Recombinant polypeptides

Abstract

The present invention relates generally to an isolated polypeptide from Hylaeus nubilosus, a proteinaceous polymer comprising the polypeptide or a portion thereof, a composition comprising the polypeptide and / or polymer or a portion thereof, and a method of recombinantly producing the polypeptide and / or polymer or a portion thereof.

Description

Technical Field

[0001] The present invention generally relates to a polypeptide from the honey bee Hylaeus nubilosus of the family Hylaeus. The present invention also relates to the recombinant production of the polypeptide, methods for preparing the polypeptide, and the use of the polypeptide in preparing various products having desired properties. Background Art

[0002] Synthetic polymers (the building blocks of "plastics") are a particularly useful group of materials. Many of the everyday items we wear, sleep in, and create contain synthetic polymers. In many cases, synthetic polymers are used to create various materials to impart desirable properties. Examples of such properties include imparting mechanical strength, heat resistance, wicking properties, or the ability to resist water or moisture.

[0003] Industries that commonly use synthetic polymers include textiles, biomedical devices, and cosmetics. In one example, mass-produced synthetic clothing is composed of various polymers that have been spun into synthetic fibers, including polyester, nylon, vinyl, and acrylic. In some cases, these synthetic fibers are hydrophobic and poorly absorb water or sweat, affecting their comfort. In other cases, the fibers are hydrophilic and too absorbent, which again affects wear resistance.

[0004] In other examples, synthetic polymers can be used to impart wicking, hygroscopic, or hydrophilic properties to textiles or other materials. Current synthetic polymers used in such applications are not environmentally friendly. For example, polyetheramines are used to create hydrophilic coatings on nylon garments. Unfortunately, these polymers degrade over time, releasing harmful byproducts into the environment that are toxic to aquatic life.

[0005] Environmental concerns related to textile finishing chemicals have shifted the focus of major manufacturers toward eco-friendly (bio-based) chemicals. Green chemicals are produced using animal and vegetable fats / oils, making them environmentally friendly and cost-effective compared to their traditional counterparts. However, the added weight of oil-based products, the need to reapply them, and fluctuations in raw material availability and prices have made achieving profitability and economies of scale challenging for market participants.

[0006] Another important use of such coatings is in the medical device industry. Examples of various biomedical devices currently coated with hygroscopic / hydrophilic coatings include catheters, implants, tubes, lenses, and disposable plastic slides. In many instances, these coatings provide excellent biocompatibility, hydrophilicity, hydrophobicity, and / or frictional resistance to the coated biomedical devices (particularly those used in situ), resulting in effective performance.

[0007] Typically, these coatings are composed of polyurethane, silicone, and polyethylene terephthalate materials.

[0008] Synthetic polymers are also used in the production of cosmetics / personal care products to impart properties such as lubricity and viscosity. Phthalates are a class of chemicals known as "ubiquitous chemicals" found in products such as nail polish, perfume, deodorant, hairspray, shampoo, soap, hairspray, and body lotion. Phthalates have been identified as endocrine disruptors in humans, causing hormone imbalances and a variety of reproductive health and developmental problems. Phthalates also bioaccumulate in fish, proving toxic to aquatic ecosystems and posing a hazard to humans.

[0009] The increasing public awareness of the environmental problems associated with the use of synthetic polymers has led to an urgent need in this industrial field to provide non-toxic and environmentally friendly alternatives.

[0010] The object of the present invention is to provide a naturally occurring polypeptide or a derivative thereof which can be used as an environmentally friendly alternative to at least some synthetic polymers currently used in the various industries mentioned above, and / or to provide a method for preparing such a polypeptide or a derivative thereof, and / or to at least provide the public with a useful choice.

[0011] In this specification, references to patent specifications, other external documents or other sources of information are generally made for the purpose of providing a context for discussing the features of the invention. Unless expressly stated otherwise, the reference to such external documents is not to be construed as an admission that such documents or such sources of information are, in any rights whatsoever, prior art or form part of the common general knowledge in the art. Summary of the Invention

[0012] Disclosed herein is a Hylaeus nubilosus polypeptide, also referred to as a "FUN" polypeptide. Also disclosed are protein polymers comprising a H. nubilosus "FUN" polypeptide, or at least a portion of a Hylaeus nubilosus polypeptide. In some embodiments, the protein polymer comprises a quasi-repeated domain. In some embodiments, the polymer is capable of assembling into fibers. Also disclosed are compositions of such polypeptides and protein polymers, as well as methods of producing and using the polypeptides, protein polymers, and compositions.

[0013] Thus, in one aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:2.

[0014] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:2.

[0015] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:2.

[0016] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide having at least 70% amino acid sequence identity to SESAVQSSKSSSGMSSQAQSQQQQX1QLQQSQAQSQEQESQSAX2 (SEQ ID NO: 6).

[0017] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SESAVQSSKSSSGMSSQAQSQQQQX1QLQQSQAQSQEQESQSAX2 (SEQ ID NO: 6).

[0018] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SESAVQSSKSSSGMSSQAQSQQQQX1QLQQSQAQSQEQESQSAX2 (SEQ ID NO: 6).

[0019] In another aspect, the present invention relates to a vector encoding the isolated polypeptide described herein.

[0020] In another aspect, the present invention relates to a vector comprising the isolated polynucleotide described herein.

[0021] In another aspect, the present invention relates to an isolated host cell comprising an isolated polypeptide, an isolated polynucleotide and / or a vector as described herein.

[0022] In another aspect, the present invention relates to a protein polymer comprising a FUN polypeptide as described herein or a portion thereof, wherein the protein polymer comprises at least one quasi-repeat domain.

[0023] In another aspect, the present invention relates to a composition comprising an isolated polypeptide, isolated polynucleotide, carrier and / or proteinaceous polymer as described herein, and a carrier, diluent or excipient.

[0024] In another aspect, the present invention relates to a method of preparing an isolated FUN polypeptide selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 50 and 52, or a portion thereof, comprising heterologously expressing the FUN polypeptide in an isolated host cell and optionally purifying the FUN polypeptide.

[0025] In another aspect, the present invention relates to a method for preparing a protein polymer comprising a FUN polypeptide selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 50 and 52, or a portion thereof, the method comprising heterologously expressing the FUN polypeptide or a portion thereof in an isolated host cell under conditions resulting in the expression of the FUN polypeptide or a portion thereof, and optionally purifying the protein polymer.

[0026] In another aspect, the invention relates to a polypeptide as described herein, produced by the method described herein.

[0027] Various embodiments of the different aspects of the invention as discussed above are also set forth in the following detailed description of the invention, but the invention is not limited thereto.

[0028] Other aspects of the invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0030] Figure 1: Glutamine-rich sequences TRINITY_DN0_c0_g1_i18 (SEQ ID NO: 34), TRINITY_DN0_c0_g1_i13 (SEQ ID NO: 33), TRINITY_DN0_c0_g1_i19 (SEQ ID NO: 35), TRINITY_DN0_c0_g1_i20 (SEQ ID NO: 36), TRINITY_DN220917_c0_g1_i1 (SEQ ID NO: 37), TRINITY_DN0_c0_g1_i9 (SEQ ID NO: 38), TRINITY_DN0_c0_g1_i1 (SEQ ID NO: 39), TRINITY_DN0_c0_g1_i2 (SEQ ID NO: 40), TRINITY_DN0_c0_g1_i16 (SEQ ID NO: 41), TRINITY_DN0_c0_g1_i11_a (SEQ ID NO: 42), TRINITY_DN0_c0_g1_i11_b (SEQ ID NO: 43), TRINITY_DN0_c0_g1_i10 (SEQ ID NO: 44), TRINITY_DN16733_c0_g1_i2 (SEQ ID NO: 45) and TRINITY_DN0_c4_g1_i2 (SEQ ID NO: 46), which were important sequences identified from different entries of the transcript database (version 2).

[0031] Figure 2 : Consensus sequence alignment. Alignment of the FUN_069765.2 sequence (from long read sequence assembly) SEQ ID NO: 2 with the assembled proteome sequence SEQ ID NO: 33 from mass spectrometry analysis of nest material. Consensus sequence: X = mismatch, Z = glutamic acid or glutamine.

[0032] Figure 3 Flowchart of a high-level overview of genetic characterization of nest materials. Data and samples are indicated by uninterrupted borders. Analyses and software programs are bolded within dashed boxes. Research results are outlined within dotted boxes. Arrows indicate directional relationships between boxes. Dashed arrows emphasize relationships between research results.

[0033] Figure 4 : SDS-PAGE showing purified FUN polypeptide. The last three lanes show IMAC elution of a FUN polypeptide band with an apparent molecular weight of ∼100 kDa on SDS-PAGE, with an estimated purity of >95%.

[0034] Figure 5: FTIR FUN peptide coating on foil. FTIR of FUN peptide and silk fibroin coated on aluminum foil with and without ethanol treatment.

[0035] Figure 6 : FTIR-FUN peptide coating on glass. FTIR of FUN peptide coated on glass substrate with and without ethanol treatment.

[0036] Figure 7 : Scanning electron microscopy (SEM) images of FUN polypeptide coatings. The image on the right is a magnified cross-section of the first image, depicting the filamentous structure along the plane of the film.

[0037] Figure 8 : Wettability - Water contact angle profiles of FUN polypeptide immediately after contact and after 30 seconds. Comparison of water contact angle profiles of FUN polypeptide and silk fibroin coated on glass substrates with and without ethanol treatment.

[0038] Figure 9 Washability - The washability of the FUN polypeptide coating and its ability to withstand water and PBS were evaluated. After immersion in water or PBS solution for 24 hours, the FUN polypeptide coating remained intact (as shown by Coomassie staining coverage).

[0039] Figure 10 : Sequence coverage with Glu-C digestion: Bold text indicates high confidence peptide matches, normal text indicates medium confidence, and italics indicates low confidence. Underline Indicates unobserved areas.

[0040] Figure 11 : Combined sequence coverage with Glu-C and trypsin digestion: Reducing and alkylating trypsin digestion produced coverage in the C-terminal region with two high confidence peptides (bold) and one medium peptide match (normal), resulting in an overall coverage of 95.78%.

[0041] Figure 12 : Alkylated trypsin digestion yielded 99.81% total coverage.

[0042] Alkylated trypsin digestion obtained the entire fragment except for the final cysteine ​​residue, resulting in 99.81% total coverage.

[0043] Figure 13 : Photographs and confocal images of tubes of A) oil / buffer emulsion (control); B) oil / buffer emulsion containing HnM1M7-01 after 1 and 2 days.

[0044] Figure 14: A) Oil / buffer emulsion in which no spheroids were observed in the aqueous phase (control); B) Photograph and confocal image of a tube of oil / buffer emulsion containing HnM1M7-01 showing stable spheroids at day 7, reflecting the emulsion-stabilizing effect of HnM1M7-01.

[0045] Figure 15 : Water contact angles of glass slides with and without HnM1M7-03 coating (measured in degrees).

[0046] Figure 16 : Water contact angles of glass slides with and without HnM7-06 coating (measured in degrees).

[0047] Figure 17 : SEM surface morphology of wet-spun nylon and HnP1-nylon (1, 5 and 10 wt.%) fibers.

[0048] Figure 18 : SEM cross-sectional morphologies of wet-spun nylon and HnP1-nylon (1, 5, and 10 HnP1 wt.%) fibers.

[0049] Figure 19 : SEM surface morphology of wet-spun and HnP1-silk (1, 2, 5 and 10 wt.% HnP1) fibers.

[0050] Figure 20 : SEM cross-sectional morphologies of wet-spun and HnP1-silk (1, 2, 5, and 10 wt.%) fibers.

[0051] Figure 21 : Water contact angles (measured in degrees) of wet-spun nylon and HnP1-nylon (1, 2, 5 and 10 wt. %) fibers.

[0052] Figure 22 : Water contact angles (measured in degrees) of wet-spun and HnP1-silk (1, 2, 5 and 10 HnP1 wt. %) fibers. DETAILED DESCRIPTION

[0053] definition

[0054] The following definitions are set forth to better define the present invention and to serve as a guide for those of ordinary skill in the art in practicing the present invention.

[0055] Unless defined otherwise, all technical and scientific terms used herein should be understood to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0056] As used in this specification and claims, the term "comprising" means "consisting at least in part of"; that is, when interpreting statements in this specification and claims that include "comprising," the features beginning with the term in each statement need to be present, but additional features may also be present. Related terms such as "comprise" and "comprises" should be interpreted in a similar manner.

[0057] As used herein, the term "consisting essentially of" refers to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.

[0058] As used herein, the term "consisting of" refers to the specified materials or steps of the claimed invention and excludes any elements, steps, or ingredients not specified in the claims.

[0059] The term "vector" as used herein refers to any type of polynucleotide molecule that can be used to manipulate genetic material so that it can be amplified, replicated, manipulated, partially replicated, modified and / or expressed, but is not limited thereto. In some embodiments, a vector can be used to transport the polynucleotide contained in the vector into a cell or organism.

[0060] As used herein, the term "polynucleotide" refers to a single- or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length, including, by way of non-limiting example, coding and non-coding sequences of a gene, sense and antisense sequences, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polynucleotides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers, fragments, genetic constructs, vectors, and modified polynucleotides. References to nucleic acids, nucleic acid molecules, nucleotide sequences, and polynucleotide sequences should be understood similarly.

[0061] As used herein, the term "gene" refers to a gene that is a biological unit that is inherited, self-replicated, and located at a defined position (locus) on a specific chromosome. In one embodiment, the specific chromosome is a eukaryotic or bacterial chromosome. The term bacterial chromosome can be used interchangeably with the term bacterial genome in this article.

[0062] As used herein, the term "endogenous" refers to a component of a cell, tissue, or organism that originates from or is naturally produced in that cell, tissue, or organism. An "endogenous" component can be any component, including but not limited to a polynucleotide, a polypeptide including a non-ribosomal polypeptide, a fatty acid, or a polyketide.

[0063] As used herein, the term "exogenous" refers to any component of a cell, tissue, or organism that does not originate from or is not naturally produced in that cell, tissue, or organism. An exogenous component can be, for example, a polynucleotide sequence that has been introduced into a cell, tissue, or organism, or a polypeptide expressed in that cell, tissue, or organism from that polynucleotide sequence.

[0064] As used herein, "naturally occurring" with respect to the polynucleotide sequences according to the present invention refers to a primary polynucleotide sequence found in nature. For the purposes of the present invention, a synthetic polynucleotide that is identical to a wild-type polynucleotide sequence is considered a naturally occurring sequence. What is important about a naturally occurring polynucleotide is that the actual sequence of nucleotide bases comprising the polynucleotide is found or known in nature.

[0065] For example, a wild-type polynucleotide sequence is a naturally occurring polynucleotide sequence, but is not limited thereto. A naturally occurring polynucleotide sequence also refers to a variant polynucleotide sequence found in nature that differs from the wild-type sequence. For example, allelic variants resulting from hybridization or horizontal gene transfer and naturally occurring recombinant polynucleotide sequences are examples, but are not limited thereto.

[0066] As used herein, "non-naturally occurring" with respect to the polynucleotide sequences according to the present invention refers to polynucleotide sequences that are not found in nature. Examples of non-naturally occurring polynucleotide sequences include, but are not limited to, artificially generated mutant and variant polynucleotide sequences prepared, for example, by point mutations, insertions, or deletions. Non-naturally occurring polynucleotides also include chemically evolved sequences. Importantly, for non-naturally occurring polynucleotide sequences according to the present invention, the actual sequence of nucleotide bases comprising the polynucleotide is not found or is unknown in nature.

[0067] As used herein with respect to polynucleotides, the term "wild type" refers to a naturally occurring, non-mutated form of a polynucleotide. A mutant polynucleotide refers to a polynucleotide having continuous mutations known in the art, such as point mutations, insertions, deletions, substitutions, amplifications, or translocations, but is not limited thereto.

[0068] When used herein with respect to a polypeptide, the term "wild-type" refers to the naturally occurring, non-mutated form of the polypeptide. A wild-type polypeptide is a polypeptide capable of being expressed by a wild-type polynucleotide.

[0069] The term "coding sequence" (CDS) or "open reading frame" (ORF) refers to a genomic DNA sequence or the sense strand of a cDNA sequence that is capable of producing a transcript and / or polypeptide under the control of appropriate regulatory sequences. A CDS is identified by the presence of a 5' translation start codon and a 3' translation stop codon. When inserted into a gene construct or expression cassette, a "coding sequence" (CDS) is capable of being expressed when operably linked to a promoter sequence and / or other regulatory elements.

[0070] "Operably linked" means that the sequence to be expressed is under the control of regulatory elements.

[0071] As used herein, "regulatory element" refers to any nucleic acid sequence element that controls or influences the expression of a polynucleotide insert from a vector, gene construct, or expression cassette, including promoters, transcriptional control sequences, translational control sequences, origins of replication, tissue-specific regulatory elements, temporal regulatory elements, enhancers, polyadenylation signals, repressors, and terminators. A regulatory element can be "homologous" or "heterologous" to the polynucleotide insert to be expressed from a gene construct, expression cassette, or vector described herein. When a gene construct, expression cassette, or vector described herein is present in a cell, a regulatory element can be "endogenous," "exogenous," "naturally occurring," and / or "non-naturally occurring" with respect to the cell.

[0072] The term "non-coding region" refers to the untranslated sequences upstream of the translation start site and downstream of the translation stop site. These sequences are also referred to as 5' UTR and 3' UTR, respectively. These regions include elements required for transcription initiation and termination, as well as for regulating translation efficiency.

[0073] A terminator is a sequence that terminates transcription and is found at the 3' untranslated end of a gene, downstream of the translated sequence.Terminators are important determinants of mRNA stability and, in some cases, have been found to have spatial regulatory functions.

[0074] The term "promoter" refers to a non-transcriptional cis-regulatory element upstream of the coding region that regulates the transcription of a polynucleotide sequence. Promoters include cis-initiation elements that specify a transcription start site and a conserved box. In a non-limiting example, bacterial promoters can include a "Pribnow box" (also referred to as the -10 region), and other motifs that are combined with and promote transcription by transcription factors. Promoters can be homologous or heterologous to the polynucleotide sequence to be expressed. When the polynucleotide sequence is expressed in a cell, the promoter can be an endogenous or exogenous promoter. Promoters can be constitutive promoters, inducible promoters, or regulatable promoters known in the art.

[0075] As used herein, "homologous" with respect to polynucleotide regulatory elements refers to polynucleotide regulatory elements that are native and naturally occurring polynucleotide regulatory elements. Homologous polynucleotide regulatory elements can be operably linked to a polynucleotide of interest such that the polynucleotide of interest can be expressed from a vector according to the invention.

[0076] As used herein, "homologous" with respect to a polynucleotide or polypeptide in a host organism means that the polynucleotide or polypeptide is a polynucleotide or polypeptide native and naturally occurring in the host organism. The homologous polynucleotide can be operably linked to homologous or heterologous regulatory elements such that the homologous polypeptide can be expressed from a vector comprising the homologous polynucleotide as described herein.

[0077] As used herein, "heterologous" with respect to polynucleotide regulatory elements refers to polynucleotide regulatory elements that are not native and naturally occurring polynucleotide regulatory elements. A heterologous polynucleotide regulatory element is not typically associated with the CDS to which it is operably linked. A heterologous regulatory element can be operably linked to a polynucleotide of interest such that the polynucleotide of interest can be expressed from a polynucleotide or vector according to the present invention. Such promoters can include promoters typically associated with other genes, ORFs, or coding regions, and / or promoters isolated from any other bacteria, viruses, eukaryotic cells, or mammalian cells.

[0078] As used herein, "heterologous" (i.e., "heterologous polynucleotide" or "heterologous polypeptide") with respect to a polynucleotide or polypeptide in a host organism refers to a polynucleotide or polypeptide that is not native and naturally occurring in the host organism. The heterologous polynucleotide can be operably linked to heterologous or homologous regulatory elements such that the heterologous polypeptide can be expressed from a vector comprising the heterologous polynucleotide as described herein.

[0079] The term "heterologously expressing" or "heterologous expression" refers to the expression of a heterologous polypeptide in a host cell.

[0080] A "functional variant or fragment thereof" of a polypeptide is a subsequence of the polypeptide that performs the biological activity of the polypeptide or binds a desired function and / or provides the three-dimensional structure of the polypeptide. The term can refer to a polypeptide, a polypeptide aggregate such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or a functional polypeptide derivative thereof that is capable of performing the activity of the polypeptide.

[0081] As used herein, "isolated" with respect to a polynucleotide or polypeptide sequence describes a sequence that has been removed from its natural cellular environment. An isolated molecule can be obtained by any method or combination of methods known and used in the art, including biochemical, recombinant, and synthetic techniques. A polynucleotide or polypeptide sequence can be prepared by at least one purification step.

[0082] When used herein with respect to a cell or host cell, "isolated" describes a cell or host cell that has been obtained or removed from an organism or its natural environment and subsequently maintained in laboratory settings as known in the art. The term encompasses single cells per se as well as cells or host cells contained in cell culture, and can include single cells or single host cells.

[0083] The term "recombinant" refers to a polynucleotide sequence that is removed from the sequences surrounding it in its natural environment and / or recombined with sequences not found in its natural environment. A "recombinant" polypeptide sequence is produced by translation of a "recombinant" polynucleotide sequence.

[0084] As used herein, the term "variant" refers to a polynucleotide or polypeptide sequence that is different from a specifically identified sequence in which one or more nucleotides or amino acid residues are deleted, substituted, or added. Variants can be naturally occurring allelic variants or non-naturally occurring variants. Variants can be from the same species or from other species and can encompass homologs, paralogs, and orthologs. In certain embodiments, variants of the polypeptides useful in the present invention have the same or similar biological activity as the corresponding wild-type molecule (i.e., the parent polypeptide or polynucleotide).

[0085] In certain embodiments, variants of the polypeptides described herein have similar or substantially similar biological activities as their corresponding wild-type molecules. In certain embodiments, the similarity is similar activity and / or binding specificity.

[0086] In certain embodiments, variants of the polypeptides described herein have a biological activity that is different from that of their corresponding wild-type molecules. In certain embodiments, the difference is an altered activity and / or binding specificity.

[0087] The term "variant" with reference to polynucleotides and polypeptides encompasses all forms of the polynucleotides and polypeptides as defined herein.

[0088] The variant polynucleotide sequences preferably exhibit at least 50%, at least 60%, preferably at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, and preferably at least 99% identity to the sequences of the present invention. Identity is found over a comparison window of at least 8 nucleotide positions, preferably at least 10 nucleotide positions, preferably at least 15 nucleotide positions, preferably at least 20 nucleotide positions, preferably at least 27 nucleotide positions, preferably at least 40 nucleotide positions, preferably at least 50 nucleotide positions, preferably at least 60 nucleotide positions, preferably at least 70 nucleotide positions, preferably at least 80 nucleotide positions, preferably over the entire length of the polynucleotide used or identified according to the method of the invention.

[0089] Polynucleotide variants also encompass those that exhibit similarity to one or more of the specifically identified sequences, are likely to retain functional equivalence to those sequences, and are unlikely to have arisen by chance.

[0090] Polynucleotide sequence identity and similarity can be readily determined by one skilled in the art.

[0091] Variant polynucleotides also encompass polynucleotides that differ from the polynucleotide sequences described herein, but which, due to the degeneracy of the genetic code, encode polypeptides having similar activities to the polypeptides encoded by the polynucleotides of the present invention. Sequence changes that do not alter the amino acid sequence of a polypeptide are "silent variations." With the exception of ATG (methionine) and TGG (tryptophan), other codons for the same amino acid can be altered by techniques recognized in the art, for example, to optimize codon expression in a particular host organism.

[0092] Polynucleotide sequence changes that result in conservative substitutions of one or more amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also encompassed by the present invention. Methods for performing phenotypically silent amino acid substitutions are known to those skilled in the art (see, e.g., Bowie et al., 1990, Science 247, 1306).

[0093] The term "variant" with respect to polypeptides also encompasses naturally occurring, recombinant and synthetically produced polypeptides. Variant polypeptide sequences preferably exhibit at least 35%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% and preferably at least 99% identity to the sequences of the present invention. Identity is found over a comparison window of at least 2 amino acid positions, preferably at least 3 amino acid positions, preferably at least 4 amino acid positions, preferably at least 5 amino acid positions, preferably at least 7 amino acid positions, preferably at least 10 amino acid positions, preferably at least 15 amino acid positions, preferably at least 20 amino acid positions, preferably over the entire length of the polypeptide used or identified according to the method of the invention.

[0094] Polypeptide variants also encompass those that exhibit similarity to one or more specifically identified sequences, are likely to retain functional equivalence to those sequences, and are unlikely to have arisen by chance.

[0095] Polypeptide sequence identity and similarity can be readily determined by one skilled in the art.

[0096] Variant polypeptides include polypeptides whose amino acid sequences differ from those of the polypeptides herein by one or more conservative amino acids or non-conservative substitutions, deletions, additions, or insertions that do not affect the biological activity of the peptide.

[0097] Conservative substitutions generally involve substituting one amino acid for another amino acid having similar properties, as known and used in the art.

[0098] Analysis of evolved biological sequences shows that not all sequence variations are equally likely, which at least in part reflects the difference between conservative and non-conservative substitutions at the biological level. For example, some amino acid substitutions may occur frequently, while others are very rare. Evolutionary changes or substitutions in amino acid residues can be modeled using a scoring matrix (also referred to as a substitution matrix). Such matrices are used in bioinformatics analysis to identify relationships between sequences and are known to those skilled in the art.

[0099] Other variants include peptides with modifications that affect peptide stability. Such analogs may contain, for example, one or more non-peptide bonds in the peptide sequence (which replace peptide bonds). Also included are analogs containing residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring synthetic amino acids, such as β or γ amino acids and cyclic analogs.

[0100] Substitutions, deletions, additions or insertions can be made by mutagenesis methods known in the art. The skilled artisan is aware of methods for making phenotypically silent amino acid substitutions. See, for example, Bowie et al., 1990, Science 247, 1306.

[0101] The term "peptide" includes a chain of amino acids linked by peptide bonds. The term "peptide" may also refer to a "protein" or "polypeptide," a molecule comprising amino acids arranged in a linear chain that can also fold into a globular shape. As used herein, a protein generally refers to a molecule comprising more than about 200 amino acids, up to the full-length sequence translated from a gene; a polypeptide generally refers to a molecule comprising more than about 100 amino acids; and a peptide generally refers to a molecule comprising from about 2 to about 100 amino acids. For convenience, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein. As contemplated herein, a protein may represent any macromolecule containing an amine or thiol. In this regard, a "protein or peptide" may comprise an amino acid sequence comprising at least one common amino acid found in naturally occurring proteins, or at least one modified or unusual amino acid. Proteins, polypeptides, and peptides, including the FUN polypeptides described herein and portions thereof, can be prepared by a variety of techniques known to those skilled in the art, including expressing the protein, polypeptide, or peptide by standard molecular biology techniques, isolating the protein or peptide from a natural source, or chemically synthesizing the protein or peptide.

[0102] As used herein, polypeptides may also refer to polypeptides that have been modified during or after synthesis, for example, by biotinylation, benzylation, glycosylation, phosphorylation, amidation, derivatization with blocking / protecting groups, etc. Such modifications may increase the stability or activity of the polypeptide.

[0103] The term "modulate(s) expression / modulated expression / modulating expression" of a polynucleotide or polypeptide is intended to encompass situations in which the genomic DNA corresponding to the polynucleotide to be expressed according to the present invention is modified, thereby resulting in the regulated expression of the polynucleotide or polypeptide of the present invention. Modification of the genomic DNA can be carried out by genetic transformation or other methods known in the art for inducing mutations. "Modulated expression" may be associated with an increase or decrease in the amount of the messenger RNA and / or polypeptide produced, and may also result in an increase or decrease in the activity of the polypeptide due to changes in the sequence of the polynucleotide and polypeptide produced.

[0104] The terms "modulate(s)activity / modulated activity / modulating activity" of a polynucleotide or polypeptide are intended to encompass situations in which the genomic DNA corresponding to the polynucleotide to be expressed according to the present invention is modified so as to result in regulated expression of the polynucleotide of the present invention or regulated expression or activity of the polypeptide. Modification of the genomic DNA can be carried out by genetic transformation or other methods known in the art for inducing mutations. "Modulating activity" may be associated with an increase or decrease in the amount of the messenger RNA and / or polypeptide produced, and may also result in an increase or decrease in the functional activity of the polypeptide due to changes in the sequence of the polynucleotide and polypeptide produced.

[0105] In the context of a protein polymer described herein, the phrase "polypeptide or portion thereof" refers to a polypeptide described herein or a portion of the polypeptide that is incorporated into the protein polymer. The polypeptide or portion thereof comprises a quasi-repeated domain.

[0106] As used herein, the term "quasi-repeated domain" and its grammatical variations refer to imperfect repeats.

[0107] Reference to a numerical range disclosed herein (e.g., 1 to 10) is intended to also include reference to all related numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) as well as any rational number range within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and thus, all subranges of all ranges explicitly disclosed herein are explicitly disclosed. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between the lowest and highest values ​​recited are to be considered to be expressly stated in this application in a similar manner.

[0108] The present invention generally relates to a polypeptide of a nesting material protein from the solitary honey bee Hylaeus nubilosus, and portions, functional analogs, variants, and / or derivatives thereof. The present invention also generally relates to a polynucleotide encoding such a polypeptide, a protein polymer comprising such a polypeptide or a portion thereof comprising a quasi-repeated domain, and methods for preparing such a polypeptide and a protein polymer comprising such a polypeptide or a portion thereof, comprising heterologously expressing the polynucleotide in a suitable isolated host cell.

[0109] Bees of the genus Hylaeus (Hymenoptera: Colletidae) produce nesting material that has been described as "cellophane-like" (Almeida, EABC Colletidae nesting biology (Hymenoptera: Apoidea). Apidologie 39, 16–29 (2008)).

[0110] Previously published results on nesting material produced by closely related honey bees indicated that this material is a unique composite of lipid polymers and protein biopolymers. To better understand the basis for the observed properties, the inventors investigated the nesting material to try to identify the components of the material that are responsible for its surprising properties.

[0111] Molecular analysis of the Dunaliella, labial, mandibular glands, and nesting material of H. nubilosus identified a single major protein component (>10%) of the hive material of H. nubilosus, designated FUN_069765-T1 (referred to herein as the "FUN" polypeptide). FUN is a silk-like protein, but is not similar to or homologous to the silk known to be produced by Hymenoptera (ants, wasps, and bees). This difference is due to the nature of FUN and the way FUN is produced. For example, larval honey bees (Apis mellifera) produce protein-rich silk in their labial glands to seal the hive prior to pupation. Honey bee silk is made of proteins that fold primarily into α-helices and then assemble into larger supersecondary structures called coiled coils. The honey bee protein consists of four small fibroin subunits, ~30 kDa long, and contains ~30% alanine.

[0112] In contrast, FUN from H. nubilosus has a β-sheet-rich structure, which is identical to the structure of silk fibers made by the silkworm (Bombyx mori) or the dragline silk of the spider, but different from the α-helical structure that dominates larval honeybee silk ( Figure 5 and Figure 6FUN polypeptides are rich in glutamine and serine, which are not characteristic of bee silk, silkworm silk, or spider silk. Although an asparagine-rich silk protein has been reported from a distantly related parasitic wasp (Cotesia glomerata) (asparagine and glutamine are similar amino acids), glutamine-rich silk proteins are unknown.

[0113] Using a combination of genomic sequencing, transcriptome sequencing, and proteomic approaches, the inventors have determined the nucleic acid and amino acid sequences of the FUN polypeptide, including numerous sequences rich in glutamine and serine. Without wishing to be bound by theory, the applicants believe that the FUN polypeptide is contained within a protein polymer that is itself contained within or comprises the Hylaeus nubilosus nesting material. Furthermore, without wishing to be bound by theory, the inventors believe that the polypeptide and / or protein polymer comprising the polypeptide or at least a portion thereof imparts important structural and functional properties to the Hylaeus nubilosus nesting material.

[0114] Applicants believe that the FUN polypeptides described herein, and variants, analogs, and derivatives thereof, have numerous applications in materials that generally utilize known synthetic polymers and biopolymers, including silk proteins.

[0115] As described herein, applicants have provided, for the first time, silk or silk-like proteins from H. nubilosus. To achieve this goal, applicants analyzed the genomic sequence of H. nubilosus and identified a single predicted coding sequence (CDS) for the major protein associated with nest material: FUN_069765-T1 genomic DNA (SEQ ID NO:25) and FUN_069765-T1 cDNA (SEQ ID NO:1). The predicted amino acid sequence of the FUN_069765-T1 polypeptide from H. nubilosus is set forth in SEQ ID NO:2. This full-length polypeptide (i.e., SEQ ID NO:2), as well as other amino acid subsequences and functional variants disclosed herein and described by primary sequences including, but not necessarily limited to, SEQ ID NOs:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 50, and 52, are referred to herein as FUN polypeptides.

[0116] In addition, the polypeptide produced according to SEQ ID NO: 12 described herein is referred to in this document by different names, including: "FSS with cleaved secretion tag (FUNSecSf9)"; "FSS"; "HBB B3.0"; and "HnM1M7-01". All names describe the same polypeptide and are used synonymously.

[0117] The polypeptide produced according to SEQ ID NO: 4 described herein is referred to in this document by "consensus 43mer" or "HnP1." Both names describe the same polypeptide and are used synonymously.

[0118] The applicant also provided for the first time a method for heterologously expressing a silk-like protein from H. nubilosus in an isolated host cell.

[0119] As described in the Examples herein, the inventors have for the first time identified the gene for the full-length FUN polypeptide from H. nubilosus.

[0120] Analysis of the predicted coding sequence (i.e., cDNA) of the full-length FUN polypeptide (SEQ ID NO: 2) using an evolutionary failure mode (EFM) calculator revealed a highly diverse array of hundreds of repetitive DNA motifs, ranging in frequency and length from two copies of a 770 bp motif to 117 copies of a 16 bp sequence.

[0121] The predicted primary amino acid sequence of the full-length FUN polypeptide (SEQ ID NO: 2) reveals numerous repetitive amino acid sequence motifs—perfect (or identical) repeats and imperfect (or quasi) repeats—organized in a complex manner. The RADAR (Rapid Automated Detection and Alignment of Repeats) tool, which allows for mismatches and gaps, identified several classes of repeats, but the imperfect and fragmented nature of many of these repeats led to a complex picture of the protein's overall architecture.

[0122] Using these initial surveys as the basis for more extensive analysis, it was found that almost the entire protein could be organized as a continuous series of peptide domains (also referred to herein as FUN polypeptides), each built around a basic, repetitive 43 amino acid motif found only in the QS repeat region and having the following consensus sequence (with a threshold of >50%): SESAVQSSKSSSGMSSQAQSQQQQX1QLQQSQAQSQEQESQSAX2 (SEQ ID NO: 6).

[0123] In some embodiments, X1 and X2 are independently any amino acid. By accommodating a limited number of substitutions at key residues and gaps, the entire protein can be organized according to the repetition of this 43-mer consensus sequence, except for the first 137 amino acids (non-repeated region) and the spacer repeat sequence.

[0124] In one embodiment, X1 = A or S. In one embodiment, X2 = E, G or Q.

[0125] 75 peptides from the QS repeat region conformed to the strict 43 residue length of the consensus sequence, and these 75 repeats were referred to as class A repeats.

[0126] Interspersed between these class A repeats are shorter versions of the consensus sequence, also located in the QS repeat region, 35-36 residues in length, which can be roughly divided into two categories; those with a tetraglutamine (QQQQ) motif (referred to as class B) and restricted to the second QS repeat region, see, for example, amino acid residues 635-670 of SEQ ID NO: 2 having the following amino acid sequence:

[0127] SESAVQSSKSSSGMSSQAQSQQQQAQSQEQESQSAQ (SEQ ID NO: 18),

[0128] and those that lack the tetraglutamine motif (Category C) and are dispersed throughout the molecule, see, for example, amino acid residues 1508-1543 of SEQ ID NO: 2 having the following amino acid sequence:

[0129] SESDVQSSKSSRGMSSHAQSQQSQAQSQEQESQSAQ (SEQ ID NO: 20).

[0130] By definition, class A, B, and C repeats are found only within the QS repeat region, interspersed with a small number of irregular repeats called spacer repeats (class X), which can align with each other but not with the QS repeat region sequence. These irregular spacer repeats contain most of the proline and cysteine ​​residues found in proteins.

[0131] Except for the final spacer repeat sequence that forms the C-terminus of the protein, all other spacer repeat sequences are followed by a variable length repeat sequence ranging from 23 to 38 residues in length, referred to as class D, for example, amino acid residues 3087-3109 of SEQ ID NO: 2 having the following amino acid sequence: MSSRAQSQQSQAQSQEQESQSAQ (SEQ ID NO: 22).

[0132] In addition, the spacer repeat sequence is preceded by another variable length repeat sequence ranging from 5 to 40 residues in length, referred to as class E; see, for example, amino acid residues 2249-2259 of SEQ ID NO: 2 having the following amino acid sequence: SESAVQSSKSS (SEQ ID NO: 24).

[0133] Although the lengths of class D and class E repeats are highly variable, they can all align to a 43-mer consensus sequence.

[0134] The repeat region of the FUN polypeptide comprises amino acid residues 138 to 5451 of SEQ ID NO: 2 and is organized as a series of contiguous peptide domains aligned with the 43-mer and spacer repeat consensus motifs.

[0135] Following the non-repetitive N-terminal domain (NTD or N, amino acid residues 18-137 of SEQ ID NO: 2) is a 21-mer peptide (amino acid residues 138-158 of SEQ ID NO: 2) that, although unable to be assigned to any other repeat sequence class, can still be aligned with the 43-mer consensus sequence (referred to as class F).

[0136] protein synthesis

[0137] The FUN polypeptides described herein and / or portions thereof can be prepared by chemical synthesis using methods such as solution phase synthesis or solid phase peptide synthesis, with or without chemical ligation, followed by purification of the resulting peptides. In some embodiments, the FUN polypeptides described herein or portions thereof are prepared by chemical synthesis.

[0138] Protein expression

[0139] To confirm the function of the FUN polypeptide and characterize its properties, the inventors constructed a series of expression vectors and transformed these vectors into suitable hosts for heterologous production of the FUN_069765-T1 protein.

[0140] As described herein, after transformation and expression of the FUN polypeptide and its various portions in a suitable host, the inventors initially determined the chemical phenotype of the transformants by normal phase thin layer chromatography (TLC, results not shown) and subsequently by reverse phase liquid chromatography-mass spectrometry (LC-MS) analysis of cell extracts. The inventors purified the newly expressed metabolites as determined by high resolution mass spectrometry (HRMS), by semi-preparative reverse phase high performance liquid chromatography (HPLC), and performed nuclear magnetic resonance (NMR) spectroscopy analysis on the compounds ( 1 H. 13 C and HSQC, HMBC, COSY) for final identification.

[0141] Based on the work described herein, the inventors disclose the use of heterologous expression to produce recombinant FUN polypeptides. In summary, the inventors' work described herein confirms that heterologous expression of at least a portion of a complete FUN polypeptide from H. nubilosus in a heterologous host is a viable method for artificially producing such polypeptides, including portions, variants, analogs, and derivatives thereof, using recombinant biosynthetic systems. As described above, the polypeptides described herein by SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 50, and 52 are referred to as FUN polypeptides.

[0142] In one aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:2.

[0143] In one embodiment, the isolated polynucleotide encodes a polypeptide having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:2.

[0144] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. In one embodiment, the isolated polynucleotide encodes a polypeptide consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: 2.

[0145] In one embodiment, the polypeptide comprises 1 to 75 copies of SEQ ID NO:6.

[0146] In one embodiment, the polypeptide comprises at least one copy of SEQ ID NO:18.

[0147] In one embodiment, the polypeptide comprises at least one copy of SEQ ID NO:20.

[0148] In one embodiment, the polypeptide comprises at least one copy of SEQ ID NO:22.

[0149] In one embodiment, the polypeptide comprises at least one copy of SEQ ID NO:24.

[0150] In one embodiment, the isolated polypeptide comprises at least one copy of SEQ ID NO:52.

[0151] In one embodiment, the polypeptide comprises at least two copies of SEQ ID NO: 6 separated by at least one copy of SEQ ID NO: 24 and at least one copy of SEQ ID NO: 22. In one embodiment, the polypeptide has the following organization: SEQ ID NO: 6 or SEQ ID NO: 20 - SEQ ID NO: 24 - spacer - SEQ ID NO: 22 - SEQ ID NO: 6 or SEQ ID NO: 20.

[0152] In one embodiment, the isolated polypeptide comprises, consists essentially of, or consists of at least 25% serine, 25% glutamine, and 5% glutamic acid residues.

[0153] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0154] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 1. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 1.

[0155] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:2.

[0156] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 1. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 1.

[0157] Specifically contemplated as embodiments of this aspect of the invention are all embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:2.

[0158] The isolated polynucleotide molecules described herein can be separated from biological samples using a variety of techniques known to those of ordinary skill in the art. As an example, such polynucleotides can be separated using polymerase chain reaction (PCR) known in the art. The nucleic acid molecules can be amplified using primers defined herein that are derived from polynucleotide sequences described herein.

[0159] Other methods for isolating polynucleotides include using all or part of the polynucleotides described herein as hybridization probes. The technology of hybridizing the polynucleotide probes labeled with polynucleotides immobilized on solid supports such as nitrocellulose filters or nylon membranes can be used to screen genomic libraries or cDNA libraries. Similarly, the probes can be coupled to beads and hybridized with the target sequence. Separation can be affected using known techniques such as magnetic separation. The selection of suitable stringent hybridization and washing conditions is considered to be within the skill of those skilled in the art.

[0160] Polynucleotide fragments can be produced by techniques well known in the art, such as restriction endonuclease digestion and oligonucleotide synthesis.

[0161] In methods well known in the art, partial polynucleotide sequences can be used as probes to identify the corresponding full-length polynucleotide sequences in a sample. These methods include methods known in the art based on PCR, methods based on 5' RACE and hybridization, and methods based on computers / databases. Detectable labels such as radioisotopes, fluorescent labels, chemiluminescent labels, and bioluminescent labels can be used to assist in detection. Inverse PCR also allows, starting from primers based on known regions, to obtain unknown sequences flanking the polynucleotide sequences disclosed herein, as known and used in the art. This method uses several restriction enzymes to generate suitable fragments in the known regions of the gene. The fragments are then cyclized by intramolecular ligation and used as PCR templates. Divergent primers are designed from known regions. In order to physically assemble full-length clones, standard molecular biology methods known in the art can be utilized. Primers and primer pairs that allow amplification of the polynucleotides of the present invention are also contemplated as embodiments disclosed herein.

[0162] Variants (including orthologs) can be identified by the described method. Variant polynucleotides can be identified using PCR-based methods known in the art. Typically, the polynucleotide sequence of primers that can be used for PCR amplification of polynucleotide molecule variants can be based on the sequence of the conserved region encoding the corresponding amino acid sequence.

[0163] Other methods for identifying variant polynucleotides include using all or part of a given polynucleotide to screen a genomic library or cDNA library as described above as a hybridization probe. Typically, probes based on sequences in the conserved regions of the corresponding amino acid sequences can be used. Hybridization conditions may also be less stringent than those used when screening the sequence identical to the probe.

[0164] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:2.

[0165] In one embodiment, the isolated polypeptide has at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:2.

[0166] In one embodiment, the isolated polypeptide comprises SEQ ID NO: 2. In one embodiment, the isolated polypeptide consists of or consists essentially of SEQ ID NO: 2.

[0167] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0168] In one embodiment, the isolated polypeptide comprises 1 to 75 copies of SEQ ID NO:6.

[0169] In one embodiment, the isolated polypeptide comprises at least one copy of SEQ ID NO:18.

[0170] In one embodiment, the isolated polypeptide comprises at least one copy of SEQ ID NO:20.

[0171] In one embodiment, the isolated polypeptide comprises at least one copy of SEQ ID NO:22.

[0172] In one embodiment, the isolated polypeptide comprises at least one copy of SEQ ID NO:24.

[0173] In one embodiment, the isolated polypeptide comprises at least one copy of SEQ ID NO:52.

[0174] In one embodiment, the polypeptide comprises at least two copies of SEQ ID NO: 6 separated by at least one copy of SEQ ID NO: 24 and at least one copy of SEQ ID NO: 22. In one embodiment, the polypeptide has the following organization: SEQ ID NO: 6 or SEQ ID NO: 20 - SEQ ID NO: 24 - spacer - SEQ ID NO: 22 - SEQ ID NO: 6 or SEQ ID NO: 20.

[0175] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 1. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 1.

[0176] In one embodiment, the nucleic acid sequence further comprises a heterologous regulatory element. In one embodiment, the regulatory element comprises a nucleic acid sequence encoding a signal peptide.

[0177] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide having at least 70% amino acid sequence identity to SESAVQSSKSSSGMSSQAQSQQQQX1QLQQSQAQSQEQESQSAX2 (SEQ ID NO: 6).

[0178] In one embodiment, the isolated polynucleotide encodes a polypeptide having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:6.

[0179] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment, the isolated polynucleotide encodes a polypeptide consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: 6.

[0180] In one embodiment, X1 and X2 are independently any amino acid. In one embodiment, X1 = A or S. In one embodiment, X2 = E, G or Q.

[0181] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0182] In one embodiment, the isolated polypeptide is contained in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 6. In one embodiment, the polymer comprises from two to 75 copies of SEQ ID NO: 6.

[0183] In one embodiment, the polypeptide comprises at least two copies of SEQ ID NO: 6 separated by at least one copy of SEQ ID NO: 24 and at least one copy of SEQ ID NO: 22. In one embodiment, the polypeptide has the following organization (SEQ ID NO: 6 or SEQ ID NO: 20 - SEQ ID NO: 24 - spacer - SEQ ID NO: 22 - SEQ ID NO: 6 or SEQ ID NO: 20).

[0184] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 5. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 5.

[0185] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SESAVQSSKSSSGMSSQAQSQQQQX1QLQQSQAQSQEQESQSAX2 (SEQ ID NO: 6). In one embodiment, X1 or X2 are independently any amino acid. In one embodiment, X1 = A or S. In one embodiment, X2 = E, G or Q.

[0186] Specifically contemplated as embodiments of this aspect of the invention are all embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO: 6. Those skilled in the art will also recognize that the above disclosure relating to isolated polynucleotides comprising, consisting essentially of, or consisting of SEQ ID NO: 1 equally and appropriately applies to this aspect of the invention.

[0187] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SESAVQSSKSSSGMSSQAQSQQQQX1QLQQSQAQSQEQESQSAX2 (SEQ ID NO: 6).

[0188] In one embodiment, the isolated polypeptide has at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:6.

[0189] In one embodiment, the isolated polypeptide comprises SEQ ID NO: 6. In one embodiment, the isolated polypeptide consists of or consists essentially of SEQ ID NO: 6.

[0190] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0191] In one embodiment, the polypeptide is contained in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 6. In one embodiment, the polymer comprises from two to 75 copies of SEQ ID NO: 6.

[0192] In one embodiment, the polypeptide comprises at least two copies of SEQ ID NO: 6 separated by at least one copy of SEQ ID NO: 24 and at least one copy of SEQ ID NO: 22. In one embodiment, the polypeptide has the following organization: SEQ ID NO: 6 or SEQ ID NO: 20 - SEQ ID NO: 24 - spacer - SEQ ID NO: 22 - SEQ ID NO: 6 or SEQ ID NO: 20.

[0193] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 5. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 5.

[0194] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide having at least 70% amino acid sequence identity to MSSQAQSQQQQSQLQQSQAQSQEQESQSAX1SGSDVX2X3SKSSSG (SEQ ID NO: 4).

[0195] In one embodiment, X1, X2 and X3 are independently any amino acid. In one embodiment, X1 = G or Q. In one embodiment, X2 = Q or E. In one embodiment, X3 = S or V.

[0196] In one embodiment, the isolated polynucleotide encodes a polypeptide having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:4.

[0197] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4. In one embodiment, the isolated polynucleotide encodes a polypeptide consisting essentially of or consisting of the amino acid sequence of SEQ ID NO:4.

[0198] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0199] In one embodiment, the polypeptide is comprised in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 4.

[0200] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 3. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 3.

[0201] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising MSSQAQSQQQQSQLQQSQAQSQEQESQSAX1SGSDVX2X3SKSSSG (SEQ ID NO: 4). In one embodiment, X1, X2, and X3 are independently any amino acid. In one embodiment, X1 = G or Q. In one embodiment, X2 = Q or E. In one embodiment, X3 = S or V.

[0202] Specifically contemplated as embodiments of this aspect of the invention are all embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO: 4. Those skilled in the art will also recognize that the above disclosure relating to isolated polynucleotides comprising, consisting essentially of, or consisting of SEQ ID NO: 1 equally and appropriately applies to this aspect of the invention.

[0203] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to MSSQAQSQQQQSQLQQSQAQSQEQESQSAX1SGSDVX2X3SKSSSG (SEQ ID NO: 4).

[0204] In one embodiment, X1, X2 and X3 are independently any amino acid. In one embodiment, X1 = G or Q. In one embodiment, X2 = Q or E. In one embodiment, X3 = S or V.

[0205] In one embodiment, the isolated polypeptide has at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:4.

[0206] In one embodiment, the isolated polypeptide comprises SEQ ID NO: 4. In one embodiment, the isolated polypeptide consists of or consists essentially of SEQ ID NO: 6.

[0207] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0208] In one embodiment, the polypeptide is comprised in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 6.

[0209] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 3. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 3.

[0210] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:8.

[0211] In one embodiment, the isolated polynucleotide encodes a polypeptide having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:8.

[0212] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 8. In one embodiment, the isolated polynucleotide encodes a polypeptide consisting essentially of or consisting of the amino acid sequence of SEQ ID NO:8.

[0213] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0214] In one embodiment, the polypeptide is comprised in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 8.

[0215] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 7. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 7.

[0216] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:8.

[0217] Specifically contemplated as embodiments of this aspect of the invention are all embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO: 8. One skilled in the art will also recognize that the above disclosure relating to isolated polynucleotides comprising, consisting essentially of, or consisting of SEQ ID NO: 1 equally and appropriately applies to this aspect of the invention.

[0218] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:8.

[0219] In one embodiment, the isolated polypeptide has at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:8.

[0220] In one embodiment, the isolated polypeptide comprises SEQ ID NO: 8. In one embodiment, the isolated polypeptide consists of or consists essentially of SEQ ID NO: 8.

[0221] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0222] In one embodiment, the polypeptide is comprised in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 8.

[0223] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 7. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 7.

[0224] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:10.

[0225] In one embodiment, the isolated polynucleotide encodes a polypeptide having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO: 10.

[0226] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 10. In one embodiment, the isolated polynucleotide encodes a polypeptide consisting essentially of or consisting of the amino acid sequence of SEQ ID NO:10.

[0227] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0228] In one embodiment, the polypeptide is comprised in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 10.

[0229] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 9. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 9.

[0230] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:10.

[0231] Specifically contemplated as embodiments of this aspect of the invention are all embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO: 10. One skilled in the art will also recognize that the above disclosure relating to isolated polynucleotides comprising, consisting essentially of, or consisting of SEQ ID NO: 1 equally and appropriately applies to this aspect of the invention.

[0232] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:10.

[0233] In one embodiment, the isolated polypeptide has at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:10.

[0234] In one embodiment, the isolated polypeptide comprises SEQ ID NO: 10. In one embodiment, the isolated polypeptide consists of or consists essentially of SEQ ID NO: 10.

[0235] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0236] In one embodiment, the polypeptide is comprised in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 10.

[0237] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 9. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 9.

[0238] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide having at least 70% amino acid sequence identity to (SEQ ID NO: 12).

[0239] In one embodiment, the isolated polynucleotide encodes a polypeptide having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO: 12.

[0240] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 12. In one embodiment, the isolated polynucleotide encodes a polypeptide consisting essentially of or consisting of the amino acid sequence of SEQ ID NO:12.

[0241] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0242] In one embodiment, the polypeptide is comprised in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 12.

[0243] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 11. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 11.

[0244] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising (SEQ ID NO: 12).

[0245] Specifically contemplated as embodiments of this aspect of the invention are all embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO: 12. One skilled in the art will also recognize that the disclosure above relating to isolated polynucleotides comprising, consisting essentially of, or consisting of SEQ ID NO: 1 equally and appropriately applies to this aspect of the invention.

[0246] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to (SEQ ID NO: 12).

[0247] In one embodiment, the isolated polypeptide has at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:12.

[0248] In one embodiment, the isolated polypeptide comprises SEQ ID NO: 12. In one embodiment, the isolated polypeptide consists of or consists essentially of SEQ ID NO:12.

[0249] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0250] In one embodiment, the polypeptide is comprised in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 12.

[0251] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 11. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 11.

[0252] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:14.

[0253] In one embodiment, the isolated polynucleotide encodes a polypeptide having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:14.

[0254] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, the isolated polynucleotide encodes a polypeptide consisting essentially of or consisting of the amino acid sequence of SEQ ID NO:14.

[0255] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0256] In one embodiment, the polypeptide is comprised in a protein polymer.In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2.

[0257] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 13. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 13.

[0258] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:14.

[0259] Specifically contemplated as embodiments of this aspect of the invention are all embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO: 14. One skilled in the art will also recognize that the disclosure above relating to isolated polynucleotides comprising, consisting essentially of, or consisting of SEQ ID NO: 1 equally and appropriately applies to this aspect of the invention.

[0260] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:14.

[0261] In one embodiment, the isolated polypeptide has at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:14.

[0262] In one embodiment, the isolated polypeptide comprises SEQ ID NO: 14. In one embodiment, the isolated polypeptide consists of or consists essentially of SEQ ID NO:14.

[0263] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0264] In one embodiment, the polypeptide is comprised in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 14.

[0265] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 13. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 13.

[0266] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:16.

[0267] In one embodiment, the isolated polynucleotide encodes a polypeptide having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO: 16.

[0268] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 16. In one embodiment, the isolated polynucleotide encodes a polypeptide consisting essentially of or consisting of the amino acid sequence of SEQ ID NO:16.

[0269] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0270] In one embodiment, the polypeptide is comprised in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 16.

[0271] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 15. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 15.

[0272] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:16.

[0273] Specifically contemplated as embodiments of this aspect of the invention are all embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO: 16. One skilled in the art will also recognize that the above disclosure relating to isolated polynucleotides comprising, consisting essentially of, or consisting of SEQ ID NO: 1 equally and appropriately applies to this aspect of the invention.

[0274] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:16.

[0275] In one embodiment, the isolated polypeptide has at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:16.

[0276] In one embodiment, the isolated polypeptide comprises SEQ ID NO: 16. In one embodiment, the isolated polypeptide consists of or consists essentially of SEQ ID NO:16.

[0277] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0278] In one embodiment, the polypeptide is comprised in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 16.

[0279] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 15. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 15.

[0280] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:18.

[0281] In one embodiment, the isolated polynucleotide encodes a polypeptide having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO: 18.

[0282] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 18. In one embodiment, the isolated polynucleotide encodes a polypeptide consisting essentially of or consisting of the amino acid sequence of SEQ ID NO:18.

[0283] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0284] In one embodiment, the isolated polypeptide is contained in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 18. In one embodiment, the polymer comprises two to six copies of SEQ ID NO: 18.

[0285] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 17. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 17.

[0286] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:18.

[0287] Specifically contemplated as embodiments of this aspect of the invention are all embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO: 18. One skilled in the art will also recognize that the above disclosure relating to isolated polynucleotides comprising, consisting essentially of, or consisting of SEQ ID NO: 1 equally and appropriately applies to this aspect of the invention.

[0288] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:18.

[0289] In one embodiment, the isolated polypeptide has at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:18.

[0290] In one embodiment, the isolated polypeptide comprises SEQ ID NO: 18. In one embodiment, the isolated polypeptide consists of or consists essentially of SEQ ID NO:18.

[0291] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0292] In one embodiment, the polypeptide is contained in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 18. In one embodiment, the polymer comprises two to six copies of SEQ ID NO: 18.

[0293] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 17. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 17.

[0294] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:20.

[0295] In one embodiment, the isolated polynucleotide encodes a polypeptide having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:20.

[0296] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 20. In one embodiment, the isolated polynucleotide encodes a polypeptide consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: 20.

[0297] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0298] In one embodiment, the isolated polypeptide is contained in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 20. In one embodiment, the polymer comprises from two to 29 copies of SEQ ID NO: 20.

[0299] In one embodiment, the polypeptide comprises at least two copies of SEQ ID NO: 20 separated by at least one copy of SEQ ID NO: 24 and at least one copy of SEQ ID NO: 22. In one embodiment, the polypeptide has the following structure: SEQ ID NO: 6 or SEQ ID NO: 20 - SEQ ID NO: 24 - spacer - SEQ ID NO: 22 - SEQ ID NO: 6 or SEQ ID NO: 20.

[0300] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 19. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 19.

[0301] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:20.

[0302] Specifically contemplated as embodiments of this aspect of the invention are all embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO: 20. Those skilled in the art will also recognize that the above disclosure relating to isolated polynucleotides comprising, consisting essentially of, or consisting of SEQ ID NO: 1 equally and appropriately applies to this aspect of the invention.

[0303] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:20.

[0304] In one embodiment, the isolated polypeptide has at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:20.

[0305] In one embodiment, the isolated polypeptide comprises SEQ ID NO: 20. In one embodiment, the isolated polypeptide consists of or consists essentially of SEQ ID NO: 20.

[0306] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0307] In one embodiment, the polypeptide is contained in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 20. In one embodiment, the polymer comprises from two to 29 copies of SEQ ID NO: 20.

[0308] In one embodiment, the polypeptide comprises at least two copies of SEQ ID NO: 20 separated by at least one copy of SEQ ID NO: 24 and at least one copy of SEQ ID NO: 22. In one embodiment, the polypeptide has the following structure: SEQ ID NO: 6 or SEQ ID NO: 20 - SEQ ID NO: 24 - spacer - SEQ ID NO: 22 - SEQ ID NO: 6 or SEQ ID NO: 20.

[0309] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 19. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 19.

[0310] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:22.

[0311] In one embodiment, the isolated polynucleotide encodes a polypeptide having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:22.

[0312] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 22. In one embodiment, the isolated polynucleotide encodes a polypeptide consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: 22.

[0313] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0314] In one embodiment, the isolated polypeptide is contained in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 22. In one embodiment, the polymer comprises two to ten copies of SEQ ID NO: 22.

[0315] In one embodiment, the isolated polypeptide is contained within a protein polymer comprising at least one copy of SEQ ID NO:22 positioned between two copies of SEQ ID NO:6.

[0316] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 21. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 21.

[0317] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:22.

[0318] Specifically contemplated as embodiments of this aspect of the invention are all embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO: 22. Those skilled in the art will also recognize that the above disclosure relating to isolated polynucleotides comprising, consisting essentially of, or consisting of SEQ ID NO: 1 equally and appropriately applies to this aspect of the invention.

[0319] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:22.

[0320] In one embodiment, the isolated polypeptide has at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:22.

[0321] In one embodiment, the isolated polypeptide comprises SEQ ID NO: 22. In one embodiment, the isolated polypeptide consists of or consists essentially of SEQ ID NO: 22.

[0322] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0323] In one embodiment, the polypeptide is contained in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 22. In one embodiment, the polymer comprises from two to ten copies of SEQ ID NO: 22.

[0324] In one embodiment, the isolated polypeptide is contained within a protein polymer comprising at least one copy of SEQ ID NO:22 positioned between two copies of SEQ ID NO:6.

[0325] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 21. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 21.

[0326] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:24.

[0327] In one embodiment, the isolated polynucleotide encodes a polypeptide having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:24.

[0328] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 24. In one embodiment, the isolated polynucleotide encodes a polypeptide consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: 24.

[0329] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0330] In one embodiment, the isolated polypeptide is contained in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 24. In one embodiment, the polymer comprises two to 11 copies of SEQ ID NO: 24.

[0331] In one embodiment, the isolated polypeptide is contained within a protein polymer comprising at least one copy of SEQ ID NO:24 positioned between two copies of SEQ ID NO:6.

[0332] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 23. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 23.

[0333] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:24.

[0334] Specifically contemplated as embodiments of this aspect of the invention are all embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO: 24. Those skilled in the art will also recognize that the above disclosure relating to isolated polynucleotides comprising, consisting essentially of, or consisting of SEQ ID NO: 1 equally and appropriately applies to this aspect of the invention.

[0335] In another aspect, the invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:24.

[0336] In one embodiment, the isolated polypeptide has at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:24.

[0337] In one embodiment, the isolated polypeptide comprises SEQ ID NO: 24. In one embodiment, the isolated polypeptide consists of or consists essentially of SEQ ID NO: 24.

[0338] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0339] In one embodiment, the polypeptide is contained in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 24. In one embodiment, the polymer comprises from two to 11 copies of SEQ ID NO: 24.

[0340] In one embodiment, the isolated polypeptide is contained within a protein polymer comprising at least one copy of SEQ ID NO:24 positioned between two copies of SEQ ID NO:6.

[0341] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 23. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 23.

[0342] Specifically contemplated as embodiments of each of the above-described isolated polynucleotide aspects, the isolated polynucleotide further comprises a heterologous regulatory element. In one embodiment, the regulatory element comprises a nucleic acid sequence encoding a signal peptide. Transcription of the polynucleotide comprising a nucleic acid sequence encoding a signal peptide allows secretion of the expressed polypeptide and / or protein polymer from the host cell.

[0343] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide having at least 70% amino acid sequence identity to (SEQ ID NO: 50).

[0344] In one embodiment, the isolated polynucleotide encodes a polypeptide having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:50.

[0345] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 50. In one embodiment, the isolated polynucleotide encodes a polypeptide consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: 50.

[0346] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0347] In one embodiment, the polypeptide is comprised in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 50.

[0348] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 49. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 49.

[0349] In one embodiment, the polynucleotide further encodes a His tag. In one embodiment, the His tag is located at the N-terminus or C-terminus of the polypeptide.

[0350] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising (SEQ ID NO: 50). In one embodiment, the isolated polynucleotide encodes a polypeptide consisting essentially of or consisting of SEQ ID NO: 50.

[0351] In one embodiment, the polynucleotide further encodes a His tag. In one embodiment, the His tag is located at the N-terminus or C-terminus of the polypeptide.

[0352] Specifically contemplated as embodiments of this aspect of the invention are all embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO: 50. Those skilled in the art will also recognize that the above disclosure relating to isolated polynucleotides comprising, consisting essentially of, or consisting of SEQ ID NO: 1 equally and appropriately applies to this aspect of the invention.

[0353] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to (SEQ ID NO: 50).

[0354] In one embodiment, the isolated polypeptide has at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:50.

[0355] In one embodiment, the isolated polypeptide comprises SEQ ID NO: 50. In one embodiment, the isolated polypeptide consists of or consists essentially of SEQ ID NO: 50.

[0356] In one embodiment, the polynucleotide further encodes a His tag. In one embodiment, the His tag is located at the N-terminus or C-terminus of the polypeptide.

[0357] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0358] In one embodiment, the polypeptide is comprised in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 6.

[0359] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 49. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 49.

[0360] In one embodiment, the polynucleotide further encodes a His tag. In one embodiment, the His tag is located at the N-terminus or C-terminus of the polypeptide.

[0361] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide having at least 70% amino acid sequence identity to (SEQ ID NO: 52).

[0362] In one embodiment, the isolated polynucleotide encodes a polypeptide having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:52.

[0363] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 52. In one embodiment, the isolated polynucleotide encodes a polypeptide consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: 50.

[0364] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0365] In one embodiment, the polypeptide is comprised in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 52.

[0366] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 51. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 51.

[0367] In one embodiment, the polynucleotide further encodes a His tag. In one embodiment, the His tag is located at the N-terminus or C-terminus of the polypeptide.

[0368] In another aspect, the invention relates to an isolated polynucleotide encoding a polypeptide comprising (SEQ ID NO: 52). In one embodiment, the isolated polynucleotide encodes a polypeptide consisting essentially of or consisting of SEQ ID NO: 50.

[0369] In one embodiment, the polynucleotide further encodes a His tag. In one embodiment, the His tag is located at the N-terminus or C-terminus of the polypeptide.

[0370] Specifically contemplated as embodiments of this aspect of the invention are all embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding a polypeptide having at least 70% amino acid sequence identity to SEQ ID NO: 52. Those skilled in the art will also recognize that the above disclosure relating to isolated polynucleotides comprising, consisting essentially of, or consisting of SEQ ID NO: 1 equally and appropriately applies to this aspect of the invention.

[0371] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to (SEQ ID NO: 52).

[0372] In one embodiment, the isolated polypeptide has at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:52.

[0373] In one embodiment, the isolated polypeptide comprises SEQ ID NO: 52. In one embodiment, the isolated polypeptide consists of or consists essentially of SEQ ID NO:52.

[0374] In one embodiment, the polynucleotide further encodes a His tag. In one embodiment, the His tag is located at the N-terminus or C-terminus of the polypeptide.

[0375] In one embodiment, the polypeptide is included in a nesting material of a bee of the family Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus. In one embodiment, the bee of the family Hylaeus is a bee of the genus Hylaeus nubilosus. In one embodiment, the polypeptide or portion thereof is hygroscopic or hydrophilic. In one embodiment, the polypeptide or portion thereof is hydrophobic. In one embodiment, the polypeptide or portion thereof is amphiphilic.

[0376] In one embodiment, the polypeptide is comprised in a protein polymer. In one embodiment, the polymer comprises SEQ ID NO: 2 or at least a portion of SEQ ID NO: 2. In one embodiment, the polymer comprises at least two copies of SEQ ID NO: 6.

[0377] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 51. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO: 51.

[0378] In one embodiment, the polynucleotide further encodes a His tag. In one embodiment, the His tag is located at the N-terminus or C-terminus of the polypeptide.

[0379] In one embodiment of any and / or all of the aforementioned polypeptide aspects, the polypeptide is a FUN polypeptide or a functional part, analog or derivative thereof.

[0380] In one embodiment of any and / or all of the aforementioned polypeptide aspects, the polypeptide is a recombinant FUN polypeptide or a functional part, analog or derivative thereof.

[0381] In another aspect, the present invention relates to a vector encoding the isolated polypeptide according to the present invention.

[0382] In another aspect, the present invention relates to a vector comprising the isolated polynucleotide according to the present invention.

[0383] In one embodiment, the vector is selected from the group consisting of a plasmid, a BAC, (PAC), a YAC, a phage, a phagemid, and a cosmid. In one embodiment, the vector is a plasmid.

[0384] In one embodiment, the vector is selected from the group consisting of a plasmid, a BAC, a PAC, a YAC, a phage, a phagemid, and a cosmid. Preferably, the vector is a plasmid. In one embodiment, the vector is an expression vector. In one embodiment, the vector is pET or pFastBac.

[0385] Examples of suitable expression vectors include, but are not limited to, plasmid DNA vectors, viral DNA vectors (such as adenovirus and adeno-associated virus), or viral RNA vectors (such as retroviral vectors). In some embodiments, the plasmid and / or phage vector can be selected from the following vectors or variants thereof, including pET, pFastBac, pUC18, pU19, Mp18, Mp19, ColE1, PCR1, and pKRC; λgt10 and M13 plasmids, such as pBR322, pACYC184, pT127, RP4, p1J101, SV40, and BPV. Other non-limiting examples of vectors include cosmids, YACs, BACs, shuttle vectors such as pSA3, and PAT28 transposons.

[0386] Suitable viral vectors include, but are not limited to, vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentivirus (LV), rhabdovirus, murine leukemia virus); herpes virus, etc. The viral vectors used herein can be appropriately modified by pseudotyping with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as known and used in the art.

[0387] Vectors can be constructed to drive expression of the polypeptides described herein in vitro or in vivo. In one embodiment, the vector comprises a polynucleotide of the present invention operably linked to a 5' or 3' untranslated regulatory sequence. The design of the vector will depend on various factors, including the host cell in which the operably linked polynucleotide is to be expressed and the desired level of polynucleotide expression.

[0388] Equally, the selection of various promotor, enhancer and / or other genetic elements of carrier will depend on various factors, comprise host cell and expression level discussed above.In one embodiment, carrier comprises the homologous promoter that is operably connected with polynucleotide of the present invention.In another embodiment, carrier comprises the heterologous promoter that is operably connected with polynucleotide of the present invention.In one embodiment, homology or heterologous promoter is inducible, suppressible or regulatable promoter.Can select suitable promotor, and use under appropriate conditions, to instruct polynucleotide high level expression of the present invention.Many such elements have been described in the document, and these elements can be obtained through commercial suppliers.

[0389] As an example only, the promoter that can be used for the vector can be any suitable eukaryotic or prokaryotic promoter. In one embodiment, the eukaryotic promoter can be eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II) or RNA polymerase III (pol III). The expression level of the operably linked polynucleotide in a specific cell type will be determined by the presence (or absence) of specific gene regulatory sequences (e.g., enhancers, silencers, etc.) nearby. Any suitable promoter / enhancer combination (see: eukaryotic promoter database EPDB) can be used to drive the expression of the polynucleotide of the present invention.

[0390] Other promoters that can be used in the expression cassette include beta-lactamase, alkaline phosphatase, tryptophan, and tac promoter systems, all of which are well known in the art. Yeast promoters include, but are not limited to, 3-phosphoglycerate kinase, enolase, hexokinase, pyruvate decarboxylase, glucokinase, and glyceraldehyde-3-phosphate dehydrogenase.

[0391] Prokaryotic promoters that can be used for expression cassettes include constitutive promoters known in the art (such as the int promoter of bacteriophage lambda and the bla promoter of the β-lactamase gene sequence of pBR322) and regulatable promoters (such as lacZ, recA and gal). Expression may also require a ribosome binding site upstream of the CDS.

[0392] Enhancers that can be used in the vectors described herein include SV40 enhancer, cytomegalovirus early promoter enhancer, globulin, albumin, insulin, and the like.

[0393] In one embodiment, the vector can be driven by a T3, T7, or SP6 cytoplasmic expression system.

[0394] In another aspect, the present invention relates to an isolated host cell comprising the isolated polypeptide, isolated polynucleotide and / or vector according to the present invention.

[0395] In one embodiment, the isolated host cell is a eukaryotic cell or a prokaryotic cell.

[0396] In one embodiment, the prokaryotic cell is selected from strains of Escherichia coli (E. coli), Pseudomonas, Bacillus, Serratia, Klebsiella, Streptomyces, Listeria, Salmonella and Mycobacteria, but is not limited thereto.

[0397] In one embodiment, the eukaryotic cell is an animal cell, a plant cell, a fungal cell, or a protist cell.

[0398] In one embodiment, the eukaryotic cell is a fungal cell. In one embodiment, the fungal cell is a yeast cell. In one embodiment, the yeast cell is a Pichia pastoris or Saccharomyces cell. In one embodiment, the fungal cell is an Aspergillus cell. In one embodiment, the Aspergillus is Aspergillus niger.

[0399] In one embodiment, the animal cell is an insect cell or a mammalian cell. In one embodiment, the animal cell is a non-human animal cell. In one embodiment, the mammalian cell is a non-human mammalian cell.

[0400] In one embodiment, the insect cell comprises the polynucleotide described herein in a viral vector, preferably a baculovirus. In one embodiment, the insect cell is an Sf9 or High Five cell.

[0401] In another aspect, the present invention relates to a protein polymer comprising a FUN polypeptide as described herein or a portion thereof, wherein the protein polymer comprises at least one quasi-repeat domain.

[0402] In one embodiment, the protein polymer comprises two to about 10, 20, 30, 50, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 and about 1000 copies of a quasi-repeated domain.

[0403] In one embodiment, the quasi-repeat domain is comprised in a FUN polypeptide described herein, or a portion thereof.

[0404] In one embodiment, the protein polymer comprises a glutamine composition of >25%, 26%, 27%, 28%, 29%, preferably >30% of the amino acid sequence of the polymer, a serine composition of >25%, 26%, 27%, 28%, 29%, preferably >30% of the amino acid sequence of the polymer, and a glutamic acid composition of >2%, 3%, 4%, preferably >5% of the amino acid sequence of the polymer.

[0405] In one embodiment, the protein polymer comprises a glutamine composition >30% of the polymer amino acid sequence, a serine composition >30% of the polymer amino acid sequence, and a glutamic acid composition >5% of the polymer amino acid sequence.

[0406] In one embodiment, the protein polymer comprises a glutamine composition of about 30% of the amino acid sequence of the polymer, a serine composition of about 30% of the amino acid sequence of the polymer, and a glutamic acid composition of about 5% of the amino acid sequence of the polymer.

[0407] In another aspect, the present invention relates to a composition comprising an isolated polypeptide, isolated polynucleotide, protein polymer and / or carrier as described herein, and a carrier, diluent or excipient.

[0408] In one embodiment, the composition consists essentially of an isolated polypeptide, isolated polynucleotide, proteinaceous polymer, and / or carrier described herein.

[0409] In one embodiment, the composition is a cosmetic composition. In one embodiment, the cosmetic composition is a hair or skin care composition.

[0410] In another aspect, the present invention relates to a method of preparing an isolated FUN polypeptide selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 50 and 52, or a portion thereof, comprising heterologously expressing the FUN polypeptide in an isolated host cell and optionally purifying the FUN polypeptide.

[0411] In one embodiment, a FUN polynucleotide from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 22, 23, 25, 49, and 51, or a portion thereof, is expressed.

[0412] In one embodiment, the polynucleotide further encodes a His tag. In one embodiment, the His tag is located at the N-terminus or C-terminus of the polypeptide.

[0413] In one embodiment, the FUN polynucleotide or a portion thereof comprises at least one heterologous regulatory element. In one embodiment, the FUN polynucleotide or a portion thereof encodes a signal sequence that encodes a signal peptide. In one embodiment, the signal sequence is a homologous sequence. In one embodiment, the signal sequence is a nonhomologous sequence.

[0414] In one embodiment, the signal peptide directs secretion of the FUN polypeptide or portion thereof.

[0415] In one embodiment, the method includes optionally purifying the FUN polypeptide after secretion from the isolated host cell.

[0416] In one embodiment, the method comprises optionally purifying the FUN polypeptide from the isolated host cells.

[0417] In one embodiment, the isolated host cell is a fungal cell, a bacterial cell, or an insect cell. In one embodiment, the FUN polypeptide or portion thereof is expressed by a FUN polynucleotide selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 22, 23, 25, 49, and 51.

[0418] In one embodiment, the FUN polynucleotide is contained in a vector, preferably a baculovirus vector.

[0419] In one embodiment, the polynucleotide further encodes a His tag. In one embodiment, the His tag is located at the N-terminus or C-terminus of the polypeptide.

[0420] In one embodiment, the isolated host cell is a bacterial cell. In one embodiment, the bacterial cell is Escherichia coli.

[0421] In one embodiment, the isolated host cell is a fungal cell. In one embodiment, the fungal cell is an Aspergillus niger cell.

[0422] In another aspect, the present invention relates to an isolated FUN polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 50 and 52 prepared by a method of the present invention.

[0423] In another aspect, the present invention relates to a method for preparing a protein polymer comprising a FUN polypeptide selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 50 and 52, or a portion thereof, the method comprising heterologously expressing the FUN polypeptide or a portion thereof in an isolated host cell under conditions resulting in the expression of the FUN polypeptide or a portion thereof, and optionally purifying the polymer from the host cell.

[0424] In one embodiment, the protein polymer is an isolated protein polymer.

[0425] In one embodiment, the FUN polynucleotide or a portion thereof is expressed from a polynucleotide sequence comprising at least one heterologous regulatory element.

[0426] In one embodiment, the FUN polynucleotide or a portion thereof is expressed from a polynucleotide sequence comprising at least one signal sequence.

[0427] In one embodiment, the signal sequence encodes a signal peptide or a portion thereof. In one embodiment, the signal peptide or a portion thereof directs secretion of the protein polymer from an isolated host cell.

[0428] In one embodiment, the method comprises purifying the polymer after secretion from the host cell.

[0429] In one embodiment, the method comprises purifying the polymer from the isolated host cell.

[0430] In one embodiment, the isolated host cell is an isolated host cell contemplated in the previously described aspects and embodiments of the invention.

[0431] In one embodiment, the polymer comprises at least two copies of a FUN polypeptide.

[0432] In one embodiment, the isolated host cell is a fungal cell, a bacterial cell, or an insect cell. In one embodiment, the FUN polypeptide or portion thereof is expressed by a FUN polynucleotide selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 22, 23, 25, 49, and 51.

[0433] In one embodiment, the FUN polynucleotide is contained in a vector, preferably a baculovirus vector.

[0434] In one embodiment, the isolated host cell is a bacterial cell. In one embodiment, the bacterial cell is Escherichia coli.

[0435] In one embodiment, the isolated host cell is a fungal cell. In one embodiment, the fungal cell is an Aspergillus niger cell.

[0436] In another aspect, the present invention relates to the use of a FUN polypeptide or portion thereof as described herein for coating an article or forming a coating on an article. In one embodiment, the coating is hydrophilic, hydrophobic, or amphiphilic. In one embodiment, the coating is hydrophobic. In one embodiment, the coating is hydrophilic. In one embodiment, the coating is amphiphilic.

[0437] In another aspect, the present invention relates to the use of the FUN polypeptide described herein or a portion thereof for preparing a film on an article of manufacture.

[0438] In one embodiment, the film is hydrophilic, hydrophobic, or amphiphilic. In one embodiment, the film is hydrophobic. In one embodiment, the film is hydrophilic. In one embodiment, the coating is amphiphilic.

[0439] In one embodiment, the article is selected from a textile or textile component or portion thereof and a biomedical device or component or portion thereof.

[0440] In one embodiment, the article or a component or portion thereof is a synthetic fiber. In one embodiment, a component or portion thereof is a synthetic polymer. In one embodiment, the synthetic fiber or synthetic polymer is selected from polyester, spandex, rayon, nylon, acrylic, microfiber, neoprene, polyamide, acetate, polyvinyl chloride (PVC), and synthetic or "faux" leather or fur fibers and polymers.

[0441] In one embodiment, the synthetic fiber is nylon.

[0442] In one embodiment, the article or component or portion thereof is a natural fiber.In one embodiment, the natural fiber is selected from the group consisting of cotton, wool, silk, coir, alpaca, flax, bamboo, sisal and jute.

[0443] In one embodiment, the natural fiber is silk.

[0444] In one embodiment, the article is a textile, preferably a natural textile or a synthetic textile.

[0445] In one embodiment, the article or component or portion thereof is a biomedical device or is included in or on a biomedical device. In one embodiment, the biomedical device is an implantable biomedical device. In one embodiment, the implantable biomedical device is selected from cardiovascular devices, including cardiac defibrillators, pacemakers, and left ventricular assist devices, breast implants, cochlear implants, intraocular lenses, joint replacements, including hip implants, catheters, dialysis tubing, contraceptive intrauterine devices, stents, sutures, staples, bandages, and wound dressings.

[0446] In one embodiment, the article is an air filtration device, a component or part thereof. In one embodiment, the component or part thereof is an air filter. In one embodiment, the component or part thereof is a synthetic or natural fiber and / or polymer. In one embodiment, the synthetic or natural fiber and / or polymer is contained in an air filter. In one embodiment, the synthetic or natural fiber and / or polymer is in the form of nanofibers.

[0447] Specifically contemplated as embodiments of the methods and uses of the present invention are the various embodiments described above with respect to the selection of FUN polypeptides and portions thereof, polynucleotides encoding FUN polypeptides or portions thereof, suitable polynucleotide regulatory sequences including signal sequences, host cells and / or vectors that allow for the expression and purification of the FUN polypeptides or portions thereof described herein.

[0448] The invention will now be illustrated in a non-limiting manner by reference to the following examples.

[0449] Example

[0450] Example 1

[0451] Source, collection and transportation of Hylaeus nubilosus bees and their nesting materials

[0452] Hylaeus nubilosus was collected in Queensland, Australia. A total of 13 specimens of Hylaeus nubilosus were collected. The Duchenne glands, venom glands, and salivary glands, as well as the entire head, were dissected from all 13 specimens and preserved in either dichloromethane (DCM) or ethanol.

[0453] Nest material from Hylaeus nubilosus was collected from vacant (after larvae hatched and left the nest) paper nesting straw tubes (~10-20 cm long, ~0.525 cm inner diameter). Bees emerging from nesting straw tubes were confirmed as H. nubilosus by visual observation by a trained entomologist. After emergence and identification, the straw tubes were frozen for at least 48 hours and stored frozen until needed. Nest material was collected from the inside of the straw tubes using a scalpel and forceps, and any debris was carefully removed.

[0454] Example 2

[0455] Identification of genes encoding honeycomb material proteins using a multiplex sequencing approach

[0456] Transcriptome: RNA extraction and sequencing

[0457] Hylaeus samples were fixed at -20°C for 3 minutes. Hylaeus sample dissection was performed as follows: the heads of the Hylaeus samples were cut off and transferred to RNAlater, which served as an indicator of transcripts enriched in the mandibular gland. The salivary glands and Duchenne glands were dissected into RNAlater. After dissection, the samples were stored at -80°C. RNA was extracted from the Duchenne glands, mandibular glands, and salivary gland pooled samples. Triplicate sets were used for the Duchenne glands and mandibular glands (2 groups of 10 individual bees each and 1 group of 11 individual bees), and duplicate sets were used for the salivary glands (1 group of 10 individual bees and 1 group of 11 individual bees). Tissue was homogenized using Tissulyser II. RNA was extracted from each pooled sample using the RNeasy mini kit. Sequencing was performed using an Illumina NextSeq sequencer to generate 150bp double-end reads. RNA sequencing generated a total of 451,975,167 paired-end reads (136.5 Gb of total data).

[0458] Proteomics: mass spectrometry sequencing of wild Hylaeus nest material

[0459] Mass spectrometry analysis was performed on the washed and enzymatically digested nesting material to identify the proteins present. The nesting material was initially washed with water, diluted organic solvent, and 8M urea solution, followed by protein digestion with trypsin / chymotrypsin to generate a peptide fragment fingerprint. All samples were analyzed by liquid chromatography coupled to tandem mass spectrometry using an LTQ-Orbitrap and a 5600+ TripleTOF mass spectrometer. Data analysis was performed using the in-house Mascot server and the ProteomeDiscoverer 2.4 software package.

[0460] The mass spectra were searched against both the transcript nucleotide sequence and the predicted amino acid sequence. Both searches identified several high confidence matches to peptide sequences rich in glutamine and serine. A BLAST search revealed that the glutamine-rich sequence had no homology to any other known protein sequence in the NCBI database (see Sequence Listing 4).

[0461] Genomics: gDNA and RNA extraction, cDNA generation, and sequencing

[0462] Genomic DNA extraction and sequencing: Bees were quickly frozen using liquid nitrogen and stored at -80°C. DNA was extracted using the following protocol. Lysis buffer was prepared by adding 1.5 μL of RNase A (100 mg / mL) to 1438.5 μL of G2 buffer. Bee tissue was disrupted by crushing with a DNAse-free pestle. Lysis buffer was added to the crushed tissue. The samples were incubated at 37°C for 30 minutes while nutating the tube. 60 μL of 20 mg / ml proteinase K was added to the sample tube. The samples were then incubated at 50°C for 2 hours while nutating. The sample tube was then centrifuged at maximum speed (12,000 g). The supernatant was then transferred to a 15 ml Falcon tube. The sample was then diluted with G2 buffer to a total volume of 3 ml. A QIAGEN Genomic-tip 20 / G was equilibrated with 1 ml of QTB buffer. The lysate from the falcon tube was then applied to the Genomic-tip, the Genomic-tip was allowed to drain, and any additional remaining lysate was then added to the Genomic-tip. The QIAGEN Genomic-tip was then washed four times with 1 ml of QC buffer. The DNA in the QC buffer was then aliquoted into three DNA Lobind 1.5 ml tubes. 666 μL of QF buffer was added to each tube. 666 μL of SPRI reagent and 3 μL of SPRIselect beads were then added to each of the three tubes (make sure the beads are brought to room temperature before use and vortexed immediately before use). The sample was then nutated on a shaker at room temperature for 10 minutes. The tube was then placed on a magnet until the liquid cleared and the beads aggregated into a pellet. The supernatant was then discarded. The beads were then washed with 70% ethanol and incubated at room temperature for 30 seconds. The wash step was then repeated. The beads were then air-dried at room temperature for 30 seconds. The tube was then gently flicked at 50°C, and the pellet was eluted with 42 μL of DNAse-free water. The beads were then collected using a magnet. The sample was then pipetted into a fresh DNA Lobind 1.5 ml tube. The gDNA concentration was then measured using a Nanodrop. The size distribution of the gDNA extraction was verified by running 100 ng on a 1% agarose gel and measured against a high MW DNA marker. The sample was then snap-frozen in liquid nitrogen and then stored at -80°C. DNA extraction yielded 3 ug of high MW DNA. gDNA sequencing was performed using an Oxford Nanopore PromethION sequencer. Subsequent analysis was performed using fastq formatted sequences, which passed the default quality filter cutoff.

[0463] RNA extraction, cDNA generation, and sequencing: Bees were flash-frozen in liquid nitrogen and stored at -80°C. RNA was extracted from one intact female Hylaeus using a Trizol-based extraction method. First, the cuticle was removed from the bee from which RNA was to be extracted, and the remaining tissue was transferred to a 1.5ml DNA Lobind tube. 50 μL of TRIzol reagent was transferred to the tube containing the sample. The tissue was then disrupted by crushing with a DNAse-free pestle. The pestle was rinsed with an additional 200 μL of TRIzol reagent, and the remaining tissue was washed into the tube. The sample was then incubated at room temperature for 2 minutes. 50 μL of chloroform was added to the tube containing the sample. The tube containing the sample was shaken vigorously for 15 seconds, followed by incubation at room temperature for 5 minutes. The sample tube was then centrifuged at 12,000 g for 15 minutes at 4°C. The upper aqueous phase containing the RNA was transferred to a new 1.5ml DNA Lobind tube. 0.125 mL of isopropanol was then added to the tube containing the aqueous phase. The sample was mixed by inversion to precipitate the RNA. The samples were then incubated at room temperature for 2 minutes. The samples were then centrifuged at 12,000 g for 10 minutes at 4°C. The supernatant was then removed and discarded. The samples were then washed with 250 μL of 75% ethanol. The samples were centrifuged at 7,500 g for 5 minutes at 4°C. The supernatant was then removed and discarded. The samples were then air-dried to allow the RNA pellet to settle until all droplets had evaporated. The RNA pellet was then gently resuspended in 32 μL of RNAse-free water by flicking the tube. RNA concentration was assessed by analyzing the A260 / 280 and A260 / 230 ratios on a Nanodrop. The samples were then snap-frozen in liquid nitrogen and stored at −80°C. After poly A RNA sequence enrichment, cDNA was generated from the RNA samples using reverse transcription. The cDNA read data were base-called using Guppy version 4 to generate fastq sequence files. Subsequent analysis was performed using the fastq-formatted sequences, which passed the default quality filter cutoff.

[0464] Genome assembly from gDNA sequencing

[0465] Genome assembly is the process by which DNA sequencing reads are pieced together to accurately represent the nucleotide sequence and structure of an organism's genome. Genome sequencing and assembly have helped identify genes, including the nest material gene in Hylaeus nubilosus.

[0466] Adapters were trimmed from the gDNA sequence files using Porechop (version 0.2.4; Wick et al., 2017). The "--discard_middle" parameter was used with Porechop. The Hylaeus nubilosus genome was assembled using Flye (version 2.8; Kolmogorov et al., 2019). The "--nano-raw" parameter was used in the assembly, consistent with the input Oxford Nanopore sequence data. Flye was executed with a predicted "genome-size" parameter of 250 MB. This size was used because other Hymenoptera genomes have a size of ∼250 MB. The Hylaeus nubilosus genome is 360 MB, which is surprisingly large for a honey bee. Genome assembly statistics indicate that the assembly is highly contiguous and therefore an accurate representation of the Hylaeus nubilosus genome. The genome assembly consists of 2,259 relatively contiguous scaffolds. The N50 of the Hylaeus nubilosus assembly is 7, and the L50 is 17,978,421 bp, indicating a contiguous assembly. Analysis of the assembly with Busco (version 5.4.2; Siminto et al., 2015) using the Hymenoptera database (hymenoptera_odb10) yielded a Busco score of 95.7%, strongly indicating that the genome was assembled to a high level of completeness.

[0467]

[0468] Annotation of the Hylaeus genome

[0469] The Hylaeus genome was assembled using sequence data compiled from RNA sequencing, genomic DNA sequencing, and cDNA sequencing datasets, as well as proteomic analysis (peptide fragment sequencing). The "funannotate" (version 1.7.4; Palmer & Stajich, 2022) genome annotation software was used, which utilizes the ab-initio gene prediction tools Augustus and GeneMark. The genome was prepared for annotation by masking uninformative regions of the genome using the "funannotate mask" script (Palmer & Stajich, 2022). The "funannotate mask" script was executed using default parameters, which were soft-masked using the program "tantan" (Frith, 2011). All RNA sequence reads, including sequence reads from salivary glands, mandibular glands, and Duchenne glands, were combined into forward and reverse groups for training "funannotate". cDNA reads for annotation were prepared using Porechop (version 0.2.4; Wick et al., 2017) by trimming adapters. Porechop was executed using the "--discard_middle" parameter. The processed cDNA was used as input for funannoate annotation training.

[0470] 'funannotate-train' is a packaged tool for transcriptome assembly using Trinity (Palmer & Stajich, 2022; Grabherr et al., 2011). The 'funannotate-train' script uses the masked genome assembly, Nanopore cDNA, and forward and reverse RNA-seq data as input. The 'funannotate-train' script is executed using the '--stranded' switch set to 'RF' (reverse-forward). The maximum intron length is set to 10,000 using the '--max_intronlen' parameter. 'funannotate-train' generates a file containing transcripts in gff3 format, which is used as input to 'funannotate-predict'. 'funannotate-predict' trains and executes Augustus and GeneMark to produce gene predictions (version 1.7.4; Palmer & Stajich, 2022).

[0471] 'funannotate predict' then uses the Evidence Modeler to generate consensus gene models from Augustus and GeneMark gene predictions. The masked Hylaeus nubilosus genome assembly, the transcript gff3 generated by 'funannotatetrain', and 'fundb_20200227' (generated by 'Funanotate setup') were used as input to 'funannotate predict'. 'funannotate predict' was executed with the '--augustus_species' hn, the '--optimize_augustus' switch, the '--busco_seed_species' set to honeybee1, the '--buso_db' set to hymenoptera, the '--organism' set to other, the '--repeats2evm' switch, and the '--max_intronlen' set to 50,000. 'funannotate predict' generates Hylaeus gff3, mRNA transcripts, and protein annotation files as output. `funannotate update` updates annotations using RNA-seq data (version 1.7.4; Palmer & Stajich, 2022). `funannotate update` is executed with predicted mRNA transcripts as input. `funannotate annotate` is used for functional annotation of genes.

[0472] ‘funannotate-annotate’ performs functional annotation of the Hylaeus genome annotation using input generated by EGGNO, IPRSCAN, and BUSCO (version 1.7.4; Palmer & Stajich, 2022; Huerta-Cepas et al., 2019; version 5.44-79.0; Jones et al., 2014; version 5.4.2; Eggnog uses the Hylaeus protein database and the "eggnog_proteins" diamond database. Diamond was selected as the search method using the "-m diamond" option of eggnog. Functional annotation was provided using iprscan (interproscan) (Jones et al., 2014). iprscan was executed with default parameters to generate functional annotations, except that precomputation was disabled ("-dp" switch).

[0473] 75,295 protein-coding genes were predicted by funannotate. The predicted proteins were analyzed using the Hymenoptera database (hymenoptera_odb10) using Busco (version 5.4.2; et al., 2015) analysis yielded a busco score of 72.4% (15.8% fragmentation).

[0474] Identification of genes encoding honeycomb material proteins

[0475] Nest material genes from Hylaeus were identified using a hidden Markov model constructed using mass spectrometry peptide sequence data from Hylaeus nest material. Peptide sequences from the mass spectrometry sequence data were aligned using Muscle, and hidden Markov models were generated using hmmbuild in the "Hmmer" software suite (version v3.8.1551; Edgar, 2004; version 3.3.2; Johnson et al., 2010). The Hylaeustrinity transcriptome protein database was searched using hmmsearch, with nest material genes as the highest-scoring matches. High expression of our target genes was identified using Salmon and Deseq2; genes encoding honeycomb material proteins were highly expressed in the salivary glands, accounting for ~10% of the genes expressed in this tissue.

[0476] result

[0477] A combination of transcriptome, genome, and proteome sequencing enabled the identification of a highly unique nest material gene. This gene has an uncharacterized gene structure, comprising 14 exons and unstructured introns, is highly repetitive, and has an extremely high ratio of glutamine (30.5%) to serine (32.1%) content.

[0478] The longest uninterrupted sequence assembled from peptide identifications assigned to the same sequence database entry is 86 amino acids in length:

[0479] ...SSSGMSSQAQSQQQQAQLQQSQDQRQEQESQSAGSESAVKSSKSSSGMSSQAQSQQQQAQLQQSQAQSQEEESESAQSESEVHV SK… (SEQ ID NO: 33).

[0480] Searches against the cDNA sequence (mRNA transcript) of Hylaeus nubilosus revealed various highly significant matches to glutamine- and serine-rich sequences, as well as to several other protein sequences.

[0481] A BLAST search against all protein sequences in nrNCBI did not reveal significant homology of the glutamine-rich sequence to any other known protein sequence.

[0482] Based on the above findings and those described elsewhere herein, the inventors believe they have identified a novel nest material polypeptide from H. nubilosus. This novel polypeptide comprises 5451 amino acid residues, has a predicted molecular weight of 578.4 kDa, and contains 30.5% glutamine (Q) and 32.1% serine (S) residues, respectively.

[0483] Figure 1 The alignment of the FUN module 7 sequence (from the long read sequence) SEQ ID NO 14 with the assembled proteome sequence is shown, and the alignment result is relatively ideal (note that the fidelity of proteome sequencing is lower than that of gene sequencing and is challenging).

[0484] The amino acid sequence of the predicted long read FUN_069765 sequence was mapped to the assembled proteome sequence generated by mass spectrometry of the nest material = 85%.

[0485] Identification of amino acid composition and sequence in proteomic analysis.

[0486] SEQ ID NO: 25 is the genomic sequence (20,870 bp) of the H. nubilosus gene putatively encoding a nesting material polypeptide (designated FUN_069765-T1, also referred to as "FUN polypeptide"). Without wishing to be bound by theory, the inventors believe that the FUN polypeptide forms the proteinaceous biopolymer component of the H. nubilosus nesting material. The genomic sequence has annotated exons, introns, and untranslated regions (UTRs), and shows a predicted transcript (also referred to as FUN_069765-T1) and a conceptual amino acid translation.

[0487] FUN_069765cDNA

[0488] SEQ ID NO: 1 (16,353 bp) is derived from the FUN_069765-T1 transcript sequence (ie, after intron splicing) and shows the FUN cDNA; ie, only the predicted coding sequence for the FUN_069765-T1 polypeptide; and does not include any 5' or 3' UTRs.

[0489] SEQ ID NO: 2 is the predicted amino acid sequence of the FUN_069765-T1 polypeptide (5451 residues; theoretical MW = 578.4 kDa), proposed to form a proteinaceous biopolymer component of H. nubilosus honey bee nesting material. SEQ ID NO: 2 is the amino acid sequence translation of SEQ ID NO: 1, which was derived by splicing and removing introns from SEQ ID NO: 25.

[0490] Bioinformatics analysis of protein sequences

[0491] Translation of SEQ ID NO: 1 (FUN_069765 cDNA) and subsequent bioinformatics analysis of the polypeptide (FUN) yielded the following observations:

[0492] The FUN polypeptide consists of 5451 amino acids and has a theoretical molecular weight of 578,350 Da.

[0493] Most proteins (aa138-5451) are characterized by the presence of many repeated sequence motifs; therefore, this region of the protein is referred to as the repetitive region (see Figure 2 The repeat region can be further divided into QS repeat region ( Figure 2 The genomic DNA of the present invention is labeled QS in the genome, which contains repeat sequences rich in glutamine (Q) and serine (S). Separating the QS repeat regions are spacer repeat sequences rich in proline (P) and cysteine ​​(C). The details of these repeat sequences will be discussed in more detail below.

[0494] In contrast, the first 137 amino acids of the protein lack any repetitive sequence features. Using the SignalP 5.0 server (see Almagro Armenteros et al., 2019), the first 17 residues were predicted to be a signal peptide, consistent with a secreted protein. The predicted signal peptide is followed by an N-terminal domain (NTD or N, aa 18-137) rich in serine (S, 16.67%), histidine (H, 15.83%), lysine (K, 11.67%), and glutamic acid (E, 10.00%).

[0495] Predicted amino acid composition of FUN_069765-T1

[0496] The full-length mature protein is rich in serine (S, 32.14%) and glutamine (Q, 30.49%), and lacks tyrosine (Y, 0.04%), phenylalanine (F, 0.07%), tryptophan (W, 0.28%), cysteine ​​(C, 0.29%) and asparagine (N, 0.31%).

[0497] There is a clear glutamine / asparagine bias, with glutamine occurring almost 100 times more frequently than asparagine. Asparagine is completely absent from the QS repeat region.

[0498] Amino acids with large aromatic side chains (Y, F, and W) are all present in low amounts and are virtually absent from the QS repeat region, being present only in the signal peptide, N-terminal domain, or spacer repeats. Similarly, cysteine ​​and proline residues are absent from the QS repeat region, being present only in the signal peptide, N-terminal domain, or spacer repeats.

[0499] There are 16 cysteine ​​residues. There is one potential N-linked glycosylation site (Asn 109-His-Thr) located within the N-terminal domain.

[0500] Example 3 - Recombinant Expression and Purification of FUN Polypeptides

[0501] The initial goal was to express a synthetic protein whose properties could be compared with those of the nest material. For this purpose, the inventors chose the insect cell line Sf9 (derived from the fall armyworm Spodoptera frugiperda) for expression, since insects belong to the same phylum as bees. The design is based on a secreted synthetic protein (SEQ No: 10) fused to two modules (module 1 and module 7), which should provide the essential elements of the nest material. Module 1 consists of a natural signal peptide (SEQ No: 47), which can be recognized by the Sf9 cell machinery and targeted for secretion into the culture medium. The N-terminal histidine-rich region of the native module 1 was replaced by a hexahistidine tag for affinity purification.

[0502] The cDNA of SEQ No: 10 was cloned into the pFastBac Dual entry vector from Invitrogen. The plasmid is optimally propagated in E. coli DH5α cells or similar cells. Selection is preferably performed in liquid LB or LB agar supplemented with 75 μg ml⁻¹ ampicillin. The entry vector was transformed into E. coli DH5α cells using a basic chemical heat shock protocol. The propagated entry vector was recovered using a chemical alkaline lysis protocol. Recombinant bacmids were generated using competent E. coli DH10 Multibac cells (Geneva Biotech) containing the bacmid (baculovirus genome vector plasmid) and helper plasmids according to the manufacturer's protocol (Invitrogen). Recombinant bacmids (FUN polypeptide bacmids) from confirmed white DH10 Multibac cells were recovered from the cells by alkaline lysis. Gene insertion into the bacmid was verified by PCR.

[0503] Sf9 cells were transfected with recombinant bacmid DNA in 6-well plates using insect genejuice transfection reagent (Merck). Sf9 cells were propagated at 28°C in Sf900-III serum-free insect cell culture medium (Thermo Fisher). Sf9 cells were grown as a monolayer on coverslips in 6-well plates or in shake flasks agitated at 130 rpm. The cells were incubated at 28°C for 4 hours, rinsed, and incubated for another 72 hours. After 72 hours of incubation, the transfection mixture was removed to a sterile falcon tube, the residual Sf9 cells were precipitated by centrifugation (500g), and the clarified medium containing the baculovirus was placed in a new 6-well plate. The primary amplification plate was incubated at 28°C for another 72 hours. The amplified virus was harvested, the cells were removed by centrifugation at 500g, and the clarified virus stock was supplemented with 2% fetal bovine serum and stored at 4°C. To express the FUN polypeptide, the generated recombinant virus was used to infect Sf9 cells (~2.0-2.5×10 6 cells / ml).

[0504] Small-scale protein expression was performed in 200 ml volumes of Sf9 cells in serum-free medium. Cultures were infected with an empirically determined virus ratio and incubated at 28°C with shaking at 130 rpm. Cultures were routinely supplemented with sterile D-glucose, L-serine, and L-glutamine to 5 mM at t48 and t72. Cultures were typically removed 72-96 hours post-infection and centrifuged at 1500 g before protein harvesting.

[0505] FUN polypeptide expression was scaled up to a total volume of 4.4 L in 250 ml shake flasks. The culture medium was harvested and stored frozen at -20°C for several weeks prior to downstream processing. 4.4 L of culture medium was treated using the NaCl-isopropanol method for biomaterial generation (B2.0).

[0506] 4.4L culture medium is stored frozen in Schott bottles at -20 ℃. The culture medium is then thawed at 4 ℃ for 2 days for further processing. As described below, NaCl-isopropanol fractionation is used to process culture medium (pH~5.8). 500ml culture medium is measured in a polypropylene graduated cylinder and poured into a 1L Schott bottle. By gradually adding salt under stirring, it is titrated to 2M NaCl concentration. When NaCl is completely dissolved, 500ml of 100% isopropanol is added to the mixture (1M final NaCl concentration) under sufficient stirring. The mixture is stirred for 30 minutes and rotated at 6750g for 45 minutes. Repeat the above operation for the whole 4.4L culture medium, reclaim the rotation precipitation, and resuspend it in 1x PBS (pH 7.4) containing 5% glycerol. The precipitation in the PBS is placed in a falcon tube, rotated again at 4000g for 30 minutes, and discard the supernatant (PBS washing). The pellet was mixed in NuPAGE SDS loading buffer, boiled to 90°C for 10 minutes, and loaded onto a precast 12% NuPAGE Bis-Tris gel (Thermo Fisher scientific). Electrophoresis was performed at 160 V for 40 minutes using 1X MOPS running buffer (Thermo Fisher scientific), and protein bands were visualized by staining with Coomassie Brilliant Blue R-250.

[0507] The pellet was resuspended in PBS, and the slurry was then quickly frozen in liquid nitrogen and stored at -80°C. The pellet was thawed on ice and centrifuged at 4000 g for 30 minutes, after which the supernatant was discarded. The product (wet weight ~11 g) was then shipped cold chain.

[0508] The purity of the expressed FUN polypeptide 2.0 was determined to be quite low (estimated purity < 30%) using SDS page gel analysis of NaCl-isopropanol precipitated material (not shown). Further purification of the FUN polypeptide was performed as described below.

[0509] The culture medium (harvested at t120) was spun at low g to precipitate cells and debris. In order to reduce the working volume, NaCl-isopropanol fractionation was included as described above. The precipitate containing the FUN polypeptide was resuspended in PBS+5% glycerol (1 / 5 of the starting culture medium volume), at least 6M urea, pH=7.5 or 8. It was then subjected to ultrasonic treatment to disperse the protein and further release the His tag. Ultrasonic treatment was performed for a total of 1 minute (1 second on, 1 second off), with an amplitude of 10 (the energy applied was approximately 1600 J). The culture medium was then taken and centrifuged at 10,000 x g for 20 minutes. The clarified supernatant was loaded onto a 5mL Ni-HisTrap FF (fast flow) column balanced with wash buffer (25mM sodium phosphate, 300mM NaCl, 10mM imidazole, 5% glycerol, 6M urea). The protein on the column was eluted with elution buffer (25 mM sodium phosphate, 300 mM NaCl, 300 mM imidazole, 5% glycerol, 6 M urea) and dialyzed overnight with stirring against buffer (containing 25 mM NaP, 150 mM NaCl and 5% glycerol, pH = 7.5) to remove urea and imidazole.

[0510] composition

[0511] The NaCl-isopropanol precipitated, centrifuged and filtered FUN polypeptide protein was further characterized to clarify the nature of the sample. In addition to monitoring the protein molecular weight using SDS-PAGE ( Figure 4 ) and chemical characterization using FT-IR ( Figure 5 and Figure 6 ).

[0512] Preliminary coating

[0513] Before attempting to spin fibers, the target protein is typically cast into coatings and films, and the chemical and physical properties of the materials are evaluated. Using drop casting, coatings containing FUN peptide materials are cast onto various substrates. Drop casting is a deposition method suitable for forming thin films over small areas. This method is performed by dropping 50-100 μL of FUN peptide sample (~1 mg / mL) onto the desired substrate and allowing the solution to evaporate for the desired amount of time. The FUN peptide coatings are then characterized using a range of different techniques.

[0514] Fourier transform infrared spectroscopy (FTIR)

[0515] 50 μL of FUN peptide sample (~1 mg / mL) was drop-cast on a 1×1 cm aluminum ( Figure 5 ) and coverslip ( Figure 6) and subsequently mounted on a solid support (whole glass slide). Fourier transform infrared (FTIR) spectra of these samples were measured on a Bruker Lumos FTIR spectrophotometer in attenuated total reflectance (ATR) mode. IR spectra were acquired in the absorption range of 400-3900 cm-1 with a resolution of 2 cm-1 and a total of 128 scans. Significant β-sheet formation was observed from the FTIR spectra of FUN polypeptide drop-cast films. The presence of β-sheets renders the protein substrate insoluble in water, which is crucial for coating and fiber-related applications. In contrast, the FTIR spectra of silk fibroin cast films showed the presence of predominantly α-helical / coiled structures. Upon treatment with ethanol, α-helices were converted to β-sheets (peak shifts were observed in the FTIR spectra). As shown in this work, the FUN polypeptide formed β-sheets even before any ethanol treatment. These results provide structural characterization of FUN polypeptides as expected in H. Nubilosus nesting materials and demonstrate that the FUN polypeptides provided herein (and various compositions comprising FUN polypeptides) have great potential for use as functional coatings and fiber-forming materials.

[0516] Scanning electron microscopy (SEM) characterization

[0517] The surface morphology of the FUN protein coating was observed using scanning electron microscopy (SEM). For SEM analysis, microfilms of FUN polypeptide (5 μl, 0.5 mg / ml) were cast onto silicon wafers and analyzed using a Supra 55-VP field emission scanning electron microscope (SEM, Zeiss, Germany) at an accelerating voltage of 3 keV. Images were captured at 10,000x and 100,000x at a working distance of 7-8 mm.

[0518] Figure 7 A typical SEM image of a FUN polypeptide coating is shown, showing a smooth, clean surface with the exception of a fibril or reticular pattern. The results of the SEM work are consistent with the surface morphology of currently known honeybee nesting materials, i.e., coatings and films constructed with the FUN polypeptides described herein exhibit the characteristic patterns of honeycomb materials (as expected).

[0519] Water contact

[0520] Using the drop casting technique, a recombinant protein film of FUN polypeptide was cast on a surface area of ​​1 × 1 cm by dropping 100 μL of concentrated protein solution (about 2 mg / ml). 2The piranha solution was cleaned on a glass slide (from Westlab, Australia). Water contact angle measurements were performed to investigate the hydrophobicity of the film surface. Using a static sessile drop technique analyzed using a BiolinAttention Theta flow tensiometer, measurements were performed on FUN polypeptide and silk coatings (Sigma) and ethanol-treated FUN polypeptide and silk coatings (Sigma). Water droplets were applied to a protein film cast glass substrate. Images were captured immediately after contact and after incubation for 30 seconds at room temperature. Surfaces with contact angle measurements below 90° are considered hydrophilic. The glass substrate was hydrophilic and exhibited an angle of 14°, which remained unchanged after 30 seconds of equilibrium. The FUN polypeptide-coated surface exhibited a contact angle of approximately 74°, which decreased to approximately 62° after 30 seconds of equilibrium, while the sigma silk exhibited a contact angle of approximately 48°, which decreased to approximately 22° after 30 seconds of equilibrium. The coating made the glass more hydrophobic, as indicated by the better spreading of the droplets on the uncoated glass substrate than after the protein coating. The ethanol-treated FUN polypeptide exhibited a hydrophobic angle of 96° immediately upon contact, which decreased to 75° after 30 seconds of equilibration. Thus, it is clear from the results presented herein that the FUN polypeptide, or at least a portion thereof, is relatively more hydrophobic than sigma silk when measured under the same conditions.

[0521] Washability

[0522] A simple test was also performed to confirm the stability of the FUN polypeptide coating. 50 μL of FUN polypeptide sample (~1 mg / mL) was drop-cast onto a 1×1 cm glass slide. The slide was soaked in a pure ethanol solution for 2 hours and then vacuum dried until further use. Washability testing was performed by soaking the FUN polypeptide-coated slide in water and / or 0.1 M PBS buffer (pH 7.4) for 24 hours. The soaked slide was rinsed with deionized water to remove excess salt. The dried "soaked" slide was stained with Coomassie Brilliant Blue dye for 3-4 hours and decolorized in deionized water overnight. After soaking the coating in water or PBS for 24 hours, the coating remained intact, as indicated by complete coverage of the Coomassie stain ( Figure 9 ).

[0523] Example 4 - Proteomic or other analyses supporting the conclusions of the previous examples.

[0524] Proteomics

[0525] The target bands were excised from the purified SDS-PAGE gel and used for mass spectrometry fingerprint analysis. The highlighted box points to the putative FUN polypeptide band. The excised FUN polypeptide bands were combined and sent to the University of Auckland Mass Spectrometry Facility for fingerprint analysis.

[0526] Mass spectrometry fingerprint analysis

[0527] The goal was to achieve sequence coverage and ensure that the 100 kDa band of interest on the denaturing gel was the expected protein and the exact sequence designed in the construct. GluC digestion of the FUN polypeptide protein band yielded ~83% sequence coverage ( Figure 10 ), where the underlined peptides depict regions where no observations were made. The signal peptide is expected to be cleaved following secretion from Sf9 cells. In theory, it is quite possible to obtain the "SKSTHTAHKSSGGKSSQME" peptide (SEQ ID NO: 26) in the GluC digest; however, this peptide did not produce coverage. Due to the presence of two cysteines at the C-terminus, a certain proportion of post-translational modifications is expected here, which may result in unconfident matches. To achieve complete sequence coverage, trypsin digestion was used along with reduction and alkylation, which is believed to provide better coverage in the C-terminal region ( Figure 11 and Figure 12 ).

[0528] Reduction and alkylation trypsin digestion yielded a fragment that covered almost the entire C-terminal region ( Figure 11 ), except for the final cysteine ​​residue, which was likely cleaved by trypsin. The first segment of the C-terminus matched two high-quality peptides ("PTTTSSTPTVPSSEPR" (SEQ ID NO: 27) and "TGIPICSIWIR" (SEQ ID NO: 28)), and a medium-scoring peptide covered SSQWNEQPSSK (SEQ ID NO: 29). Therefore, the combined trypsin and GluC digestion gave a total coverage of ∼96%. Trypsin digestion is likely to obtain NLYFQGAK (SEQ ID NO: 30) and KHHGHPNHHK (SEQID NO: 31), but between these peptides " TLK The suspected glycopeptides surrounding "FHPHH" (SEQ ID NO: 32) may hinder its cleavage.

[0529] Alkylated trypsin digestion ( Figure 12 ) to obtain the entire fragment except the last cysteine ​​residue, resulting in an overall coverage of 99.81%.

[0530] Example 5 - HnM1M7-01

[0531] HnM1M7-01 construct

[0532] The secreted synthetic protein (SEQ No. 10) based on the fusion of two modules (HnM1M7-01) begins with a proposed native signal peptide (SEQ No. 47), which is recognized by the Sf9 cellular machinery and targeted for secretion into the culture medium. In addition to containing the secretory signal peptide, the construct was designed to incorporate a hexahistidine tag for affinity purification and an rTEV protease recognition motif for tag removal. This example describes an optimized protocol for the expression and purification of HnM1M7-01 in the SF9 baculovirus system.

[0533] Generation of recombinant bacmid

[0534] Competent E. coli DH10 Multibac cells (Geneva Biotech) containing the bacmid (baculoviral genome vector plasmid) and helper plasmids were transformed with the entry vector according to the manufacturer's protocol (Invitrogen) to generate recombinant bacmids. Briefly, 100 ng of the entry vector (HnM1M7-01 in pFastBac-DUAL) was transformed into chemically competent DH10 Multibac cells and plated on KGTIX plates (kanamycin, gentamicin, tetracycline, IPTG, and X-gal). Successful recombinants (white colonies) were confirmed by replating a number of white colonies onto fresh KGTIX plates and ensuring a stable color phenotype (relative to control non-recombinant blue colonies plated on the same selective plate). Recombinant bacmids (HnM1M7-01 bacmids) from confirmed white DH10 Multibac cells were recovered from the cells by chemical alkaline lysis and used to transfect logarithmic phase Sf9 cells.

[0535] Sf9 transfection and virus amplification

[0536] In a 6-well plate, Sf9 cells were transfected with recombinant bacmid DNA using insect GeneJuice transfection reagent (Merck). 6Logarithmically phase Sf9 cells (~80% confluency) were plated into the wells of a 35 mm 6-well cell culture plate at 4 μg / ml and allowed to adhere to the plate for 60 minutes at 28°C. Four micrograms of recombinant bacmid were incubated with 10 μl of transfection reagent (Merck) in a total volume of 200 μl of Sf900-III medium at room temperature for 30 minutes, then made up to 1 ml with fresh Sf900-III medium. The medium in the 6-well plate was aspirated from the adherent Sf9 cells, and 1 ml of HnM1M7-01 bacmid / GeneJuice solution was overlaid on the cells. The cells were incubated at 28°C for 4 hours, rinsed, and supplemented with an additional 1 ml of Sf900-III medium. After 72 hours of incubation, the transfection mixture was removed from a sterile falcon tube, residual Sf9 cells were pelleted by centrifugation (500 g), and the clarified medium containing the baculovirus was placed in a new 6-well plate. Then use 1.6 ml of 1.5×10 6 Each well was overlaid with Sf9 cells at 100 cells / ml and incubated for an additional 72 hours at 28°C. The amplified virus was harvested, centrifuged at 500 g to remove cells, and the clarified viral stock was supplemented with 2% fetal bovine serum. The amplified viral stock was stored at 4°C prior to titration and expression. For expression of HnM1M7-01, the number of amplification cycles was kept to a minimum (no more than one) to avoid deletion events of the target gene.

[0537] HnM1M7-01 expression

[0538] For expression of HnM1M7-01, 2.0-2.5×10 6 Small-scale protein expression was performed in 200 ml of Sf9 cells in serum-free medium. Cultures were infected with recombinant virus at a ratio of 1:5k to 1:10k and incubated at 28°C with shaking at 130 rpm. Cultures were routinely supplemented with sterile D-glucose, L-serine, and L-glutamine to 5 mM at t48 and t72, and harvested typically 96 hours after infection. The culture medium was clarified by centrifugation at 1500 g before protein harvesting.

[0539] HnM1M7-01 purification

[0540] The culture medium of clarification is adjusted to 50mM Tris.Cl pH 8.0 and 2M NaCl. When NaCl dissolves completely, under sufficient stirring, isopyknic 100% isopropanol is added in the mixture (final NaCl concentration of 1M). The mixture is stirred 30 minutes, and rotated 45 minutes with 6750g. The precipitation after the rotation is resuspended in ultrasonic treatment buffer (25mM Tris pH 8.0, 300mM NaCl, 6M urea and 5% glycerol), until 1 / 5 of the starting culture medium volume. On ice, ultrasonic treatment is carried out with 75% amplitude, pulse 1 second, pulse interval 1 second, until applying minimum 80,000J (Qsonix medium-sized probe). With 10,000xg centrifugal 20 minutes, solution clarification is made. Clarifying supernatant is adjusted to 15mM imidazoles, and continues to be loaded on the 5mL Ni-HisTrap FF (Cytiva) of ultrasonic treatment buffer balance. After protein loading, the column was washed with 5×CV wash buffer (25 mM Tris pH 8.0, 300 mM NaCl, 6 M urea, and 5% glycerol), and the bound protein was eluted stepwise with elution buffer (25 mM Tris pH 8.0, 300 mM NaCl, 15 mM imidazole, 6 M urea, and 5% glycerol). The eluted protein fractions were collected and analyzed on a 10% PAGE Tris-glycine gel to estimate protein purity. The protein fractions were combined and dialyzed overnight at 4°C against 2 L of dialysis buffer (25 mM Tris pH 8.0, 150 mM NaCl, and 5% glycerol).

[0541] Emulsifying properties of HnM1M7-01

[0542] This example aims to demonstrate the emulsion stabilizing effect of HnM1M7-01 protein. To this end, the inventors used HnM1M7-01 as a self-emulsifier to prepare an oil / water emulsion without adding any other surfactants.

[0543] method :

[0544] HnM1M7-01 solution was prepared as described in Example 5.1. Oil-in-water (O / W) emulsions were prepared in a final ratio of 1:1 using MCT oil (caprylic / capric triglyceride, NewDirections Australia) as the oil phase and buffer (control) or protein solution (treated) as the aqueous phase. Samples were prepared by mixing the protein aqueous solution with MCT oil containing 5 μg / mL Nile Red (Merck). The mixture was emulsified using an Omi Sonic Ruptor 400 ultrasonic homogenizer with a 3.8 mm very high intensity processing probe. Emulsification consisted of applying 10×1 second pulses at 50% power limit. The emulsions were imaged using a Nikon Eclipse Ti-S inverted microscope in fluorescence mode using a Texas Red filter. Samples were imaged on day 1, day 2, and day 7 to assess emulsion stability. The tubes were also photographed to assess overall phase separation.

[0545] result :

[0546] If the buffer contains oil droplets, spheres will be seen (Nile red dissolved in oil, Figure 13 Confocal images of the samples on day 1 and day 2 showed the presence of spheres consisting of the emulsified oil-protein solution ( Figure 13 B), reflecting that the HnM1M7-01 modified mixture exhibited some surface activity. Although the buffer / oil mixture (untreated) also showed the formation of oil droplets / spheres in the aqueous phase during the first two days of the experiment ( Figure 13 A, Days 1 and 2), but these spheres were unstable and could not be seen after day 7 ( Figure 14 A). Meanwhile, spheroids observed in the HnM1M7-01-stabilized mixture were found to be stable even after 7 days of equilibration.

[0547] Based on these observations, the inventors demonstrated that HnM1M7-01 exhibits surface activity. Without wishing to be bound by theory, the inventors believe this surface activity may be due to its inherent amphiphilic structure. Therefore, in an oil / HnM1M7-01 buffer system, protein molecules present at the oil / buffer interface are adsorbed there, minimizing interfacial tension. The HnM1M7-01 at the interface rearranges, exposing the hydrophilic chains to the buffer phase and the hydrophobic chains to the oil phase, thus promoting the stability of the oil / buffer emulsion in the mixture.

[0548] Example 6 - HnM1M7-03 - (SEQ ID NO: 50)

[0549] Expression and purification of HnM1M7-03

[0550] This example describes the production of polypeptides in E. coli BL21(DE3) using a kanamycin resistance plasmid, and the resulting production of purified polypeptide HnM1M7-03.

[0551] The nucleic acid sequence encoding the synthesis of HnM1M7-03 protein was synthesized using non-template PCR. In brief, the virtual nucleic acid sequence was converted to an oligonucleotide sequence using the software suite LIMS (DNA TwoPoint®, Inc., Newark, CA, USA). The full-length nucleic acid sequence was synthesized by assembling oligonucleotides using non-template PCR. Standard cloning methods were used to purify and clone amplicons (Molecular Cloning: A Laboratory Manual, 2012, Green and Sambrook).

[0552] The gene encoding the synthesis of the HnM1M7-03 protein was cloned into the expression vector pD451-SR (DNA TwoPoint, Inc., Newark, CA, USA) containing a T7 inducible promoter. The purified plasmid containing the gene was transformed into heat-shocked chemically competent Escherichia coli BL21 (DE3) cells and plated on non-inducing agar containing 0.1 mg / L kanamycin. The plates were incubated overnight at 37°C. Single colonies were selected from the transformation plates and grown in non-inducing medium. The cells were then suspended in a glycerol-containing medium and stored at -80°C to prepare a glycerol stock.

[0553] Escherichia coli BL21(DE3) containing a plasmid expressing the HnM1M7-03 protein was grown in a 100-L fermentor. A culture medium was prepared and autoclaved in the fermentor. The culture medium components and concentrations were as follows: casein hydrolysate, 12 g / L; yeast extract, 24 g / L; NaCl, 10 g / L; K₂HPO₄, 8 g / L; and glycerol, 30 g / L. After the culture was cooled, 50 mg / L of kanamycin was added. Preculture 1 flask was grown at 37°C for approximately 6 hours. Preculture 2 flask was inoculated from preculture 1 and grown at 28°C for approximately 12 hours. The fermentor was inoculated from preculture 2 flask and the temperature was controlled at 37°C for the initial growth phase. Dissolved oxygen was controlled at 30% of air saturation, and the fermentor was maintained at a pH of 6.8. 5 g / L of Pluronic antifoam was added to control foaming.

[0554] Immediately before induction, the fermentor was cooled to 20°C and 0.2 mM IPTG was used to induce expression of the HnM1M7-03 protein at an OD600 of approximately 2. Approximately 22 hours after induction, the biomass was concentrated by tangential flow filtration (TFF) and harvested by centrifugation. The biomass was frozen at -20°C until further processing.

[0555] The biomass was thawed overnight and resuspended in lysis buffer (25 mM Tris, 2 mM MgCl 2 , 0.5% (w / v) Triton X-100, pH 8.0) using a Miccra D-9 rotor-stator. 2 mg of lysozyme per gram of biomass was used for lysis at room temperature for 40 minutes. 25 units of benzonase were used for DNA degradation per gram of biomass. The insoluble material was collected by centrifugation at 17,000 g for 20 minutes. The lysate pellet (i.e., the "insoluble" fraction) was washed in 25 mM Tris, 2 mM MgCl 2 , 0.5% (w / v) Triton X-100 pH 8.0 for 40 minutes. The insoluble material was collected by centrifugation at 17,500 g for 40 minutes. The washed pellet was further washed in 0.05 M sodium phosphate pH 11.5. The insoluble material was collected by centrifugation at 17,000 g for 40 minutes. The washed precipitation was extracted at room temperature for 40 minutes in 10mM Tris, 4M guanidine pH 8.0. The fraction containing the HnM1M7-06 protein extracted was centrifuged at 17,000xg for 20 minutes to remove debris, and filtered supernatant. The extracted fraction was diluted to fixed metal affinity chromatography (IMAC) loading conditions (10mM Tris, 4M guanidine, 500mM NaCl, 20mM imidazoles, pH 8.0). The diluted material was loaded on the HiScale post that is filled with IMAC Sepharose 6Fast Flow resin (Cytiva), and nickel was housed in the resin. The IMAC column was washed with loading buffer (10 mM Tris, 2 M guanidine, 0.5 M NaCl, 20 mM imidazole, pH 8.0), and the HnM7-06 protein was recovered with elution buffer (10 mM tris, 2 M guanidine, 0.5 M NaCl, 500 mM imidazole, pH 8.0).

[0556] The HnM1M7-03 protein was precipitated from the eluted fraction using 2M ammonium sulfate. The precipitated protein was recovered by centrifugation at 12,000 g for 10 minutes. The precipitated protein pellet was resuspended in ultrapure water and centrifuged at 12,000 g for 10 minutes to collect the washed HnM1M7-03 protein pellet.

[0557] Physical properties of HmM1M7-03

[0558] Surface water contact angle of HnM1M7-03 protein-coated glass surface

[0559] Protein solution (1 mg / mL HnM1M7-03, dissolved in 98% formic acid) was drop-cast onto a glass slide and then dried overnight at room temperature. The coated slide was mounted in a Theta flow tensiometer (Biolin Scientific, UK). Water contact angle (WCA) was recorded continuously over 30 seconds.

[0560] The surface of untreated glass was hydrophilic, exhibiting a water contact angle of 22-24°, which remained unchanged after 30 seconds of equilibration. Glass coated with HnM1M7-03 exhibited a contact angle of 60-65°, which remained unchanged after 30 seconds of equilibration. The inventors have thus demonstrated that HnM1M7-03 is a useful material for modifying the relative hydrophobicity of materials, providing the ability to impart useful properties to a variety of articles when applied as a film or coating.

[0561] Example 6 - HnM7-06 (SEQ ID NO: 52)

[0562] Expression and purification of HnM7-06

[0563] This example describes the use of a kanamycin resistance plasmid to produce polypeptides in E. coli BL21(DE3) and thereby produce the purified polypeptide HnM7-06.

[0564] The synthetic nucleotide sequence encoding HnM7-06 protein was synthesized using non-template PCR. In brief, the virtual nucleic acid sequence was converted to an oligonucleotide sequence using the software suite LIMS (DNA TwoPoint®, Inc., Newark, CA, USA). The full-length nucleic acid sequence was synthesized by assembling oligonucleotides using non-template PCR. Standard cloning methods were used to purify and clone amplicon (Molecular Cloning: A Laboratory Manual, 2012, Green and Sambrook).

[0565] The gene encoding the HnM7-06 protein was cloned into the expression vector pD451-SR (DNA TwoPoint, Inc., Newark, CA, USA) containing a T7 inducible promoter. The purified plasmid containing the gene was transformed into heat-shocked, chemically competent Escherichia coli BL21 (DE3) cells and plated on non-inducing agar containing 0.1 mg / L kanamycin. The plates were incubated overnight at 37°C. Single colonies were selected from the transformation plates and grown in non-inducing medium. The cells were then suspended in a glycerol-containing medium and stored at -80°C to prepare a glycerol stock.

[0566] Escherichia coli BL21(DE3) containing a plasmid expressing the HnM7-06 protein was grown in a 100-L fermentor. A culture medium was prepared and autoclaved in the fermentor. The culture medium components and concentrations were as follows: casein hydrolysate, 12 g / L; yeast extract, 24 g / L; NaCl, 10 g / L; K₂HPO₄, 8 g / L; and glycerol, 30 g / L. After the culture was cooled, 50 mg / L of kanamycin was added. Preculture 1 flask was grown at 37°C for approximately 7 hours. Preculture 2 flask was inoculated from preculture 1 and grown at 28°C for 17 hours. The fermentor was inoculated from preculture 2 flask and the temperature was controlled at 37°C for the initial growth phase. Dissolved oxygen was controlled at 30% of air saturation, and the fermentor was maintained at a pH of 6.8. 5 g / L of Pluronic antifoam was added to control foaming.

[0567] Immediately before induction, the fermentor was cooled to 20°C and 0.2 mM IPTG was used to induce expression of the HnM7-06 protein at an OD600 of approximately 2. The biomass was concentrated by tangential flow filtration (TFF) and harvested by centrifugation approximately 22 hours after induction. The biomass was frozen at -20°C until further processing.

[0568] The biomass was thawed overnight and resuspended in lysis buffer (25 mM Tris, 2 mM MgCl2, 0.5% (w / v) TritonX-100, pH 8.0) using a Miccra D-9 rotor-stator. Lysis was performed at room temperature for 40 minutes using 2 mg of lysozyme per gram of biomass. DNA was degraded using 25 units of benzonase per gram of biomass. Insoluble material was collected by centrifugation at 12,000 g for 20 minutes. The lysate pellet (i.e., the "insoluble" fraction) was washed in 25 mM Tris, 2 mM MgCl2, 0.5% (w / v) TritonX-100 pH 8.0 for 40 minutes. Insoluble material was collected by centrifugation at 17,500 g for 40 minutes. The washed pellet was further washed in 0.05 M NaOH. Insoluble material was collected by centrifugation at 17,500 g for 20 minutes. The precipitate that 0.05M NaOH was washed was extracted at room temperature for 40 minutes in 10mM Tris, 4M guanidine pH 8.0. The fraction containing HnM7-06 protein extracted was centrifuged for 20 minutes at 17,500xg to remove debris, and filtered supernatant. The fraction extracted was diluted to fixed metal affinity chromatography (IMAC) loading conditions (10mM Tris, 4M guanidine, 500mM NaCl, 20mM imidazoles, pH 8.0). The diluted material was loaded on the HiScale post that is filled with IMAC Sepharose 6Fast Flow resin (Cytiva), and this resin is equipped with nickel. The IMAC column was washed with loading buffer (10 mM Tris, 2 M guanidine, 0.5 M NaCl, 20 mM imidazole, pH 8.0), and the HnM7-06 protein was recovered with elution buffer (10 mM tris, 2 M guanidine, 0.5 M NaCl, 500 mM imidazole, pH 8.0).

[0569] The HnM7-06 protein was precipitated from the eluted fraction using 2.5 M ammonium sulfate. The precipitated protein was recovered by centrifugation at 12,000 g for 10 minutes. The precipitated protein pellet was resuspended in ultrapure water and centrifuged at 12,000 g for 10 minutes to collect the washed HnM7-06 protein pellet.

[0570] Physical properties of HnM7-06

[0571] Surface water contact angle of HnM7-06 protein-coated glass surface

[0572] Protein solution (1 mg / mL HnM7-06, dissolved in 98% formic acid) was drop-cast onto a glass slide and then dried overnight at room temperature. The coated slide was mounted in a Theta flow tensiometer (Biolin Scientific, UK). The water contact angle was recorded continuously over 30 seconds.

[0573] The surface of the untreated glass was hydrophilic, exhibiting a water contact angle of 22-24°, which remained unchanged after 30 seconds of equilibration. The glass coated with HnM7-06 exhibited a contact angle of 76-80°, which remained unchanged after 30 seconds of equilibration. The inventors thus demonstrated that HnM7-06 is a promising material for modifying the relative hydrophobicity of a material, such as may be desirable when used as a thin film or coating.

[0574] Co-spinning of HnM7-06 protein and nylon

[0575] HnM7-06-nylon fibers were wet-spun using a nylon dope solution containing 15 wt.% nylon in 98% formic acid (FA) using the same method as described in Example 8. HnM7-06 was dissolved in FA, followed by the addition of nylon, using continuous mixing and shaking until a clear solution was obtained. Wet-spun fibers made from these solutions were tested for tensile strength.

[0576] Tensile strength of co-spun HnM7-06-nylon fiber

[0577] The mechanical properties of HnM7-06-nylon fibers were evaluated using a universal testing machine (UTM) (Agilent T150 USA) with a 0.5 N sensor. The fibers were mounted on a paper frame (10 mm window) and then subjected to a 0.01 mm / s -1 The strain rate was set at 1000 s. The nominal gauge length was fixed at 10 mm. Two samples were measured from each fiber.

[0578] Table 2. Tensile strength measurements of HnM7-06-nylon fibers

[0579]

[0580] As shown in Table 2, when HnM7-06 protein is included in nylon co-spun fibers, modulus, tensile strength, and toughness are all increased. Elongation at break is also increased when HnM7-06 protein is included in nylon co-spun fibers at levels up to and including 10 wt.%. The inventors have thus demonstrated that HnM7-06 protein improves the strength of nylon fibers, imparting desirable properties to the fibers for use in a variety of applications, including textiles, wovens, and nonwovens.

[0581] Example 8 - HnP1 (SEQ ID NO: 4)

[0582] This example describes the production of the peptide HnP1 by chemical solid phase synthesis of oligopeptides.

[0583] The peptide (SEQ ID NO: 4) was synthesized on a pre-loaded 2-Cl-Trt resin using a standard Fmoc synthesis protocol with DIC / HOBt coupling on an APEX 396 automated synthesizer. The resin was swollen in DMF for 30 min, treated with 20 v% piperidine-DMF for 8 minutes at room temperature to remove the Fmoc protecting group, and washed three times with DMF. For the coupling reaction, Fmoc-protected amino acids, HOBt, DIC, and NMP were added to the resin. The mixture was vortexed at room temperature for 20 minutes. The resin was then washed once with DMF. The cycle of deprotection and coupling steps was repeated until the last amino acid residue was assembled. The resin was then washed with DMF, DCM, and air-dried. The peptide was cleaved using a TFA mixture (95 v% TFA, 2.5 v% water, and 2.5 v% TIS) for three hours. The crude peptide was precipitated by adding ice-cold anhydrous ether, washed three times with anhydrous ether, and dried in vacuo. After synthesis, peptide purification and salt conversion (HCl or acetate) were performed using conventional preparative HPLC.

[0584] Co-spinning of HnP1 peptide and nylon

[0585] Nylon 6 / 6 pellets (molecular weight 252.35 Daltons, Sigma-Aldrich, St Louis, MO, USA) were dissolved in 98% formic acid (FA) and stirred overnight to obtain a clear solution (15% w / w nylon) to prepare nylon spinning dope solutions. HnP1 was first dissolved in FA (various concentrations), and then nylon was dissolved in the HnP1 solution to prepare the nylon-HnP1 combination dope. The solution was filled into a syringe and then wet-spun in a hydrogel bath to produce single fibers for testing.

[0586] The morphology of the as-spun fibers was characterized using scanning electron microscopy (SEM). Pristine nylon (0% HnP1) and HnP1-nylon fibers were coated with a 5 nm Pt conductive coating and then imaged using a Zeiss Supra 55VP field emission gun at an accelerating voltage of 5 kV, an aperture size of 20 μm, and a working distance of 7 to 8 mm.

[0587] Co-spinning of HnP1 and silk

[0588] Regenerated silk was prepared using undegummed fibers reeled by Bombyx mori (SRR Silk Reeling Unit; Ramanagara, Karnataka, India). The silk fibers were dissolved, degummed, and coagulated using lithium bromide (LiBr, 99%), sodium carbonate (Na2CO3, 99.5%), and ammonium sulfate ((NH4)2SO4, 99%) (Sigma-Aldrich), respectively. B. mori silk fibers were degummed using 0.2% sodium carbonate at a ratio of 1:50 raw silk (g): liquid (ml) in an Ahiba IR Pro rotary dyer (Datacolor, Lawrenceville, USA) in a 500 ml aluminum can at 98°C for 30 minutes. The degummed silk was washed several times with deionized water and then dried in a fan-forced oven at 60°C. For regenerated silk fibroin (RSF), sodium carbonate-degummed silk was dissolved in a 9.3 M LiBr solution at a raw silk (g) to liquid (ml) ratio of 1:7.5 and reacted at 60°C for 40 minutes. The dissolved solution was then dialyzed in a deionized water bath and concentrated to yield a concentrated silk solution. The silk solution was freeze-dried for 48 hours to yield pure solid regenerated silk crystals.

[0589] A similar method as used for co-spinning with nylon was used for wet spinning of 12 wt% silk with different concentrations of HnP 1. The morphology of the as-spun fibers was examined using SEM.

[0590] Discussion of SEM results:

[0591] The measured fiber diameters of wet-spun nylon, silk, HnP1-silk, and HnP1-nylon fibers were found to be consistent, ranging from approximately 60-70 μm. For nylon and silk, fiber roughness gradually increased as the HnP1 content increased from 1 wt.% to 10 wt.%. Increased surface roughness increases the surface area to volume ratio. Without wishing to be bound by theory, the inventors believe these properties could be used in applications such as functional textiles, which benefit from enhanced reactive sites, adsorption, surface adhesion, and heat transfer.

[0592] The cross-sectional morphology of nylon and HnP1-nylon and HnP1-silk fibers demonstrates a highly porous structure, as shown in Figures x and y. Interestingly, as the amount of HnP1 in the mixed matrix of HnP1-nylon and HnP1-silk increases from 1 wt.% to 10 wt.%, smaller, more regular pores form. Smaller pores can lead to higher adsorption rates for other chemicals, higher interfacial energy, and improved wettability. Without wishing to be bound by theory, the inventors believe that these properties will be beneficial in applications requiring cell adhesion or water-based coating formulations, for example, in the production of functional textile materials.

[0593] Surface water contact angle measurement of fibers containing HnP1.

[0594] The water contact angle (WCA) of single fibers co-spun with nylon or silk and HnP1 was measured using a Theta Flow Tensiometer (Biolin Scientific, UK). The WCA was recorded continuously for 30 s.

[0595] WCA Results Discussion

[0596] like Figure 21 and Figure 22 As shown, the WCA of nylon and silk fibers decreased when more HnP1 was included in the fiber composition, indicating an HnP1 concentration-dependent change in surface hydrophilicity (wettability).

[0597] These results indicate that the incorporation of HnP1 acts to increase the hydrophilicity of both nylon and silk fibers. Without wishing to be bound by theory, the inventors believe that these properties will provide useful benefits in applications requiring wettable fibers, such as in the manufacture of absorbent materials.

[0598] Tensile strength of HnP1-nylon co-spun fibers

[0599] The mechanical properties of HnP1-nylon fibers were evaluated using a universal testing machine (UTM) (Agilent T150 USA) with a 0.5 N sensor. The fibers were mounted on a paper frame (10 mm window) and then subjected to a 0.01 mm / s -1 The strain rate was set at 1000 s. The nominal gauge length was fixed at 10 mm. Two samples were measured from each fiber.

[0600] Table 3. Tensile strength measurements of HnP1-nylon fibers

[0601]

[0602] Discussion of tensile strength results

[0603] As shown in Table 3, the tensile strength of nylon was improved by including the HnP1 peptide in the co-spun fibers.

[0604] The inventors have thus demonstrated that the HnP1 peptide improves the strength of nylon fibers, imparting useful and desirable properties to the fibers for use in a variety of applications, such as in textiles, woven and nonwoven materials.

[0605] Table 4 - Nucleic acid and amino acid sequences

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615]

[0616]

[0617]

[0618]

[0619]

[0620]

[0621]

[0622]

[0623]

[0624]

[0625]

[0626]

[0627]

[0628]

[0629]

[0630]

[0631]

[0632]

[0633]

[0634]

[0635]

[0636]

[0637]

[0638]

[0639]

[0640] Industrial Applicability

[0641] The present invention has industrial applications in the production of polypeptides that can be used to provide amphiphilic, hydrophobic, hygroscopic and / or hydrophilic coatings to a variety of articles, including but not limited to synthetic fibers, textiles and components thereof, and biomedical devices and components thereof.

Claims

1. An isolated polynucleotide encoding an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO: 50, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 52.

2. An isolated polynucleotide having at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, preferably 99% nucleic acid sequence identity to 49, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 51.

3. An isolated polypeptide having 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, preferably 99% amino acid sequence identity to SEQ ID NO: 50, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 52.

4. A protein polymer comprising at least a portion of the polypeptide according to any one of claims 1 to 3.

5. A vector comprising the isolated polynucleotide according to claim 1 or claim 2.

6. A vector comprising an isolated polynucleotide encoding the isolated polypeptide according to claim 3 or the protein polymer according to claim 4.

7. An isolated host cell comprising the isolated polynucleotide according to claim 1 or claim 2, the isolated polypeptide according to claim 3, the protein polymer according to claim 4, and / or the vector according to claim 5 or claim 6.

8. The isolated host cell according to claim 7, which is a bacterial host cell, preferably Escherichia coli.

9. The isolated host cell according to claim 7, which is a fungal host cell, preferably a Pichia or Aspergillus cell, preferably Aspergillus niger.

10. The isolated host cell according to claim 7, which is an insect cell, preferably an Sf / 9 cell or a High Five cell.

11. A composition comprising the isolated polynucleotide of claim 1 or claim 2, the isolated polypeptide of claim 3, the protein polymer of claim 4, and / or the vector of claim 5 or claim 6, and / or the isolated host cell of claim 7, 8, 9 or 10.

12. A method for producing at least one FUN polypeptide or a portion thereof, the method comprising heterologously expressing the FUN polynucleotide of claim 1 or claim 2 or the vector of claim 5 or 6 in an isolated host cell of claim 7, 8, 9 or 10.

13. A method for producing at least one protein polymer comprising at least one polypeptide or a portion thereof, said method comprising heterologously expressing the FUN polynucleotide according to claim 1 or claim 2 or the vector according to claim 5 or 6 in an isolated host cell according to claim 7, 8, 9 or 10.

14. Use of the FUN polypeptide or a portion thereof according to claim 3 or the composition according to claim 11 for coating an article.

15. Use of the FUN polypeptide or a portion thereof according to claim 3 or the composition according to claim 11 for preparing a thin film on an article.

16. Use according to claim 14 or 15, wherein the article is selected from textiles or components or parts thereof and biomedical devices or components or parts thereof.

17. Use according to claim 14 or 15, wherein the article or component or part thereof is a synthetic fiber.

18. Use according to any one of claims 14 to 17, wherein the component or part thereof is a synthetic polymer.

19. The use according to claim 17 or claim 18, wherein the synthetic fibers or synthetic polymers are selected from polyester, spandex, rayon, nylon, acrylic resins, microfibers, neoprene, polyamide, acetate, polyvinyl chloride (PVC) and synthetic or "artificial" leather or fur fibers and polymers, preferably nylon.

20. Use according to claim 14 or 15, wherein the article or component or part thereof is a natural fiber.

21. Use according to claim 20, wherein the natural fiber is selected from the group consisting of cotton, wool, silk, coir, alpaca, flax, bamboo, sisal and jute, preferably silk.

22. Use according to claim 14 or 15, wherein the article is a textile, preferably a natural textile or a synthetic textile.

23. The use according to claim 14 or 15, wherein the article or component or part thereof is a biomedical device or is comprised in or on a biomedical device.

24. The use according to claim 23, wherein the biomedical device is an implantable biomedical device.

25. The use according to claim 23, wherein the implantable biomedical device is selected from cardiovascular devices, including cardiac defibrillators, pacemakers and left ventricular assist devices, breast implants, cochlear implants, intraocular lenses, joint replacements, including hip implants, catheters, dialysis tubing, contraceptive intrauterine devices, stents, sutures, staples, bandages and wound dressings.

26. Use according to claim 14 or claim 15, wherein the article is an air filtration device or a component or part thereof.

27. Use according to claim 26, wherein the component or part thereof is an air filter.

28. Use according to claim 26 or 27, wherein the components or parts thereof are synthetic or natural fibers and / or polymers.

29. Use according to claim 28, wherein the synthetic or natural fibers and / or polymers are contained in the air filter.

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