence.
• the modified spidroin may comprise the sequence of SEQ ID NO: 5, or a variant thereof having at least 50% sequence identity to SEQ ID NO: 5. In another embodiment, the variant may have at least 80% identity to SEQ ID NO: 5. The variant may have at least 90% identity to SEQ ID NO: 5. The variant may have at least 95% identity to SEQ ID NO: 5. The variant may have at least 98% identity to SEQ ID NO: 5. The variant may have at least 99% identity to SEQ ID NO: 5.
• the modified spidroin may comprise the sequence of SEQ ID NO: 3 or 4, further modified to comprise one or more non-natural amino acid residues comprising azide, alkyne, allyl or amine moieties in place of the one or more amino acid residues.
• the modified spidroin may comprise the sequence of SEQ ID NO: 3 or 4, further modified to comprise one or more non-natural amino acid residues comprising azide, alkyne, allyl or amine moieties in place of the one or more methionine residues.
• sequence identity or similarity may be across the entire length of the sequence of SEQ ID NO: 5. Sequence identity may be determined using BLAST alignment (NCBI) under standard parameters.
• the spidroin may be spidroin 1 or 2. It is understood that spidroin 2 has an additional lysine handle in addition to the azide. This would allow two sites of possible modification if desired.
• the skilled person may select an appropriate spidroin for modification according to the invention.
• the spidroin may be from an unmapped species, having differing physical properties than other known spidroins. The skilled person may determine the properties required for any particular application and apply the modification of the invention to the spidroin.
• the spidroin may comprise a spidroin capable of self-assembly, for example with or without a modification according to the invention.
• the spidroin may comprise any spidroin without structurally important (e.g. secondary or tertiary structurally important) methionine residues.
• the spidroin may comprise non-natural modified spidroin, for example, spidroin comprising one or more non-natural amino acids.
• the spidroin may be encoded in a gene with an amber codon, for a specific incorporation of a non-natural amino acid comprising a linking moiety.
• the spidroin may comprise a lysine residue, which provides an amine group to modify. For example, for at least two points of linkage/functionalisation.
• the lysine amine may be modified using standard peptide coupling reagents to form an amide.
• the lysine residue may be oxidised in the presence of a nitrous acid to convert the amine to an azide.
• the lysine side chain may also be reacted with aldehydes/ketones to form Imines that can then be reduced to amines or the amine can act as a nucleophiles reacting with alpha-halo acetate or acetamide to give an amine linkage.
• Other reactions of amines in proteins will be well known to those skilled in the art.
• the spidroin may be fused to a fusion partner.
• Fusion partners according to the invention include any protein fragment which improves the solubility and/or stability of its partner protein fragment, here the spidroin protein according to the invention.
• the fusion partner may also provide a suitable handle for affinity purification.
• examples of fusion partners according to the invention include thioredoxin, maltose-binding protein, glutathione S-transferase (GST), MTB32-C, Gbl, ZZ, His-tag, Avi-tag and Nus A.
• GST glutathione S-transferase
• MTB32-C glutathione S-transferase
• Gbl glutathione S-transferase
• ZZ His-tag
• Avi-tag Avi-tag
• Nus A Nus A.
• the fusion partner may be a thioredoxin moiety (ThrX).
• the fusion partner may be a thioredoxin moiety (ThrX) in combination with a His tag and/or an S tag.
• the spidroin and fusion partner may comprise a cleavage enzyme recognition site.
• the cleavage enzyme recognition site may be situated between the spidroin and the fusion partner such that cleavage at the recognition site results in a spidroin protein and a separated fusion partner.
• Examples of the cleavage agent recognition site according to the invention include an enterokinase recognition site, a Lys-C recognition site, or a thrombin recognition site. The skilled person will recognise that alternative cleavage enzymes are available, which will recognise their appropriate cleavage enzyme recognition site.
• the cleavage agent recognition site may comprise an enterokinase recognition site.
• enterokinase acts at a DDDK cleavage site and will not break down sensitive groups such as peptides that may be attached to the spidroin.
• the azide moiety may be arranged to react in a 'click' reaction.
• the azide moiety may be arranged to react with an alkyne moiety, for example presented on an active molecule to be linked, and form a triazole linkage.
• the alkyne moiety may be arranged to react in a 'click' reaction.
• the alkyne moiety may be arranged to react with an azide moiety, for example presented on an active molecule to be linked, and form a triazole linkage.
• Such triazole linkages are formed through the process of chemical ligation methods, such as a Cul-catalysed [3+2] Azide-Alkyne Cycloaddition (CuAAC) reaction, Iodine or heat catalysed Huisgen cycloaddition or the Strain-Promoted Alkyne Azide Cycloaddition (SPAAC) reaction between azide and cyclooctyne-modified molecules (copper-free click ligation).
• the triazole linkage may be a result of a copper-free SPAAC click reaction, which advantageously does not require copper catalysis.
• the copper free click ligation is compatible with large biomolecules and can be carried out in any biologically compatible buffer. The presence of copper causes degradation of long DNA and RNA molecules, so the avoidance of copper is advantageous. Copper-free click ligation can also be carried out in conditions under which some molecules may be unstable.
• the alkyne group may be strained by being part of a ring, for example a cycloalkyne such as a cyclooctyne.
• a dibenzocyclooctyne is highly suitable for use attached to biomolecules that are used in fast SPAAC reactions with azides.
• the conjugated aromatic rings impose ring strain and electron withdrawing properties on the alkyne, making it react very quickly with azides. Substituents to the ring may be used to increase or decrease the reactivity of the alkyne.
• the strained alkyne group may comprise a substituted cyclooctyne.
• the strained alkyne used in the present invention may be any strained alkyne group.
• the strained alkyne may be within a ring structure, for example a cycloalkyne or a cyclic compound where the ring comprises carbon atoms and one or more heteroatoms, for example, one or more nitrogen, sulphur or oxygen atoms in the ring.
• the strained alkyne group may comprise a 7 to 9 membered ring.
• the strained alkyne group may comprise a substituted 7 to 9 membered ring.
• the strained alkyne may comprise an 8 membered ring, or a substituted 8 membered ring.
• the substituted or unsubstituted 8 membered ring may comprise 8 carbon atoms in the ring or may comprise one or more nitrogen, sulphur or oxygen atoms in the ring in addition to carbon atoms.
• the strained alkyne group may comprise a cyclooctyne.
• the strained alkyne group may comprise a substituted cyclooctyne.
• the strained alkyne group may comprise a cyclic compound having at least one heteroatom in the ring.
• the strained alkyne group may comprise a dibenzocyclooctyne (DIBO) group.
• DIBO dibenzocyclooctyne
• the heteroatom may be at any position in the ring. In one embodiment there is one heteroatom in the ring.
• a strained alkyne that is reactive enough to perform the SPAAC reaction under laboratory conditions, for example because it can react in water or buffer at normal room temperature, for example 20°C, but that is also stable under laboratory conditions, for example it is stable in water, oxygen stable and stable at normal room temperature.
• the azide moiety may be arranged to form a triazole linkage with an alkyne group provided by a molecule, such as an active molecule.
• the azide moiety may be arranged to provide a Staudinger ligation with a phosphine or phosphite group provided on a molecule, such as an active molecule.
• the alkyne moiety may be arranged to form a triazole linkage with an azide group provided by a molecule, such as an active molecule.
• the non-natural amino acid may comprise an amino acid modified with an azide group, an alkyne group, an allyl group, or an amine group.
• the non-natural amino acid may comprise an amino acid azide.
• the non-natural amino acid may comprise an alkyne- comprising amino acid.
• the non-natural amino acid may comprise an allyl-comprising amino acid.
• the non-natural amino acid may comprise an amine-comprising amino acid.
• the non-natural amino acid may be a methionine analogue.
• the non-natural amino acid may be an analogue of any one of the 20 natural amino acids.
• the amino acid azide may be a methionine analogue or derivative having an azide group.
• the non-natural amino acid may comprise L-azidohomoalanine (L-Aha), azidoalanine, L- homopropargylglycine (L-Hpg), L-homoallylglycine (L-Hag),Jra «s-crotonylglycine (L- Tcg), Azidonorvaline.
• a range of non-natural amino acids may be provided by the mutagenesis methods of Wang et al. (2001. Science, Vol. 292 pp498-500 - incorporated herein by reference) or Chin J.W. et al. (2002. PNAS Vol. 99 No. 17, ppl 1020-11024- incorporated herein by reference), which allow the incorporation of a large range of un-natural/non-canonical amino acids.
• the modified spidroin may be formed by incorporation of a non-natural amino acid comprising azide moieties into spidroin.
• the modified spidroin may be formed by incorporation of a non-natural amino acid comprising alkyne moieties into spidroin.
• the modified spidroin may be formed by incorporation of a non-natural amino acid comprising allyl moieties into spidroin.
• the modified spidroin may be formed by incorporation of a non-natural amino acid comprising amine moieties into spidroin.
• the non-natural amino acids may substitute one or more natural amino acid residues in spidroin.
• the non-natural amino acids may substitute one or more methionine residues in spidroin.
• the spider silk material may comprise a plurality of modified spidroins arranged in a fibre.
• Spider silk material may comprise a plurality of fibres.
• the plurality of fibres may be aligned, meshed, interlaced, entwined, braided, weaved or twisted together, or produced by 3D printing.
• a spider silk material comprising
• the linking moiety may be a triazole, amine, amide, ether, or carbon-carbon linkage.
• the active molecule may be linked to the spidroin by a triazole or amide linkage.
• the active molecule may be linked to the spidroin by a triazole linkage.
• the triazole linkage may be a 1,4- or 1,5-triazole linkage.
• the linkage may be provided by any one of a range of reactions known to those skilled in the art including oxidation, epoxidation, 'ene' and Diels-Alder reaction, 1,3-dipolar cycloaddition 'click' reactions cyclopropanation, light dependent 2+2 cycloaddition.
• a conjugate of spidroin and an active molecule linked by a triazole, amine, amide, ether or carbon- carbon linkage is provided.
• the active molecule may be linked by a triazole or amide.
• the active molecule may be linked by a triazole, such as a 1,4- or 1,5-triazole linkage.
• the linkage or linking moiety on the spidroin may be located on the conserved c- terminal domain of the spidroin.
• the active molecule may be a therapeutically, prophylactically or diagnostically active substance.
• the active agent may be a bioactive substance.
• the active molecule may be selected from the group comprising a drug, pro-drug, peptide, protein, and nucleic acid, or combinations thereof.
• the active molecule may be a label, such as a fluorescent label.
• the active molecule may comprise or consist of a biomolecule.
• the active molecule may be a drug, a cell, signalling molecule, such as a growth factor, or any other suitable active agent.
• the active molecule may comprise amino acids, peptides, proteins, sugars, antibodies, nucleic acid, antibiotics, such as levofloxacin, antimycotics, growth factors, nutrients, enzymes, hormones, steroids, synthetic material, synthetic polymers, ceramic beads or surfaces, glass beads or surface, organometallic agents/metal chelates, PEG, PLGA, drugs, nanoparticles (such as gold, silver, quantum dots, and/or carbon nanotubes), adhesion molecules, small molecule and protein based fluorophores, colourants/dyes (which may be used for identification), radioisotopes (which may be for X-ray detection and/or monitoring of degradation), spectroscopic probes (such as gadolinium, manganese and iron contract agents), nitroxyl single electron paramagnetic agents, PET agents and other suitable constituents, or combinations thereof.
• the active molecule may comprise levofloxacin. Combinations of active molecules may be linked to the spidroin. Additionally or alternatively different spidroins may be linked to different active molecules and the different spidroins combined into the same fibre.
• the active molecule may be heat stable, for example, the active molecule may be capable of autoclaving under sterilising conditions.
• the active molecule may be heat sensitive and/or pH sensitive.
• the active molecule may be labile, degraded, inactivated, or denatured at temperatures above at least about 30°C.
• the active molecule may be labile, degraded, inactivated, or denatured at temperatures above at least about 50°C.
• the active molecule may be labile, degraded, inactivated, or denatured at temperatures above at least about 70°C.
• the active molecule may be labile, degraded, inactivated, or denatured at temperatures above at least about 100°C.
• the active molecule may be labile, degraded, inactivated, or denatured at temperatures above at least about 150°C.
• the active molecule may be labile, degraded, inactivated, or denatured at a pH ⁇ 6.
• the active molecule may be labile, degraded, inactivated, or denatured at a pH >8.
• the active molecule may be released from the fibre by exposure to light, heat, sound, enzymes, oxygen, fluoride ions and/or pH changes.
• the active molecule may comprise a linker for reacting and forming a link with the azide, alkyne, allyl or amine moieties on the modified spidroin.
• the linker may comprise a moiety capable of reacting and forming a link with one of azide, alkyne, allyl or amine moieties on the modified spidroin.
• the linker may comprise an alkyne, for example to form a link by reaction with an opposing azide group on the spidroin.
• the linker may comprise an azide, for example to form a link by reaction with an opposing alkyne group on the spidroin.
• the linker may comprise glycidyl propargyl ether.
• the active molecule may be linked to the linker by a covalent bond, for example via an ester bond.
• tissue scaffold, implant, wound dressing, or suture comprising the spider silk material or conjugate of the invention herein.
• a substrate suitable for site specific delivery and/or localisation of an active molecule comprising the spider silk material of the invention herein.
• a method of modifying spidroin with azide moieties comprising expressing a gene encoding spidroin in a methionine auxotrophic cell in the presence of methionine-analogous non- natural amino acid residues comprising an azide moiety, such that the methionine- analogous non-natural amino acids are incorporated in place of methionine residues.
• a method of modifying spidroin with a linking moiety such as an azide, alkyne, allyl or amine, comprising expressing a gene encoding spidroin in a methionine auxotrophic cell in the presence of methionine-analogous non-natural amino acid residues comprising the linking moiety, such that the methionine-analogous non-natural amino acids are incorporated in place of methionine residues.
• the methionine auxotrophic cell may be E. coli, such as, E. coli DL41.
• the cell may be cultured in a methionine-free medium.
• the methionine-free medium may comprise the methionine-analogous non-natural amino acid.
• the methionine-analogous non-natural amino acid may comprise L-azidohomoalanine (L-Aha) or azidoalanine.
• the methionine-analogous non-natural amino acid may comprise L-azidohomoalanine (L- Aha).
• the gene may encode spidroin with a fusion partner according to the invention.
• a method of modifying spidroin with a linking moiety comprising expressing a gene encoding spidroin in a cell with an orthogonal set of tRNA, aminoacyl tRNA synthetase, and codon, wherein the tRNA bears a non-natural amino acid residue comprising the linking moiety, such that the non-natural amino acid comprising the linking moiety is incorporated into the spidroin.
• the method may use the mutagenesis methods of Wang et al. (2001. Science, Vol. 292 pp498-500) or Chin J.W. et al.
• the method may comprise the use of an exogenous tRNA synthetase pair.
• the codon may be an amber codon encoded in the spidroin gene.
• a method of forming a spider silk material of the invention herein comprising:
• spidroin with a fusion partner in a methionine auxotrophic cell in the presence of methionine-analogous non-natural amino acid residues comprising a linking moiety, such that the methionine-analogous non-natural amino acids are incorporated in place of methionine residues to form modified spidroin with a fusion partner;
• the linking moiety may comprise azide, alkyne, allyl or amine.
• Extracting the modified spidroin may comprise purification of the modified spidroin and fusion partner.
• the modified spidroin and fusion partner may be affinity tagged, such that it can be purified on an affinity column.
• the affinity tag may comprise a His- tag, avi-tag, biotin, sortase tag, glutatione S-transferase (GST), maltose-binding protein (MBP).
• Cleaving the fusion partner from the modified spidroin may be by the addition of a cleavage enzyme recognising a cleavage enzyme recognition site between the modified spidroin and the fusion partner.
• the cleavage may be mediated by thrombin, enterokinase,Lys-C, TEV, Factor Xa, or Lysostaphin.
• the modified spidroin may be formed into fibres ranging from 5 nm to at least 10 cm in length.
• the method may further comprise twisting, weaving, braiding, interlacing or 3D printing the one or more fibres to form a multiple -fib ered spider silk or mesh of spider silk fibres.
• the method may further comprise reacting the linking moiety on the modified spidroin with an active molecule comprising a reactive group capable of reacting with linking moiety and forming a link between the spidroin and the active molecule.
• the linking of the active molecule may be before fibre formation, for example, before cleavage of the fusion partner, or after fibre formation. In one embodiment, the linking is carried out after fibre formation.
• a method of treatment comprising the administration of the spider silk material or conjugate according to the invention to a subject.
• the treatment may be for treatment or prevention of a disease, tissue repair or tissue replacement.
• the treatment may be wound treatment.
• the subject may be a mammal.
• the subject may be human.
• the spider silk material or the conjugate according to the invention for use in the treatment or prevention of a disease, tissue repair or tissue replacement.
• the spider silk material or conjugate of the invention may be used in an implant; medical product, such as wound closure system, band-aid, suture, wound dressing; or scaffold for tissue engineering and guided cell regeneration.
• medical product such as wound closure system, band-aid, suture, wound dressing; or scaffold for tissue engineering and guided cell regeneration.
• wound closure system such as wound closure system, band-aid, suture, wound dressing
• scaffold for tissue engineering and guided cell regeneration.
• optional features of one embodiment or aspect of the invention may be applicable, where appropriate, to other embodiments or aspects of the invention.
• the term "fibre” as used herein relates to polymers having a thickness of at least 5 nm, preferably macroscopic polymers that are visible to the human eye, i.e. having a thickness of at least 5 nm, and have a considerable extension in length compared to its thickness, preferably above 5 mm.
• the term "fibre” does not encompass unstructured aggregates or precipitates.
• Figure 1 An overview of the insertion of L-Aha into methionine sites of 4repCT AHA , subsequent click chemistry labelling with fluorophore (FAM) and processing into fibres following incubation with thrombin, enterokinase or Lys- C.
• FAM fluorophore
• Figure 3 A 15% SDS PAGE gel showing the enzymatic digestion of the
• Figure 4 A 15% SDS PAGE gel showing soluble 4RepCTAha labelled with FAM and resulting fibre after enzymatic digestion, (a) A fluorescent band at 37 kDa corresponding to 4RepCTAha fusion protein, (b) A ⁇ 3 cm fibre produced from FAM labelled 4RepCTAha glowing green upon exposure to UV.
• Figure 5 Fluorescence light microscope images of 4RepCTAha fibres and control fibres.
• the labelled 4RepCTAha fibres in the left hand panel display fine details of fibrillar bundles.
• Control fibres, located in the right hand panel, display the same fibrillar bundling with less detail and do not show any fluorescence.
• Figure 6 Structures of (a) glycidyl propargyl ether linker, (b) levofloxacin and (c) levofloxacin conjugated to the linker via an ester bond.
• Figure 7 A bar chart showing optical densities of E. coli NCTC 12242 liquid cultures after 28 hours incubation with non- functionalised (non mod silk), levofloxacin control (lev Ctrl silk) and levofloxacin-functionalised (lev mod silk) fibres. Data is the mean of 3 replicates, error bars show standard error of the mean. A two tailed t-test was used to determine significance.
• Figure 8 Muller-Hinton agar plates showing zones of inhibition of E. coli NCTC 12242 in the presence of levofloxacin control and levofloxacin- functionalised fibres. Incubation periods are displayed on the left and fibre types displayed above.
• the present study aims create biomedically applicable functionalised silk biomaterials using a simple and benign chemical method that preserves self-assembly properties.
• the non-natural amino acid L-azidohomoalanine (L-Aha) was incorporated into the methionine sites of a self-assembling miniature dragline silk spidroin, 4RepCT, to give 4RepCT AHA .
• E. coli DL41 were transformed with pJExpress401 plasmid containing the silk fusion protein gene (SEQ ID NO: 5)
• the gene was codon optimised for E. Coli and encoded the following; His6 tag/ sol tag/ Thioredoxin/ Enterokinase site/ Lys-C site/ Thrombin site/ 4RepCT.
• Transformed bacteria were streaked onto LB agar plates containing kanamycin and incubated at 37 °C.
• Das enzymatic cleavage sites that release thioredoxin, triggering fibre formation.
• Fibres were made from labelled and un-labelled silk protein by incubation with thrombin, Lys-C or Enterokinase at an enzyme: fusion protein ratio of 1 : 1000 w/w. Cleavage was achieved at a fusion protein concentration of approximately 2 mg/ml, 1 mM potassium phosphate and a total volume of 1- 3 ml. Reaction was incubated at 30°C with gentle rocking for a period of 4 hours, after which 1-10 cm long fibres were visible. Samples of the reaction were analysed by SDS PAGE.
• Soluble TRX GS MaSpl Aha + was labelled with 0.1 mM FAM alkyne (3'-Hydroxy-6'-(2- propyn-l-yloxy)-3H-spiro[2-benzofuran-l,9'-xanthen]-3-one)) or 0.1 mM Alexa Fluor 594 alkyne (carboxamido-(5-(and 6-)propargyl), bis(triethylammonium salt)).
• Cu[I] was generated in situ by addition of THPTA (3 [tris(3-hydroxypropyltriazolylmethyl)amine) and sodium ascorbate to 5 mM and CuS0 4 to 0.5 mM.
• Preformed 4RepCT Aha fibres were labelled with FAM alkyne or Alexa Fluor 594 similarly to soluble protein, however an increased concentration of Cu[I] was used (5.0 mM). Reactions were protected from light and incubated at room temperature with gentle rocking for a period of 4 hours. After the incubation period the fibres were removed to 15 ml Falcon tubes and washed thoroughly with 20 mM Tris pH 7.5 three times after which the Tris buffer was replaced with 10% DMSO in water and incubated overnight at room temperature with gentle rocking.
• L-Aha was incorporated into three methionine sites located in the C-terminal domain of 4RepCT distant from the essential cysteine residue ( Figure 2b). Incorporation of L-Aha did not affect expression of the protein which was purified as described previously yielding around 20-30 mg/L with high purity.Fibre formation was initiated by thrombin, enterokinase or Lys-C removing thioredoxin from the fusion protein. SDS PAGE analysis revealed free thioredoxin present in the thrombin and enterokinase digestions ( Figure 3a lanes 1 and 2).
• Fluorophore labelled soluble 4RepCT AHA retains the ability to form fibres
• linker-labelled and levofloxacin control fibres were placed into UPLC grade methanol containing 50 mM levofloxacin and refluxed at 65 °C for 18 hours (non-functionalised fibre was refluxed in methanol only).
• Post reflux fibres were removed and washed with 2 ml of methanol four times and 2 ml of buffer containing 20 mM Tris pH 7.5, 1 mM EDTA twice. Fibres were stored in methanol. Zone of inhibition assay
• the ability of the Levofloxacin functionalised fibres to inhibit growth of E. coli NCTC 12242 was tested by means of a zone of inhibition assay and by monitoring cell density of liquid cultures.
• a culture of E. coli NCTC 12242 was grown with shaking (180 rpm) at 37°C in Mueller Hinton broth (Oxoid) to an OD 6 oo 0.1 and diluted 100 x in the same medium.
• the diluted culture was thoroughly spread onto Mueller-Hinton agar (Oxoid) plates using a cotton swab.
• the levofloxacin functionalised and control fibre types both gave zones of inhibition after 24 hours incubation, however the non- functionalised fibre did not produce a zone ( Figure 8).
• the levofloxacin functionalised fibre yielded a 3.5 cm diameter zone whereas the control fibre yielded a 2 cm diameter zone.
• colonies were growing in the previously clear zone of the levofloxacin control fibre but not in the zone generated by the levofloxacin functionalised fibre as shown in Figure 7.
• the levofloxacin functionalised fibres displayed a sustained release of antibiotic over at least 5 days, preventing the re-colonisation of the initial zone of inhibition. This observation indicates that the inhibition zone was not simply caused by 'burst release' of adsorbed levofloxacin as also displayed by the 4RepCT control fibre, but the successful breakdown of the covalent linker to the conjugated levofloxacin over time.
• N.B. constructs with and without the Lysine (K) are available.
• an Arginine(R) takes its place.
• the extra Lysine provides an amine group to modify; it does not take the place of a methionine as azidohomoalanine does, therefore spidroin can be made to contain azides and a unique amine i.e. two points of functionalisation. Spider silks have either no or a very small number of lysine residues and these are normally non-critical hence these can be removed leaving lysines only at positions for attaching ligands.
• the lysine amine can be modified using standard peptide coupling reagents to form amides. It could also be oxidised in the presence of a nitrous acid to convert the amine to an azide.
• the lysine side chain can also react with aldehydes/ketones to form Imines that can then be reduced to amines or the amine can act as a nucleophiles reacting with alpha-halo acetate or acetamide to give an amine linkage.
• Other reactions of amines in proteins will be well known to those skilled in the art.

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