Recombinant Polypeptides

Abstract

The present invention relates generally to polypeptides isolated from the solitary bee, Hylaeus nubilosus, proteinaceous polymers comprising said polypeptides or portions thereof, compositions comprising said polypeptides and / or polymers or portions thereof, and methods for the recombinant production of said polypeptides and / or polymers or portions thereof.

Description

[Technical Field]

[0001] The present invention relates generally to polypeptides derived from the colletid wasp, Hylaeus nubilosus. The invention also relates to recombinant production of the polypeptides, methods for producing the polypeptides, and uses of the polypeptides to produce various articles of manufacture having desirable properties. [Background technology]

[0002] Synthetic polymers (a component of "plastics") are an extremely useful group of materials. They are found in many of the everyday products we wear, sleep in, and build. They are often used to manufacture various materials to impart desirable properties. Examples of such properties include mechanical strength, heat resistance, moisture absorption, or the ability to repel water or resist getting wet.

[0003] Industries where synthetic polymers are widely used include textiles, biomedical devices, and cosmetics. In one example, mass-produced synthetic clothing is composed of various polymers spun into synthetic fibers, such as polyester, nylon, vinyl, and acrylic. In some cases, these synthetic fibers are hydrophobic, meaning they have poor water and sweat absorption, which impacts comfort. In other cases, the fibers are hydrophilic and too absorbent, which also impacts wearability.

[0004] In other examples, synthetic polymers may be used to impart moisture-wicking, hygroscopic, or hydrophilic properties to textiles and other materials. Currently, the synthetic polymers used for such applications are not environmentally friendly. For example, polyetheramines are used to provide hydrophilic coatings on nylon clothing. Unfortunately, these polymers degrade over time, releasing by-products into the environment that are harmful to aquatic life.

[0005] Due to environmental concerns associated with textile finishing chemicals, major manufacturers are shifting their focus towards eco-friendly green (bio-based) chemicals. Green chemicals are produced using animal and vegetable fats / oils, making them more environmentally friendly and cost-effective than conventional chemicals. However, the additional weight of oil-based products, the need for reapplication, and fluctuations in the availability and price of raw materials pose challenges for market players in achieving profitability and economies of scale.

[0006] Another important application of such coatings is in the medical device industry. Examples of various biomedical devices currently coated with hygroscopic / hydrophilic coatings include catheters, implants, tubing, lenses, disposable plastic slides, and more. In many instances, these coatings provide the coated biomedical devices (especially those used in the field) with superior biocompatibility, hydrophilicity, hydrophobicity, and / or abrasion resistance, enabling effective performance.

[0007] These coatings are typically composed of materials such as polyurethane, silicone, and polyethylene terephthalate.

[0008] Synthetic polymers are also used in the manufacture of cosmetics and personal care products to impart properties such as lubricity and viscosity. Phthalates are a group of chemicals known as "ubiquitous" chemicals, found in products such as nail polish, perfumes, deodorants, hair gels, shampoos, soaps, hairsprays, and body lotions. Phthalates have been identified as endocrine-disrupting chemicals in humans, causing a variety of problems, including hormone imbalances, reproductive health issues, and developmental disorders. Phthalates also bioaccumulate in fish, are toxic to aquatic ecosystems, and pose a risk to humans.

[0009] As public awareness of the environmental concerns associated with the use of synthetic polymers increases, there is an urgent need for industry to provide non-toxic, environmentally friendly alternatives.

[0010] It is an object of the present invention to provide natural polypeptides or derivatives thereof that can be used as environmentally friendly replacements for at least some of the synthetic polymers currently used in the various industries mentioned above, and / or to provide methods for producing such polypeptides or derivatives thereof, and / or at least to provide a useful choice for the general public.

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

[0012] Disclosed herein are Hylaeus nuvilosus polypeptides, also referred to as "FUN" polypeptides. Also disclosed are proteinaceous polymers comprising Hylaeus nuvilosus "FUN" polypeptides, or at least a portion of a Hylaeus nuvilosus polypeptide. In some embodiments, the proteinaceous polymers comprise quasi-repeated domains. In some embodiments, the polymers are capable of assembling into fibers. Also disclosed are compositions of such polypeptides and proteinaceous polymers, as well as methods of making and using the polypeptides, proteinaceous polymers, and compositions.

[0013] Accordingly, 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 a SEQ ID NO:

[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 embodiment, the present invention provides 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 comprising 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 an isolated polynucleotide described herein.

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

[0022] In another aspect, the present invention relates to a proteinaceous polymer comprising a FUN polypeptide as described herein or a portion thereof, wherein said proteinaceous 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, vector and / or proteinaceous polymer described herein and a carrier, diluent or excipient.

[0024] In another aspect, the invention relates to a method of making 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, or a portion thereof, the method 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 of making a proteinaceous polymer comprising a FUN polypeptide or a portion thereof selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 50, and 52, the method comprising heterologously expressing the FUN polypeptide or a portion thereof in an isolated host cell under conditions resulting in expression of the FUN polypeptide or a portion thereof, and optionally purifying the proteinaceous polymer.

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

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

[0028] Other aspects of the present invention will become apparent from the following description, given by way of example only, and with reference to the accompanying drawings. [Brief explanation of the drawings]

[0029] The present invention will now be described by way of example with reference to the drawings. [Figure 1]ClustalW alignment of 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_DN N0_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) were identified as important from different entries in the Transcript Database (Version 2). [Figure 2] Consensus sequence alignment. Alignment of the FUN_069765.2 sequence (from the long read sequence assembly), SEQ ID NO: 2, with the proteome sequence assembled from nested material mass spectrometry, SEQ ID NO: 33. Consensus sequence: X = mismatch, Z = glutamic acid or glutamine. [Figure 3] Overview flowchart of nest material genetic identification. Data and samples are shown in solid boxes. Analyses and software programs are shown in bold with dashed boxes. Findings are shown in dotted boxes. Arrows indicate directional relationships between boxes. Dashed arrows indicate relationships between findings. [Figure 4] SDS-PAGE showing purified FUN polypeptide. The last three lanes show IMAC elution of the FUN polypeptide band with an apparent molecular weight of approximately 100 kDa on SDS-PAGE, estimated to be >95% pure. [Figure 5]FTIR image of FUN polypeptide coated on foil. FTIR images of FUN polypeptide and silk fibroin coated on aluminum foil with and without ethanol treatment. [Figure 6] FTIR-FUN polypeptide coated on glass substrate. FTIR images of FUN polypeptide coated on glass substrate with and without ethanol treatment. [Figure 7] Scanning electron microscope (SEM) image of a FUN polypeptide coating. The image on the right is a magnified cross-section of the first image, showing thread-like structures along the film surface. [Figure 8] Wettability - Water contact angle profile of FUN polypeptide (at the time of contact and after 30 seconds). Comparison of water contact angle profiles of FUN polypeptide and silk fibroin coated on a glass substrate (with and without ethanol treatment). [Figure 9] Washability—The washability of the FUN polypeptide coating and its resistance to water and PBS was evaluated. The FUN polypeptide coating remained intact (as indicated by Coomassie staining coverage) after 24 hours of immersion in water or PBS solution. [Figure 10] Sequence coverage by Glu-C digestion: bold text represents high-confidence peptide matches, regular text represents medium confidence, italics represents low confidence, and underlines represent unobserved regions. [Figure 11] Sequence coverage by combining Glu-C digestion and trypsin digestion: Reduced and alkylated trypsin digestion yielded two high-confidence peptides (bold) and one moderate peptide match (normal) in the C-terminal region, resulting in an overall coverage of 95.78%. [Figure 12] Alkylated trypsin digestion results in an overall coverage of 99.81%. Alkylated trypsin digestion captures the entire region except for the last cysteine ​​residue, resulting in an overall coverage of 99.81%. [Figure 13]Photographs and confocal images of tubes on days 1 and 2 of A) oil / buffer emulsion (control) and B) oil / HnM1M7-01 in buffer emulsion. [Figure 14] A) Photograph and confocal image of a tube of oil / buffer emulsion (control). No spherical particles were observed in the aqueous phase. B) Oil / HnM1M7-01 in buffer emulsion. Stable spherical particles were observed on day 7, reflecting the emulsion stabilizing effect of HnM1M7-01. [Figure 15] Water contact angle (degrees) of glass slides with and without HnM1M7-03 coating. [Figure 16] Water contact angle (degrees) of glass slides with and without HnM7-06 coating. [Figure 17] SEM surface morphology of wet-spun nylon and HnP1 nylon (1, 5, and 10 wt%) fibers. [Figure 18] SEM cross-sectional morphology of wet-spun nylon and HnP1 nylon (1, 5, and 10 HnP1 wt%) fibers. [Figure 19] SEM surface morphology of wet-spun silk and HnP1 silk (1, 2, 5, and 10 wt % HnP1) fibers. [Figure 20] SEM cross-sectional morphology of wet-spun silk and HnP1 silk (1, 2, 5, and 10 wt%) fibers. [Figure 21] Water contact angles (degrees) of wet-spun nylon and HnP1 nylon (HnP1 content 1, 2, 5, and 10 wt%) fibers. [Figure 22] Water contact angles (degrees) of wet-spun silk and HnP1 silk (HnP1 content 1, 2, 5, and 10 wt %) fibers. DETAILED DESCRIPTION OF THE INVENTION

[0030] definition The following definitions are presented to more clearly define the present invention and as a guide to those of ordinary skill in the art in practicing the present invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein are understood to have the same meaning as understood by one of ordinary skill in the relevant art to which this disclosure pertains.

[0032] The term "comprises" as used in this specification and claims means "consisting at least in part of." That is, when interpreting statements containing "comprises" in this specification and claims, all of the features beginning with this term in each statement must be present, although other features may also be present. Related terms such as "comprise" or "comprised" are to be interpreted in the same way.

[0033] As used herein, the term "consisting essentially of" means particular materials or steps, and those that do not materially affect the basic and novel characteristics of the claimed invention.

[0034] As used herein, the term "consisting of" means the specified materials or steps of a claimed invention, excluding any element, step, or ingredient not specified in the claim.

[0035] As used herein, the term "vector" refers to, but is not limited to, any type of polynucleotide molecule that can be used to manipulate genetic material so that it can be amplified, replicated, manipulated, replicated partially, modified, and / or expressed. In some embodiments, a vector can be used to transport a polynucleotide contained in the vector into a cell or organism.

[0036] As used herein, the term "polynucleotide" refers to a single- or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length, including, but not limited to, coding and non-coding regions of genes, sense and antisense sequences, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polynucleotides, isolated and purified natural DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers, fragments, gene constructs, vectors, and modified polynucleotides. References to nucleic acids, nucleic acid molecules, nucleotide sequences, and polynucleotide sequences shall be understood similarly.

[0037] As used herein, the term "gene" refers to a biological unit of heredity, a self-replicating gene located at a specific location (locus) on a specific chromosome. In one embodiment, the specific chromosome is a eukaryotic or bacterial chromosome. As used herein, the term "bacterial chromosome" is used interchangeably with the term "bacterial genome."

[0038] As used herein, the term "endogenous" refers to a component of a cell, tissue, or organism that occurs naturally or is produced within 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.

[0039] As used herein, the term "exogenous" refers to a component of a cell, tissue, or organism that does not naturally occur or is not naturally produced within that cell, tissue, or organism. An exogenous component can be, for example, a polynucleotide sequence introduced into a cell, tissue, or organism, or a polypeptide expressed in the cell, tissue, or organism from that polynucleotide sequence.

[0040] As used herein, "naturally occurring" as used with respect to polynucleotide sequences of the present invention refers to a primary polynucleotide sequence found in nature. A synthetic polynucleotide sequence identical to a wild-type polynucleotide sequence is considered a naturally occurring sequence for the purposes of this disclosure. What is important for a naturally occurring polynucleotide sequence is that the actual sequence of nucleotide bases that make up the polynucleotide is found in nature or is known.

[0041] For example, wild-type polynucleotide sequence is, but not limited to, a naturally occurring polynucleotide sequence.Naturally occurring polynucleotide sequence also refers to the variant polynucleotide sequence that is different from wild-type and found in nature.For example, it includes, but is not limited to, allelic variants, naturally occurring recombinant polynucleotide sequences due to hybridization or horizontal gene transfer, etc.

[0042] As used herein, "non-naturally occurring" in reference to the polynucleotide sequences of the present invention refers to a polynucleotide sequence that does not exist in nature. Examples of non-naturally occurring polynucleotide sequences include, but are not limited to, variants and variant polynucleotide sequences that have been artificially created, for example, by point mutation, insertion, deletion, etc. Non-naturally occurring polynucleotide sequences also include chemically evolved sequences. What is important about non-naturally occurring polynucleotide sequences of the present invention is that the actual sequence of nucleotide bases that make up the polynucleotide is not found or known in nature.

[0043] The term "wild-type" as used herein with respect to a polynucleotide refers to a naturally occurring, i.e., non-mutated, form of a polynucleotide. A variant polynucleotide refers to a polynucleotide that has undergone mutations known in the art, including, but not limited to, point mutations, insertions, deletions, substitutions, amplifications, translocations, and the like.

[0044] The term "wild-type" as used herein with respect to a polypeptide refers to a naturally occurring, non-mutated form of a polypeptide. A wild-type polypeptide is one that can be expressed from a wild-type polynucleotide.

[0045] The term "coding region" (CDS) or "open reading frame" (ORF) refers to the sense strand of a genomic DNA or 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 genetic construct or expression cassette, a "coding region" (CDS) can only be expressed when it is operably linked to a promoter sequence and / or other regulatory elements.

[0046] "Operably linked" means that the sequence to be expressed is under the control of regulatory elements.

[0047] 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, genetic construct, or expression cassette, and includes promoters, transcriptional control sequences, translational control sequences, origins of replication, tissue-specific regulatory elements, temporal control elements, enhancers, polyadenylation signals, repressors, and terminators. Regulatory elements may be "homologous" or "heterologous" to the polynucleotide insert expressed from a genetic construct, expression cassette, or vector described herein. When a genetic construct, expression cassette, or vector described herein is present in a cell, the regulatory element may be "endogenous," "exogenous," "naturally occurring," and / or "non-naturally occurring" with respect to the cell.

[0048] The term "non-coding region" refers to the untranslated sequences located upstream of the translation start site and downstream of the translation termination site. These sequences are also called the 5'UTR and 3'UTR, respectively. These regions contain elements necessary for transcription initiation and termination and for regulating translation efficiency.

[0049] Terminators are sequences that terminate transcription and are present at the 3' untranslated end of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and, in some cases, have been shown to have spatial control functions.

[0050] The term "promoter" refers to a non-transcribed cis-regulatory element upstream of a coding region that controls transcription of a polynucleotide sequence. A promoter consists of a cis-initiation element and a conserved box that specify the transcription start site. In one non-limiting example, a bacterial promoter may contain a "Pribnow box" (also known as a -10 region) and other motifs to which transcription factors bind and promote transcription. A promoter may be homologous or heterologous to the polynucleotide sequence to be expressed. When a polynucleotide sequence is expressed intracellularly, the promoter may be an endogenous or exogenous promoter. A promoter may be a constitutive promoter, an inducible promoter, or a regulatable promoter, as known in the art.

[0051] "Homologous," as used herein with respect to a polynucleotide regulatory element, refers to a native and naturally occurring polynucleotide regulatory element that is operably linked to a polynucleotide of interest such that the polynucleotide of interest can be expressed from a vector according to the invention.

[0052] "Homologous," as used herein with respect to a polynucleotide or polypeptide in a host organism, means that the polynucleotide or polypeptide is a native and naturally occurring polynucleotide or polynucleotides within that host organism. The homologous polynucleotide is operably linked to homologous or heterologous regulatory elements such that the homologous polypeptide can be expressed from a vector containing the homologous polynucleotide described herein.

[0053] As used herein, the term "heterologous" in reference to a polynucleotide regulatory element refers to a polynucleotide regulatory element that is not a native, naturally occurring polynucleotide regulatory element. A heterologous polynucleotide regulatory element is not normally associated with the CDS to which it is operably linked. A heterologous regulatory element is 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 may include promoters normally associated with other genes, ORFs, or coding regions, and / or promoters isolated from other bacterial, viral, eukaryotic, or mammalian cells.

[0054] As used herein, "heterologous" as used with reference to a polynucleotide or polypeptide in a host organism (i.e., a "heterologous polynucleotide" or "heterologous polypeptide") means a polynucleotide or polypeptide that is not a native, naturally occurring polynucleotide or polypeptide in that host organism. The heterologous polynucleotide is operably linked to heterologous or homologous regulatory elements such that the heterologous polypeptide can be expressed from a vector containing a heterologous polynucleotide described herein.

[0055] The terms "heterologously expressed" and "heterologous expression" refer to the expression of a heterologous polypeptide in a host cell.

[0056] A "functional variant or fragment thereof" of a polypeptide is a subsequence of the polypeptide that performs a function necessary for the biological activity or binding of the polypeptide and / or provides the three-dimensional structure of the polypeptide. The term can refer to a polypeptide, an aggregate of the polypeptide 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 a polypeptide activity.

[0057] As used herein, "isolated" with respect to a polynucleotide or polypeptide sequence refers to a sequence that has been removed from its natural cellular environment. Isolated molecules can be obtained by any method, or combination of methods, known and used in the art, including biochemical, recombinant, and synthetic techniques. The polynucleotide or polypeptide sequence can be prepared by at least one purification step.

[0058] "Isolated," as used herein with respect to a cell or host cell, refers to a cell or host cell that has been obtained or removed from an organism or its natural environment and then maintained in a laboratory environment as known in the art. The term includes not only a single cell by itself, but also a cell or host cell that is contained in a cell culture, and can include a single cell or a single host cell.

[0059] The term "recombinant" refers to a polynucleotide sequence that has been removed from surrounding sequences in its natural context and / or recombined with sequences that are not present in its natural context. A "recombinant" polypeptide sequence is produced by translation from a "recombinant" polynucleotide sequence.

[0060] As used herein, the term "variant" refers to a polynucleotide or polypeptide sequence that differs 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 or other species and can include homologs, alleles, or alleles. In certain embodiments, polypeptide variants useful in the present invention have biological activity that is the same as or similar to the biological activity of the corresponding wild-type molecule, i.e., the parent polypeptide or polynucleotide.

[0061] In certain embodiments, variants of the polypeptides described herein have similar or substantially similar biological activity to the corresponding wild-type molecule, hi certain embodiments, the similarity is in similar activity and / or binding specificity.

[0062] In certain embodiments, variants of the polypeptides described herein have a different biological activity than the corresponding wild-type molecule, hi certain embodiments, the difference is an altered activity and / or binding specificity.

[0063] The term "variant" with respect to polynucleotides and polypeptides encompasses all forms of the polynucleotides and polypeptides defined herein.

[0064] Variant polynucleotide sequences preferably have 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% or more, preferably 98% or more, preferably 99% or more identity to a sequence of the 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, and preferably over the entire length of the polynucleotide used in or identified according to the methods of the invention.

[0065] Polynucleotide variants include those that exhibit similarity to one or more of the specifically identified sequences, may retain the functional equivalence of those sequences, and would not reasonably be expected to occur by chance.

[0066] Polynucleotide sequence identity and similarity can be readily determined by one of ordinary skill in the art.

[0067] Variant polynucleotides also include polynucleotides that differ from the polynucleotide sequences described herein but, due to the degeneracy of the genetic code, encode polypeptides that have the same activity as the polypeptides encoded by the polynucleotides of the present invention. Sequence modifications that do not change the amino acid sequence of a polypeptide are called "silent mutations." With the exception of ATG (methionine) and TGG (tryptophan), other codons for the same amino acids can be changed by art-recognized techniques, for example, to optimize codon expression in a specific host organism.

[0068] The present invention also includes modifications of polynucleotide sequences that result in conservative substitution of one or more amino acids in the encoded polypeptide sequence, without significantly altering its biological activity. A skilled artisan will know methods for making phenotypically silent amino acid substitutions (see, for example, Bowie et al., 1990, Science 247, 1306).

[0069] The term "variant" with respect to a polypeptide includes naturally occurring, recombinantly produced, and synthetically produced polypeptides. Variant polypeptide sequences preferably have 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% or more, preferably 96% or more, preferably 97% or more, preferably 98% or more, preferably 99% or more identity to a sequence of the 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, and preferably over the entire length of the polypeptide used in or identified according to the methods of the invention.

[0070] Polypeptide variants include those that exhibit similarity to one or more of the identified sequences, may maintain functional equivalence of those sequences, and would not reasonably be expected to occur by chance.

[0071] Polypeptide sequence identity and similarity can be readily determined by one of ordinary skill in the art.

[0072] Variant polypeptides include polypeptides that differ in amino acid sequence from the polypeptides herein by one or more conservative amino acid or non-conservative substitutions, deletions, additions, or insertions that do not affect the biological activity of the peptide.

[0073] Conservative substitutions typically involve replacing one amino acid with another amino acid with similar properties as known and used in the art.

[0074] Analysis of evolved biological sequences has shown that not all sequence changes are created equal, reflecting, at least in part, the difference between conservative and non-conservative substitutions at the biological level. For example, substitutions of certain amino acids may occur frequently, while substitutions of other amino acids occur very rarely. Evolutionary changes or substitutions of amino acid residues can be modeled by scoring matrices, also known as substitution matrices. Such matrices are used in bioinformatics analysis to identify relationships between sequences and are known to skilled artisans.

[0075] Other variants include peptides with modifications that affect the stability of the peptide. Such analogs may, for example, contain one or more non-peptide bonds (replacing peptide bonds) within the peptide sequence. Also included are analogs containing residues other than naturally occurring L-amino acids (e.g., D-amino acids), or non-naturally occurring synthetic amino acids (e.g., beta- or gamma-amino acids), and cyclic analogs.

[0076] Substitutions, deletions, additions, or insertions can be made by mutagenesis methods known in the art, and those skilled in the art are familiar with methods for making phenotypically silent amino acid substitutions. See, e.g., Bowie et al., 1990, Science 247, 1306.

[0077] The term "peptide" includes a chain of amino acids linked by peptide bonds. The term "peptide" can refer to a "protein" or "polypeptide," which is a molecule consisting of a linear sequence of amino acids (which may be folded into a globular shape). As used herein, a protein generally refers to a molecule containing more than about 200 amino acids, up to the full-length sequence translated from a gene; a polypeptide generally refers to a molecule containing more than about 100 amino acids; and a peptide generally refers to a molecule containing from about 2 to about 100 amino acids. For convenience, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein. As used herein, a protein can refer to any polymer containing an amine or thiol. In this regard, a "protein or peptide" can include an amino acid sequence containing at least one of the common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid. Proteins, polypeptides, and peptides, including the FUN polypeptides and portions thereof described herein, can be produced by several methods known to those skilled in the art, including expression of the protein, polypeptide, or peptide by standard molecular biology techniques, isolation of the protein or peptide from a natural source, or chemical synthesis of the protein or peptide.

[0078] As used herein, a polypeptide can also refer to a polypeptide that has 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.

[0079] The terms "modulating expression," "modulated expression," and "modulating expression" of a polynucleotide or polypeptide are intended to encompass situations in which genomic DNA corresponding to a polynucleotide expressed in accordance with the present invention is modified, thereby modulating expression of the polynucleotide or polypeptide of the present invention. Modification of genomic DNA can be achieved by genetic recombination or other methods known in the art for inducing mutations. "Modulated expression" refers to an increase or decrease in the amount of messenger RNA and / or polypeptide produced, and may result in an increase or decrease in the activity of the polypeptide due to changes in the sequence of the produced polynucleotide and polypeptide.

[0080] The terms "modulate activity," "modulated activity," and "modulating activity" of a polynucleotide or polypeptide are intended to encompass situations in which genomic DNA corresponding to a polynucleotide expressed in accordance with the present invention is modified, thereby resulting in modulated expression of a polynucleotide or modulated expression or activity of a polypeptide of the present invention. Modification of genomic DNA can be achieved by genetic recombination or other methods known in the art for inducing mutations. "Modulated activity" relates to an increase or decrease in the amount of 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 produced polynucleotide and polypeptide.

[0081] The phrase "polypeptide or portion thereof" in the context of a proteinaceous polymer described herein refers to a polypeptide described herein, or a portion of that polypeptide that is incorporated into a proteinaceous polymer. The polypeptide or portion thereof comprises a quasi-repeated domain.

[0082] As used herein, the term "quasi-repeat domain" and grammatical variations thereof refer to imperfect repeats.

[0083] Reference to a range of numerical values ​​disclosed herein (e.g., 1 to 10) is intended to incorporate reference to all associated numerical values ​​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 range of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7); thus, all subranges of every range explicitly disclosed herein are expressly disclosed. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between the minimum and maximum values ​​recited are deemed to be expressly set forth in this application in the same manner. DETAILED DESCRIPTION

[0084] The present invention relates generally to polypeptides derived from nest material proteins of the solitary hummingbird bee, Hylaeus nubilosus, and portions, functional analogs, variants, and / or derivatives thereof. The invention also generally relates to polynucleotides encoding such polypeptides, proteinaceous polymers comprising such polypeptides or portions thereof that include quasi-repeat domains, and methods of making polypeptides and proteinaceous polymers comprising the polypeptides or portions thereof, including by heterologous expression of polynucleotides in suitable isolated host cells.

[0085] Honeybees of the genus Hylaeus (Hymenoptera: Apidae) build nesting materials that are "cellophane-like" (Almeida, EAB (Colletidae nesting biology (Hymenoptera: Apoidea). Apidologie 39, 16-29 (2008)).

[0086] Previously published studies of nest material made by related wasp species suggest that it is a unique composite of lipid polymers and protein biopolymers. To better understand the basis for the observed properties, the inventors investigated the nest material and sought to identify the components of this material responsible for its surprising properties.

[0087] Molecular analysis of the Dufour, labial, and mandibular glands and nest material of Hylaeus nubilosus identified a single major protein component (>10%) of Hylaeus nubilosus honeycomb material, designated FUN_069765-T1 (herein referred to as the "FUN" polypeptide). FUN is a silk-like protein, but it is not similar or homologous to silks known to be produced by Hymenoptera (ants, wasps, and honeybees). This difference is due to the properties of FUN and the way it is produced. For example, honeybee larvae (Apis mellifera) produce protein-rich silk in their labial glands to coat their cells before pupation. Honeybee silk is made of proteins that fold primarily into alpha helices and then form larger supersecondary structures called coiled coils. The honeybee protein is composed of four small fibroin subunits, approximately 30 kDa in length, and is composed of approximately 30% alanine.

[0088] In contrast, FUN from Hylaeus nubilosus has a β-sheet-rich structure, the same as that of silkworm (Bombyx mori) silk fibers and spider dragline silks, but differs from the α-helical structure predominantly found in honeybee larval silk (Figures 5 and 6). The FUN polypeptide is rich in glutamine and serine, a feature not found in honeybee, silkworm, or spider silks. Although asparagine-rich silk proteins (asparagine and glutamine are similar amino acids) have been reported from a distantly related parasitic wasp (Apanteles samurai braconid), no glutamine-rich silk proteins have been reported.

[0089] By combining genome sequencing, transcriptome sequencing, and proteomic techniques, the inventors have determined the nucleic acid and amino acid sequences of the FUN polypeptide, including numerous sequences rich in glutamine and serine. While not wishing to be bound by theory, the applicants believe that the FUN polypeptide is either contained within the nesting material of Hylaeus nubilosus or is contained within the proteinaceous polymer that makes up the nesting material. Furthermore, while not wishing to be bound by theory, the inventors believe that the polypeptide and / or proteinaceous polymers containing the polypeptide or at least portions thereof confer important structural and functional properties to the nesting material of Hylaeus nubilosus.

[0090] Applicants believe that the FUN polypeptides described herein and their variants, analogs and derivatives will find numerous applications in materials where known synthetic polymers and biopolymers (including silk proteins) are commonly used.

[0091] As described herein, Applicant is the first to provide a silk or silk-like protein derived from Hylaeus nuvillosus. To this end, Applicant analyzed the genome sequence of Hylaeus nuvillosus and identified a single predicted coding region for a major protein associated with nest material (CDS): 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 (derived from Hylaeus nuvillosus) is set forth in SEQ ID NO: 2. This full-length polypeptide (i.e., SEQ ID NO: 2) and all other amino acid subsequences and functional variants described by the primary sequence disclosed herein, including, but not 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.

[0092] Additionally, the polypeptide produced according to SEQ ID NO: 12 described herein is referred to herein by different names, such as "FSS with secretion tag cleaved (FUNSecSf9)," "FSS," "HBB B3.0," and "HnM1M7-01," all of which refer to the same polypeptide and are used synonymously.

[0093] The polypeptide produced according to SEQ ID NO:4 described herein is referred to in this document as the "consensus sequence 43mer" or "HnP1." Both names refer to the same polypeptide and are used synonymously.

[0094] The applicant has also proposed, for the first time, a method for heterologously expressing silk-like proteins from Hylaeus nubilosus in isolated host cells.

[0095] The gene encoding the full-length FUN polypeptide from Hylaeus nubilosus was identified for the first time by the present inventors, as described in the Examples section of this specification.

[0096] 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 reveals a highly diverse array of hundreds of repeated DNA motifs, ranging in frequency and length from two copies of a 770-bp motif to 117 copies of a 16-bp sequence.

[0097] The predicted primary amino acid sequence of the full-length FUN polypeptide (SEQ ID NO:2) shows a complex organization of numerous repetitive amino acid sequence motifs, both perfect (or identical) and imperfect (or quasi-) repeats. The RADAR (Rapid Automated Detection and Alignment of Repeats) tool, which tolerates mismatches and gaps, identifies several categories of repeats, but the incomplete and fragmentary nature of many of these repeats creates a complex picture of the overall structure of this protein.

[0098] Using these initial investigations as a basis for a more extensive analysis, we found that nearly the entire protein is organized as a contiguous series of peptide domains (also referred to here as the FUN polypeptide) built around a basic 43-amino acid repeat motif present exclusively in the QS repeat region, with the following consensus (>50% threshold): SESAVQSSKSSSGMSSQAQSQQQQX1QLQQSQAQSQEQESQSAX2 (SEQ ID NO: 6).

[0099] 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, excluding the first 137 amino acids (non-repeat region) and spacer repeats, can be constructed according to this repeating 43-mer consensus.

[0100] In one embodiment, X1=A or S. In one embodiment, X2=E, G or Q.

[0101] Seventy-five peptides from the QS repeat region conform to the strict 43-residue length of the consensus, and these 75 repeats are designated as class A repeats.

[0102] Interspersed among these class A repeats are truncated versions of the consensus sequence, 35-36 residues long, also located in the QS repeat region, which can be broadly divided into two classes: one containing a tetraglutamine (QQQQ) motif (designated class B) and restricted to the second QS repeat region, see, for example, amino acid residues 635-670 of SEQ ID NO: 2, which has the following amino acid sequence: SESAVQSSKSSSGMSSQAQSQQQQAQSQEQESQSAQ (SEQ ID NO: 18).

[0103] The other is one that lacks the tetraglutamine motif and is scattered throughout the molecule (Class C), see, for example, amino acid residues 1508 to 1543 of SEQ ID NO: 2, which has the following amino acid sequence: SESDVQSSKSSRGMSSHAQSQQSQAQSQEQESQSAQ (SEQ ID NO: 20).

[0104] Class A, B, and C repeats, by definition, occur exclusively in QS repeat regions, which are 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 sequences. These irregular spacer repeats contain the majority of the proline and cysteine ​​residues found in proteins.

[0105] With the exception of the final spacer repeat that forms the C-terminus of the protein, all other spacer repeats are followed by variable length repeats ranging in length from 23 to 38 residues, referred to as class D, see for example amino acid residues 3087 to 3109 of SEQ ID NO: 2, which has the following amino acid sequence: MSSRAQSQQSQAQSQEQESQSAQ (SEQ ID NO: 22).

[0106] Additionally, the spacer repeats are preceded by variable length repeats of 5 to 40 residues in length, designated as class E, see, for example, amino acid residues 2249 to 2259 of SEQ ID NO: 2, which has the following amino acid sequence: SESAVQSSKSS (SEQ ID NO: 24).

[0107] Class D and E repeat sequences can be aligned to a 43-base consensus sequence, despite their large length differences.

[0108] The repeat region of the FUN polypeptide consists of amino acid residues 138 to 5451 of SEQ ID NO:2 and is organized as a series of contiguous peptide domains aligned with a 43-mer and spacer repeat consensus motif.

[0109] The non-repetitive N-terminal domain (NTD or N, amino acid residues 18-137 of SEQ ID NO:2) is immediately followed by a 21-mer peptide (amino acid residues 138-158 of SEQ ID NO:2), which cannot be assigned to any of the other repeat classes but can be aligned with the 43-mer consensus (designated class F). Protein synthesis

[0110] The FUN polypeptides and / or various portions thereof described herein can be produced by chemical synthesis using methods such as solution phase synthesis or solid phase peptide synthesis, with or without chemical linkage, followed by purification of the resulting peptide. In some embodiments, the FUN polypeptides or portions thereof described herein are produced by chemical synthesis. Protein expression

[0111] To confirm the function and characterize the properties of the FUN polypeptide, we constructed a series of expression vectors, which were transformed into suitable hosts for heterologous production of the FUN_069765-T1 protein.

[0112] After transforming and expressing the FUN polypeptides and various portions thereof described herein in suitable hosts, we determined the chemical phenotype of the transformants first by normal-phase thin-layer chromatography (TLC, results not shown) and then by reverse-phase liquid chromatography-mass spectrometry (LC-MS) analysis of cell extracts. We determined the newly expressed metabolites by high-resolution mass spectrometry (HRMS), purified them by semi-preparative reverse-phase high-performance liquid chromatography (HPLC), and analyzed the compounds by nuclear magnetic resonance (NMR) spectroscopy ( 1 H, 13 C, and HSQC, HMBC, COSY) for final identification.

[0113] Based on the work described herein, the inventors disclose the use of heterologous expression to produce recombinant FUN polypeptides. Overall, the inventors' work described herein confirms that heterologous expression of at least a portion of the complete FUN polypeptide from Hylaeus nubilosus in a heterologous host is a viable method that can be used to artificially produce this polypeptide, including its related portions, variants, analogs, and derivatives, using recombinant biosynthetic systems. As noted above, the polypeptides set forth herein in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 50, and 52, are all referred to as FUN polypeptides.

[0114] In one aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising at least 70% amino acid sequence identity to SEQ ID NO:2.

[0115] In one embodiment, the isolated polynucleotide has a sequence similar to that of SEQ ID NO:2 but is at least 7 Preferably, the polypeptide encodes a polypeptide that contains at least 5%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99% amino acid sequence identity.

[0116] 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 that consists essentially of or consists of the amino acid sequence of SEQ ID NO: 2.

[0117] In one embodiment, the polypeptide comprises 1-75 copies of SEQ ID NO:6.

[0118] In one embodiment, the polypeptide comprises at least one copy of SEQ ID NO:18.

[0119] In one embodiment, the polypeptide comprises at least one copy of SEQ ID NO:20.

[0120] In one embodiment, the polypeptide comprises at least one copy of SEQ ID NO:22.

[0121] In one embodiment, the polypeptide comprises at least one copy of SEQ ID NO:24.

[0122] In one embodiment, the isolated polypeptide comprises at least one copy of SEQ ID NO:52.

[0123] 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 configuration: 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).

[0124] In one embodiment, the isolated polypeptide comprises, consists essentially of, or consists of at least 25% serine, 25% glutamine, and 5% glutamic acid residues.

[0125] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0126] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence comprising 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.

[0127] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:2.

[0128] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence comprising 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.

[0129] Embodiments of this aspect of the invention specifically contemplate all of the embodiments described in the previous aspects of the invention relating to an isolated polynucleotide encoding a polypeptide comprising at least 70% amino acid sequence identity to SEQ ID NO:2.

[0130] The isolated polynucleotide molecules described herein can be isolated from biological samples using various techniques known to those skilled in the art.For example, such polynucleotides can be isolated using polymerase chain reaction (PCR) known in the art.The described nucleic acid molecules can be amplified using primers defined herein that are derived from the polynucleotide sequences described herein.

[0131] Additional methods for isolating polynucleotides include using all or part of the polynucleotides described herein as hybridization probes. Hybridization of labeled polynucleotide probes to polynucleotides immobilized on solid supports, such as nitrocellulose filters or nylon membranes, can be used to screen genomic or cDNA libraries. Similarly, probes can be attached to beads and hybridized with target sequences. Separation can also be performed using known protocols, such as magnetic separation. The selection of appropriately stringent hybridization and washing conditions is considered within the skill of those in the art.

[0132] Polynucleotide fragments can be produced by techniques well known in the art, such as restriction endonuclease digestion and oligonucleotide synthesis.

[0133] Partial polynucleotide sequences can be used as probes to identify corresponding full-length polynucleotide sequences in a sample using methods well known in the art. These methods include PCR-based methods, 5' RACE and hybridization-based methods, as well as computer / database-based methods known in the art. Detectable labels, such as radioisotopes, fluorescence, chemiluminescence, and bioluminescence, can be used to facilitate detection. Inverse PCR also allows for the acquisition of unknown sequences adjacent to the polynucleotide sequences disclosed herein using primers based on known regions known and used in the art. This method uses several restriction enzymes to generate fragments appropriate for the known region of the gene. This fragment is then circularized by intramolecular ligation and used as a PCR template. Different primers are designed based on the known region. Standard molecular biology approaches known in the art can be used to physically assemble full-length clones. Primers and primer pairs that enable amplification of the polynucleotides of the present invention are also contemplated as embodiments disclosed herein.

[0134] Variants (including homologous genes) can be identified by the methods described. Variant polynucleotides can be identified using PCR-based methods known in the art. Typically, the polynucleotide sequences of primers useful for amplifying variants of polynucleotide molecules by PCR can be based on sequences that code for conserved regions of corresponding amino acid sequences.

[0135] Another method for identifying variant polynucleotides includes, as mentioned above, using all or part of specific polynucleotides as hybridization probes for screening genome libraries or cDNA libraries.Usually, probes based on the sequence encoding the conserved region of corresponding amino acid sequences can be used.Hybridization conditions may be less stringent than the conditions used when screening the same sequence as the probe.

[0136] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:2.

[0137] In one embodiment, the isolated polypeptide comprises 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.

[0138] 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.

[0139] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0140] In one embodiment, the isolated polypeptide comprises 1-75 copies of SEQ ID NO:6.

[0141] In one embodiment, the isolated polypeptide comprises at least one copy of SEQ ID NO:18.

[0142] In one embodiment, the isolated polypeptide comprises at least one copy of SEQ ID NO:20.

[0143] In one embodiment, the isolated polypeptide comprises at least one copy of SEQ ID NO:22.

[0144] In one embodiment, the isolated polypeptide comprises at least one copy of SEQ ID NO:24.

[0145] In one embodiment, the isolated polypeptide comprises at least one copy of SEQ ID NO:52.

[0146] 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 configuration: 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).

[0147] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence comprising 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.

[0148] 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.

[0149] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising at least 70% amino acid sequence identity to SESAVQSSKSSSGMSSQAQSQQQQX1QLQQSQAQSQEQESQSAX2 (SEQ ID NO: 6).

[0150] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising 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.

[0151] 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 that consists essentially of or consists of the amino acid sequence of SEQ ID NO: 6.

[0152] In one embodiment, X1 and X are independently any amino acid. In one embodiment, X1 is A or S. In one embodiment, X2 is E, G, or Q.

[0153] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0154] In one embodiment, the isolated polypeptide is comprised in a proteinaceous 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 between 2 and 75 copies of SEQ ID NO:6.

[0155] 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 configuration: 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).

[0156] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence comprising 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.

[0157] In another aspect, the 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 is A or S. In one embodiment, X2 is E, G, or Q.

[0158] Specifically contemplated as embodiments of this aspect of the invention are all of the embodiments set forth in the preceding aspect of the invention relating to an isolated polynucleotide encoding a polypeptide comprising at least 70% amino acid sequence identity to SEQ ID NO: 6. Those 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.

[0159] In another aspect, the present invention relates to an isolated polypeptide comprising at least 70% amino acid sequence identity to SESAVQSSKSSSGMSSQAQSQQQQX1QLQQSQAQSQEQESQSAX2 (SEQ ID NO: 6).

[0160] In one embodiment, the isolated polypeptide comprises 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.

[0161] 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.

[0162] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0163] In one embodiment, the polypeptide is comprised in a proteinaceous 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 SEQ ID NO:6.

[0164] 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 configuration: 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.

[0165] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence comprising 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.

[0166] The present invention relates to an isolated polynucleotide that encodes a polypeptide comprising at least 70% amino acid sequence identity to MSSQAQSQQQQSQLQQSQAQSQEQESQSAX1SGSDVX2X3SKSSSG (SEQ ID NO: 4).

[0167] 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.

[0168] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising 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.

[0169] 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 that consists essentially of or consists of the amino acid sequence of SEQ ID NO: 4.

[0170] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0171] In one embodiment, the polypeptide is comprised in a proteinaceous 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.

[0172] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence comprising 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.

[0173] In another aspect, the 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.

[0174] Specifically contemplated as embodiments of this aspect of the invention are all of the embodiments set forth in the preceding aspect of the invention relating to an isolated polynucleotide encoding a polypeptide comprising at least 70% amino acid sequence identity to SEQ ID NO: 4. Those 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 applies equally and appropriately to this aspect of the invention.

[0175] In another aspect, the present invention relates to an isolated polypeptide comprising at least 70% amino acid sequence identity to MSSQAQSQQQQSQLQQSQAQSQEQESQSAX1SGSDVX2X3SKSSSG (SEQ ID NO: 4).

[0176] 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.

[0177] In one embodiment, the isolated polypeptide comprises 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.

[0178] 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:4.

[0179] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0180] In one embodiment, the polypeptide is comprised in a proteinaceous 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.

[0181] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence comprising 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.

[0182] 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.

[0183] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising 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.

[0184] 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 that consists essentially of or consists of the amino acid sequence of SEQ ID NO:8.

[0185] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0186] In one embodiment, the polypeptide is comprised in a proteinaceous 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.

[0187] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence comprising 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.

[0188] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:8.

[0189] Embodiments of this aspect of the invention are specifically contemplated as all of the embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding polypeptides comprising at least 70% amino acid sequence identity to SEQ ID NO: 8. Those 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.

[0190] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:8.

[0191] In one embodiment, the isolated polypeptide comprises 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.

[0192] 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.

[0193] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0194] In one embodiment, the polypeptide is comprised in a proteinaceous 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.

[0195] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence comprising 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.

[0196] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising at least 70% amino acid sequence identity to SEQ ID NO:10.

[0197] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising 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.

[0198] 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 that consists essentially of or consists of the amino acid sequence of SEQ ID NO: 10.

[0199] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0200] In one embodiment, the polypeptide is comprised in a proteinaceous 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.

[0201] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence comprising 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.

[0202] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:10.

[0203] Embodiments of this aspect of the invention specifically contemplate all of the embodiments set forth in the preceding aspect of the invention relating to an isolated polynucleotide encoding a polypeptide comprising at least 70% amino acid sequence identity to SEQ ID NO: 10. Those 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.

[0204] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:10.

[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:10.

[0206] 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.

[0207] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0208] In one embodiment, the polypeptide is comprised in a proteinaceous 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.

[0209] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence comprising 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.

[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:12.

[0211] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising 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.

[0212] 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 that consists essentially of or consists of the amino acid sequence of SEQ ID NO: 12.

[0213] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0214] In one embodiment, the polypeptide is comprised in a proteinaceous 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. In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence comprising 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.

[0215] The present invention relates to an isolated polynucleotide that encodes a polypeptide comprising (SEQ ID NO: 12).

[0216] All of the embodiments described in the preceding aspects of the invention are contemplated as embodiments of this aspect of the invention, particularly those relating to isolated polynucleotides encoding polypeptides comprising at least 70% amino acid sequence identity to SEQ ID NO:12. 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 applies equally and appropriately to this aspect of the invention.

[0217] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to (SEQ ID NO: 12).

[0218] 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.

[0219] 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.

[0220] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0221] In one embodiment, the polypeptide is comprised in a proteinaceous 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.

[0222] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence comprising 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.

[0223] 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.

[0224] 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.

[0225] 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 that consists essentially of or consists of the amino acid sequence of SEQ ID NO: 14.

[0226] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0227] In one embodiment, the polypeptide is comprised in a proteinaceous polymer. In one embodiment, the polymer comprises SEQ ID NO:2 or at least a portion of SEQ ID NO:2.

[0228] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence comprising 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.

[0229] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:14.

[0230] Embodiments of this aspect of the invention specifically contemplate all of the embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding polypeptides comprising at least 70% amino acid sequence identity to SEQ ID NO: 14. Those 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.

[0231] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:14.

[0232] 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.

[0233] 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.

[0234] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0235] In one embodiment, the polypeptide is comprised in a proteinaceous 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.

[0236] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence comprising 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.

[0237] 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.

[0238] 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.

[0239] 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 that consists essentially of or consists of the amino acid sequence of SEQ ID NO: 16.

[0240] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0241] In one embodiment, the polypeptide is comprised in a proteinaceous 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.

[0242] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence comprising 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.

[0243] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:16.

[0244] Embodiments of this aspect of the invention specifically contemplate all of the embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding polypeptides comprising at least 70% amino acid sequence identity to SEQ ID NO: 16. Those 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.

[0245] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:16.

[0246] 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.

[0247] 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.

[0248] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0249] In one embodiment, the polypeptide is comprised in a proteinaceous 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.

[0250] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence comprising 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.

[0251] 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.

[0252] 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.

[0253] 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 that consists essentially of or consists of the amino acid sequence of SEQ ID NO: 18.

[0254] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0255] In one embodiment, the isolated polypeptide is comprised in a proteinaceous 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 2-6 copies of SEQ ID NO:18.

[0256] 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.

[0257] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:18.

[0258] Embodiments of this aspect of the invention are specifically contemplated as all of the embodiments described in the preceding aspects of the invention relating to isolated polynucleotides encoding polypeptides having at least 70% amino acid sequence identity to SEQ ID NO: 18. Those 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.

[0259] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:18.

[0260] 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.

[0261] 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.

[0262] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0263] In one embodiment, the polypeptide is comprised in a proteinaceous 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 2-6 copies of SEQ ID NO:18.

[0264] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence comprising 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.

[0265] 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.

[0266] 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.

[0267] 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 that consists essentially of or consists of the amino acid sequence of SEQ ID NO: 20.

[0268] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0269] In one embodiment, the isolated polypeptide is comprised in a proteinaceous 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 2-29 copies of SEQ ID NO:20.

[0270] 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.

[0271] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence comprising 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.

[0272] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:20.

[0273] Embodiments of this aspect of the invention specifically contemplate all of the embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding polypeptides comprising at least 70% amino acid sequence identity to SEQ ID NO: 1. Those 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.

[0274] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:20.

[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:20.

[0276] 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.

[0277] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0278] In one embodiment, the polypeptide is comprised in a proteinaceous 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 2-29 copies of SEQ ID NO:20.

[0279] 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.

[0280] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence comprising 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.

[0281] 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.

[0282] 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.

[0283] 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 that consists essentially of or consists of the amino acid sequence of SEQ ID NO: 22.

[0284] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0285] In one embodiment, the isolated polypeptide is comprised in a proteinaceous 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. The polymer comprises 2 to 10 copies of SEQ ID NO:22.

[0286] In one embodiment, the isolated polypeptide is comprised in a proteinaceous polymer, the polymer comprising at least one copy of SEQ ID NO:22 positioned between two copies of SEQ ID NO:6.

[0287] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence comprising 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.

[0288] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:22.

[0289] Embodiments of this aspect of the invention specifically contemplate all of the embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding polypeptides comprising at least 70% amino acid sequence identity to SEQ ID NO: 1. Those 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 applies equally and appropriately to this aspect of the invention.

[0290] In another aspect, the invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:22.

[0291] In one embodiment, the isolated polypeptide comprises 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.

[0292] 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.

[0293] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0294] In one embodiment, the polypeptide is comprised in a proteinaceous 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 2-10 copies of SEQ ID NO:22.

[0295] In one embodiment, the isolated polypeptide is comprised in a proteinaceous polymer, wherein the polymer has at least one copy of SEQ ID NO:22 located between two copies of SEQ ID NO:6.

[0296] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence comprising 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.

[0297] 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.

[0298] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising 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.

[0299] 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 that consists essentially of or consists of the amino acid sequence of SEQ ID NO: 24.

[0300] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0301] In one embodiment, the isolated polypeptide is comprised in a proteinaceous 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 2 to 11 copies of SEQ ID NO:24.

[0302] In one embodiment, the isolated polypeptide is comprised in a proteinaceous polymer, the polymer comprising at least one copy of SEQ ID NO:24 positioned between two copies of SEQ ID NO:6.

[0303] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence comprising 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.

[0304] In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO:24.

[0305] Embodiments of this aspect of the invention specifically contemplate all of the embodiments set forth in the preceding aspects of the invention relating to isolated polynucleotides encoding polypeptides comprising at least 70% amino acid sequence identity to SEQ ID NO: 24. Those 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.

[0306] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to SEQ ID NO:24.

[0307] 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.

[0308] 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.

[0309] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0310] In one embodiment, the polypeptide is comprised in a proteinaceous 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 2-11 copies of SEQ ID NO:24.

[0311] In one embodiment, the isolated polypeptide is comprised in a proteinaceous polymer, wherein the polymer has at least one copy of SEQ ID NO:24 located between two copies of SEQ ID NO:6.

[0312] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence comprising 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.

[0313] In a specific embodiment of each of the above-described isolated polynucleotide aspects, the isolated polynucleotide may further comprise a heterologous regulatory element. In one embodiment, the regulatory element comprises a nucleic acid sequence encoding a signal peptide. Transcription of the polynucleotide comprising the nucleic acid sequence encoding the signal peptide results in secretion of the expressed polypeptide and / or proteinaceous polymer from the host cell.

[0314] 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).

[0315] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising 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.

[0316] 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 that consists essentially of or consists of the amino acid sequence of SEQ ID NO: 50.

[0317] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0318] In one embodiment, the polypeptide is comprised in a proteinaceous 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.

[0319] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence comprising 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.

[0320] 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.

[0321] In another aspect, the 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.

[0322] In one embodiment, the polynucleotide further encodes a His tag. In one embodiment, the His tag is at the N-terminus or C-terminus of the polypeptide.

[0323] Embodiments of this aspect of the invention specifically contemplate all of the embodiments described in the preceding aspects of the invention relating to isolated polynucleotides encoding polypeptides comprising at least 70% amino acid sequence identity to SEQ ID NO: 50. Those 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 applies equally and appropriately to this aspect of the invention.

[0324] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to (SEQ ID NO:50).

[0325] In one embodiment, the isolated polypeptide comprises 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.

[0326] 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.

[0327] 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.

[0328] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0329] In one embodiment, the polypeptide is comprised in a proteinaceous 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.

[0330] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence comprising 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: 10. In one embodiment, the isolated polynucleotide comprises, consists of, or consists essentially of SEQ ID NO:49.

[0331] In one embodiment, the polynucleotide further encodes a His tag. In one embodiment, the His tag is at the N-terminus or C-terminus of the polypeptide.

[0332] 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).

[0333] In one embodiment, the isolated polynucleotide encodes a polypeptide comprising 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.

[0334] 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 that consists essentially of or consists of the amino acid sequence of SEQ ID NO: 52.

[0335] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0336] In one embodiment, the polypeptide is comprised in a proteinaceous 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.

[0337] In one embodiment, the isolated polynucleotide comprises a nucleic acid sequence comprising 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.

[0338] 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.

[0339] 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: 52.

[0340] In one embodiment, the polynucleotide further encodes a His tag. In one embodiment, the His tag is at the N-terminus or C-terminus of the polypeptide.

[0341] Embodiments of this aspect of the invention specifically contemplate all of the embodiments described in the preceding aspects of the invention relating to isolated polynucleotides encoding polypeptides comprising at least 70% amino acid sequence identity to SEQ ID NO: 52. Those 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 applies equally and appropriately to this aspect of the invention.

[0342] In another aspect, the present invention relates to an isolated polypeptide having at least 70% amino acid sequence identity to (SEQ ID NO:52).

[0343] 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.

[0344] 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.

[0345] 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.

[0346] In one embodiment, the polypeptide is comprised in nest material of a colletid wasp. In one embodiment, the colletid wasp is a Hylaeus wasp. In one embodiment, the colletid wasp is Hylaeus nubilosus. In one embodiment, the polypeptide, or a portion thereof, is hygroscopic or hydrophilic. In one embodiment, the polypeptide, or a portion thereof, is hydrophobic. In one embodiment, the polypeptide, or a portion thereof, is amphipathic.

[0347] In one embodiment, the polypeptide is comprised in a proteinaceous 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.

[0348] In one embodiment, the isolated polypeptide is encoded by a nucleic acid sequence comprising 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.

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

[0351] In one embodiment of any and / or all of the above polypeptide aspects, the polypeptide is a recombinant FUN polypeptide or a functional part, analog, or derivative thereof.

[0352] In another aspect, the present invention relates to a vector encoding the isolated polypeptide according to the invention.

[0353] In another aspect, the present invention relates to a vector comprising the isolated polynucleotide according to the present invention.

[0354] In one embodiment, the vector is selected from the group consisting of a plasmid, a BAC, a (PAC), a YAC, a bacteriophage, a phagemid, and a cosmid. In one embodiment, the vector is a plasmid.

[0355] In one embodiment, the vector is selected from the group consisting of a plasmid, a BAC, a PAC, a YAC, a bacteriophage, 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.

[0356] Examples of suitable expression vectors include, but are not limited to, plasmid DNA vectors, viral DNA vectors (such as adenoviruses and adeno-associated viruses), or viral RNA vectors (such as retroviral vectors). In some embodiments, the plasmid and / or phage vector may be selected from the following vectors or variants thereof, including pET, pFastBac, pUC18, pU19, Mp18, Mp19, ColE1, PCR1, and pKRC, lambda gt10, and M13 plasmids (e.g., pBR322, pACYC184, pT127, RP4, p1J101, SV40, and BPV). Additional non-limiting examples of vectors include cosmids, YACS, BAC, shuttle vectors such as pSA3, and PAT28 transposons.

[0357] Suitable viral vectors include, but are not limited to, vectors derived from adenovirus (AV), adeno-associated virus (AAV), retrovirus (e.g., lentivirus (LV), rhabdovirus, murine leukemia virus), herpes virus, etc. The viral vectors used in the present invention can be appropriately modified by mimicking the envelope protein or other surface antigens of other viruses, or by replacing different viral capsid proteins, as is known and used in the art.

[0358] Vectors can be constructed to drive expression of the polypeptides described herein either in vitro or in vivo. In one embodiment, the vector comprises a polynucleotide of the invention operably linked to a 5' or 3' untranslated regulatory sequence. The design of the vector depends on various factors, including the host cell in which the operably linked polynucleotide will be expressed and the desired level of polynucleotide expression.

[0359] Similarly, the selection of various promoters, enhancers, and / or other genetic elements for a vector depends on various factors, including the host cell and expression level, as described above. In one embodiment, the vector contains a homologous promoter operably linked to the polynucleotide of the present invention. In another embodiment, the vector contains a heterologous promoter operably linked to the polynucleotide of the present invention. In one embodiment, the homologous or heterologous promoter is an inducible, repressible, or regulatable promoter. An appropriate promoter can be selected and used under appropriate conditions to induce high-level expression of the polynucleotide of the present invention. Many such elements have been described in the literature and are commercially available.

[0360] For example, any suitable eukaryotic or prokaryotic promoter can be used as a promoter useful in a vector. 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 an operably linked polynucleotide in a particular cell type is determined by the presence (or absence) of specific gene regulatory sequences (e.g., enhancers, silencers, etc.) present nearby. Any suitable promoter / enhancer combination (see Eukaryotic Promoter Data Base EPDB) can be used to drive the expression of the polynucleotide of the present invention.

[0361] Additional promoters useful in expression cassettes include β-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.

[0362] Prokaryotic promoters useful in the expression cassette include constitutive promoters known in the art (e.g., the int promoter of bacteriophage lambda and the bla promoter of the β-lactamase gene sequence of pBR322) and regulatable promoters (e.g., lacZ, recA, and gal). A ribosome binding site upstream of the CDS may also be required for expression.

[0363] Enhancers useful in the vectors described herein include the SV40 enhancer, cytomegalovirus early promoter enhancer, globin, albumin, insulin, and the like.

[0364] In one embodiment, the vector may be driven by a T3, T7, or SP6 cytoplasmic expression system.

[0365] In another aspect, the present invention relates to an isolated host cell comprising an isolated polypeptide, an isolated polynucleotide, and / or a vector according to the present invention.

[0366] In one embodiment, the isolated host cell is a prokaryotic or eukaryotic cell.

[0367] In one embodiment, the prokaryotic cell is selected from the group consisting of, but not limited to, strains of E. coli, Pseudomonas, Bacillus, Serratia, Klebsiella, Streptomyces, Listeria, Salmonella, and Mycobacteria.

[0368] In one embodiment, the eukaryotic cell is an animal cell, a plant cell, a fungal cell, or a protist cell.

[0369] 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.

[0370] 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.

[0371] In one embodiment, the insect cell comprises a polynucleotide described herein in a viral vector, preferably a baculovirus. In one embodiment, the insect cell is an Sf9 cell or a HighFive cell.

[0372] In another aspect, the present invention relates to a proteinaceous polymer comprising a FUN polypeptide as described herein or a portion thereof, wherein said proteinaceous polymer comprises at least one quasi-repeat domain.

[0373] In one embodiment, the proteinaceous polymer comprises from 2 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 the quasi-repeated domain.

[0374] In one embodiment, the quasi-repeat domain is comprised in a FUN polypeptide or portion thereof described herein.

[0375] In one embodiment, the proteinaceous 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.

[0376] In one embodiment, the proteinaceous polymer comprises a glutamine composition of greater than 30% of the amino acid sequence of the polymer, a serine composition of greater than 30% of the amino acid sequence of the polymer, and a glutamic acid composition of greater than 5% of the amino acid sequence of the polymer.

[0377] In one embodiment, the proteinaceous polymer comprises about 30% glutamine composition of the amino acid sequence of the polymer, about 30% serine composition of the amino acid sequence of the polymer, and about 5% glutamic acid composition of the amino acid sequence of the polymer.

[0378] In another aspect, the present invention relates to a composition comprising an isolated polypeptide, isolated polynucleotide, proteinaceous polymer and / or vector described herein and a carrier, diluent, or excipient.

[0379] In one embodiment, the composition consists essentially of an isolated polypeptide, isolated polynucleotide, proteinaceous polymer and / or vector described herein.

[0380] In one embodiment, the composition is a cosmetic composition. In one embodiment, the cosmetic composition is a hair care composition or a skin care composition.

[0381] The present invention relates to a method for producing 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, or a portion thereof, the method comprising heterologously expressing the FUN polypeptide in an isolated host cell and optionally purifying the FUN polypeptide.

[0382] In one embodiment, expression is from 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, or a portion thereof.

[0383] 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.

[0384] 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 heterologous sequence.

[0385] In one embodiment, the signal peptide directs secretion of the FUN polypeptide or a portion thereof.

[0386] In one embodiment, the method includes secreting the FUN polypeptide from the isolated host cells, followed by optional purification.

[0387] In one embodiment, the method includes optionally purifying the FUN polypeptide from the isolated host cell.

[0388] 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 a portion thereof, is expressed from 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.

[0389] In one embodiment, the FUN polynucleotide is contained in a vector, preferably a baculovirus vector.

[0390] 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.

[0391] In one embodiment, the isolated host cell is a bacterial cell. In one embodiment, the bacterial cell is E. coli.

[0392] In one embodiment, the isolated host cell is a fungal cell. In one embodiment, the fungal cell is an Aspergillus niger cell.

[0393] In one embodiment, the 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 produced by a method of the invention.

[0394] In another aspect, the present invention relates to a method of making a proteinaceous polymer comprising a FUN polypeptide or a portion thereof selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 50, and 52, the method comprising heterologously expressing the FUN polypeptide or a portion thereof in an isolated host cell under conditions resulting in expression of the FUN polypeptide or a portion thereof, and optionally purifying the polymer from the host cell.

[0395] In one embodiment, the proteinaceous polymer is an isolated proteinaceous polymer.

[0396] In one embodiment, the FUN polynucleotide or portion thereof is expressed from a polynucleotide sequence that includes at least one heterologous regulatory element.

[0397] In one embodiment, the FUN polynucleotide or portion thereof is expressed from a polynucleotide sequence that includes at least one signal sequence.

[0398] In one embodiment, the signal sequence encodes a signal peptide or a portion thereof, hi one embodiment, the signal peptide or portion thereof directs secretion of the proteinaceous polymer from the isolated host cell.

[0399] In one embodiment, the method includes purifying the polymer after it is secreted from the host cell.

[0400] In one embodiment, the method comprises purifying the polymer from the isolated host cells.

[0401] In one embodiment, the isolated host cell is an isolated host cell as envisaged in the above aspects and embodiments of the invention.

[0402] In one embodiment, the polymer comprises at least two copies of a FUN polypeptide.

[0403] 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 a portion thereof, is expressed from a FUN polynucleotide selected from the group consisting of SEQ ID NOs: 1-3. In one embodiment, the FUN polypeptide, or a portion thereof, is expressed from a FUN polynucleotide selected from the group consisting of SEQ ID NOs: 1-3.

[0404] In one embodiment, the FUN polynucleotide is contained in a vector, preferably a baculovirus vector.

[0405] In one embodiment, the isolated host cell is a bacterial cell. In one embodiment, the bacterial cell is E. coli.

[0406] In one embodiment, the isolated host cell is a fungal cell. In one embodiment, the fungal cell is an Aspergillus niger cell.

[0407] In another aspect, the invention relates to coating an article of manufacture or forming a coating on an article of manufacture with a FUN polypeptide or portion thereof described herein. 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.

[0408] In another aspect, the invention relates to using a FUN polypeptide or portion thereof described herein to form a film on an article of manufacture.

[0409] 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.

[0410] In one embodiment, the article of manufacture is selected from the group consisting of a textile or textile component or part thereof, and a biomedical device or component or part thereof.

[0411] In one embodiment, the article of manufacture or component or part thereof is a synthetic fiber. In one embodiment, the component or part thereof is a synthetic polymer. In one embodiment, the synthetic fiber or polymer is selected from the group consisting of polyester, spandex, rayon, nylon, acrylic, microfiber, neoprene, polyamide, acetate, polyvinyl chloride (PVC), and synthetic or "faux" leather or fur fibers and polymers.

[0412] In one embodiment, the synthetic fiber is nylon.

[0413] In one embodiment, the article of manufacture or a component or part thereof is a natural fiber, hi one embodiment, the natural fiber is selected from the group consisting of cotton, wool, silk, coconut fiber, alpaca, flax, hemp, bamboo, sisal, and jute.

[0414] In one embodiment, the natural fiber is silk.

[0415] In one embodiment, the article of manufacture is a textile, preferably a natural or synthetic textile.

[0416] In one embodiment, the article of manufacture, or a component or part thereof, is contained within 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 the group consisting of a defibrillator, a pacemaker, a cardiovascular device including a left ventricular assist device, a breast implant, a cochlear implant, an intraocular lens, a joint replacement device including a hip implant, a catheter, a dialysis tubing, a contraceptive intrauterine device, a stent, a suture, a staple, a bandage, and a wound dressing.

[0417] In one embodiment, the article of manufacture is an air filtration device, 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 included in an air filter. In one embodiment, the synthetic or natural fiber and / or polymer is in the form of a nanofiber.

[0418] Specifically contemplated as embodiments in the method and use aspects of the invention are the various embodiments discussed above with respect to the selection of FUN polypeptides and portions thereof, polynucleotides encoding FUN polypeptides or portions thereof, appropriate polynucleotide regulatory sequences, including signal sequences, host cells, and / or vectors that allow for expression and purification of the FUN polypeptides or portions thereof described herein.

[0419] The present invention will now be illustrated, in a non-limiting manner, with reference to the following examples. Example Example 1 Sourcing, collecting, and transporting Hylaeus nubilosus honeybees and their nesting materials

[0420] Hylaeus nubilosus was collected in Queensland, Australia. A total of 13 Hylaeus nubilosus specimens were collected. Dufour glands, venom glands, salivary glands, and entire heads were removed from all 13 specimens and preserved in DCM or ethanol.

[0421] Hylaeus nubilosus nest material was collected from the inside of paper nest straws (approximately 10–20 cm long, with an inner diameter of approximately 0.525 cm) that had been emptied after larvae had hatched and left the nest. Bees emerging from the nest straw were identified as Hylaeus nubilosus by visual observation by a trained entomologist. After emergence and identification, the straws were frozen for at least 48 hours and stored frozen until needed. Nest material was collected from the inside of the straws using a scalpel and tweezers, and any debris was carefully removed. Example 2

[0422] Identification of genes encoding honeybee nesting materials using multiple sequencing methods Transcriptome: RNA extraction and sequencing Hylaeus samples were fixed at -20°C for 3 minutes. Hylaeus samples were dissected as follows: the heads of the hylaeus samples were removed and transferred to RNAlater. These samples were used as indicators of transcripts enriched in the mandible. The salivary and Dufour glands were dissected into RNAlater. These samples were stored at -80°C after dissection. RNA was extracted from pooled samples of Dufour, mandibular, and salivary glands. Dufour and mandibular glands were replicated in triplicate (two sets of 10 bees and one set of 11 bees), and salivary glands were replicated in single replicates (one set of 10 bees and one set of 11 bees). Tissues were homogenized using a Tissulyser II. RNA was extracted from each pooled sample using the RNeasy Mini Kit. Sequencing was performed using an Illumina NextSeq sequencer, generating 150-bp paired-end reads. A total of 451,975,167 paired-end reads (total data volume of 136.5 Gb) were generated by RNA sequencing. Proteomics: Mass spectrometric sequencing of wild Hylaeus nest material

[0423] Mass spectrometry analysis was performed on washed and enzymatically digested nest material to identify the proteins contained therein. Nest material was first washed with water, dilute organic solvent, and 8M urea solution, and then proteins were digested with trypsin / chymotrypsin to generate peptide fragment fingerprints. All samples were analyzed by liquid chromatography and tandem mass spectrometry using an LTQ-Orbitrap and a 5600+ TripleTOF mass spectrometer. Data analysis was performed using an in-house Mascot server and the ProteomeDiscoverer 2.4 software package.

[0424] The mass spectra were searched against both the nucleotide sequence and the predicted amino acid sequence of the transcript. Both searches identified several high-confidence matches to glutamine- and serine-rich peptide sequences. BLAST searches revealed that the glutamine-rich sequences shared no homology with other known protein sequences in the NCBI database (see Sequence Listing 4). Genomics: gDNA and RNA extraction, cDNA generation and sequencing

[0425] Genomic DNA extraction and sequencing: Honeybees were flash-frozen in 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 grinding with a DNase-free pestle. Lysis buffer was added to the ground tissue. The sample was incubated at 37°C for 30 minutes with rocking. 60 μL of 20 mg / mL protease K was added to the sample tube. The sample was then incubated at 50°C for 2 hours with nutation. 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-tip20 / G was equilibrated with 1 mL of QTB buffer. Next, the lysate from the Falcon tube was applied to a genome chip, the liquid was drained from the genome chip, and the remaining lysate was added to the genome chip. The QIAGEN Genomic-tip was then washed four times with 1 ml of QC buffer. The DNA in QC buffer was then dispensed into three DNA Lobind 1.5 ml tubes. 666 μL of QF buffer was added to each tube. Next, 666 μL of SPRI reagent and 3 μL of SPRIselect beads were added to each of the three tubes (the beads were allowed to warm to room temperature before use and vortexed immediately before use). The samples were then rotated on a rocker at room temperature for 10 minutes. The tubes were then placed on a magnet until the liquid became clear and the beads formed a pellet. The supernatant was then discarded. The beads were then washed with 70% ethanol and incubated at room temperature for 30 seconds. This washing procedure was repeated. The beads were then air-dried at room temperature for 30 seconds. The pellet was then eluted using 42 μL of DNAse-free water at 50°C by gently flicking the tube. The beads were then collected using a magnet. The samples were then pipetted into new DNA Lobind 1.5 ml tubes. The gDNA concentration was then measured using a Nanodrop.The size profile of the gDNA extraction was verified by running 100 ng on a 1% agarose gel and measuring against a high-molecular-weight DNA marker. Samples were then flash-frozen in liquid nitrogen and stored at -80°C. DNA extraction yielded 3 μg of HMW DNA. gDNA sequencing was performed using an Oxford Nanopore Prometheus Ion Sequencer. Subsequent analysis was performed using FastQ format sequences that passed the default quality filtering cutoff.

[0426] RNA extraction, cDNA generation, and sequencing: Honeybees were flash-frozen in liquid nitrogen and stored at -80°C. RNA was extracted from a single female hylae using a TRIzol-based extraction method. First, the cuticle was removed from the bee from which RNA was extracted, and the remaining tissue was transferred to a 1.5 ml DNALobind tube. 50 μL of TRIzol reagent was transferred to the sample tube. Next, the tissue was crushed and disrupted with a DNase-free pestle. An additional 200 μL of TRIzol reagent was used to rinse the pestle, 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 sample tube. The sample tube was shaken vigorously for 15 seconds and then incubated 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 1.5 ml DNALobind tube. Next, 0.125 mL of isopropanol was added to the tube containing the aqueous phase. The sample was mixed by inversion to precipitate the RNA. The sample was then incubated at room temperature for 2 minutes. The sample was then centrifuged at 12,000 g for 10 minutes at 4°C. The supernatant was then removed and discarded. The sample was then washed with 250 μL of 75% ethanol. The sample was centrifuged at 7,500 g for 5 minutes at 4°C. The supernatant was then removed and discarded. The sample was then air-dried until all droplets of the RNA pellet 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 using a Nanodrop™. The samples were then flash-frozen in liquid nitrogen and stored at -80°C. cDNA was generated from the RNA sample using reverse transcription after enrichment of polyA RNA sequences. cDNA read data were base-called and fastq sequence files were generated using Guppy version 4. Subsequent analysis was performed using FastQ-formatted sequences that passed the default quality filtering cutoff. Genome assembly from gDNA sequencing

[0427] Genome assembly is the process of stitching together DNA sequence reads to accurately represent the nucleotide sequence and structure of an organism's genome. Genome sequencing and assembly will facilitate the identification of genes, including nest material genes, in Hylaeus nubilosus.

[0428] Adapters were trimmed from the gDNA sequence file (version 0.2.4, Wicketal., 2017) using "Porechop." Porechop was used with the "--discard_middle" parameter. The Hylaeus genome was assembled using Flye (version 2.8, Kolmogorov et al., 2019). Considering that the input data was Oxford Nanopore sequences, the assembly used the "--nano-raw" parameter. Flye was run with a predicted "genome size" parameter of 250 MB. This size was chosen because other Hymenoptera genomes are approximately 250 MB in size. The Hylaeus nubilosus genome is 360 MB, surprisingly large for a wasp. Genome assembly statistics show that the assembly is highly contiguous and therefore accurately represents the Hylaeus nubilosus genome. The genome assembly consists of 2,259 relatively contiguous scaffolds. The Hymenoptera nubilosus assembly has an N50 of 7 and an L50 of 17,978,421 bp, indicating a contiguous assembly. The assembly was compiled using Busco (version 5.4.2, Simulink) with the Hymenoptera database (hymenoptera_odb10). JPEG2026500155000001.jpg32oetal.,2015) analysis yielded a basco score of 95.7%, strongly suggesting that the genome is assembled with a very high level of completeness.

[0429] [Table 1] Annotation of the Hylaeus genome

[0430] The H. hiraeus genome was assembled using sequence data compiled from RNA-seq, genomic DNA-seq, and cDNA-seq datasets, as well as proteomic analysis (peptide fragment sequencing). The genome annotation software "FunanAnnotate" (version 1.7.4; Palmer & Stajich, 2022), utilizing the ab initio gene prediction tools Augustus and GeneMark, was used. The genome was prepared for annotation by masking uninformative regions in the genome using the "FunanAnnotateMask" script (Palmer & Stajich, 2022). The "FunanAnnotateMask" script was run with default parameters using the program "tantan" (Frith, 2011), which performs soft masking. All RNA-seq reads, including those from the salivary, mandibular, and Dufour glands, were pooled into forward and reverse sets and used to train "FunanAnnotate." cDNA reads for annotation were prepared by trimming adapters using Porechop (version 0.2.4; Wicketal., 2017). Porechop was run with the "--discard_middle" parameter. The processed cDNA was used as input for FunanAnnotate annotation training.

[0431] 'FunannotateTrain' is a wrapper for transcriptome assembly using Trinity (Palmer & Stajich, 2022; Grabherr et al., 2011). The 'FunannotateTrain' script used the masked genome assembly, Nanopore cDNA, and RNA forward and reverse sequence data as input. The 'FunannotateTrain' script was run with the '--stranded' flag set to 'RF' (reverse). The maximum intron length was set to 10,000 using the '--max_intronlen' parameter. 'FunannotateTrain' generated a file containing gff3-formatted transcripts, which was used as input for 'Funannonatepredict'. 'FunannotatePredict' trained and ran Augustus and GeneMark to generate gene predictions (version 1.7.4, Palmer & Stajich, 2022).

[0432] Then, 'Funannotate predict' used Evidence Modeller to generate consensus gene models from the gene predictions in Augustus and GeneMark. The masked Hylaeus nubilosus genome assembly, the transcript gff3 generated by 'Funanotate train', and 'fundb_20200227' (generated by 'Funanotate setup') were used as input for 'Funanotate predict'. 'Funannotate predict' was run with the '--augustus_species'hn, '--optimize_augustus' flag, '--busco_seed_species' set to honeybee1, '--buso_db' set to hymenoptera, '--organism' set to other, '--repeats2evm' flag, and '--max_intronlen' set to 50000. "Funannotate predict" generated Hylaeus gff3, mRNA transcript, and protein annotation files as output. "Funannotate update" updates annotations using RNA-seq data (version 1.7.4; Palmer & Stajich, 2022). "Funannotate update" was run using predicted mRNA transcripts as input. "Funannotate annotate" was used for gene functional annotation.

[0433] The "Funannotate annotate" program functionally annotated the H. hilaeus genome annotation using input generated by eggnog, 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; Simao et al., 2015). Eggnog used the H. hilaeus protein database and the "eggnog_proteins" diamond database. The "-mdiamond" parameter in eggnog was used to set diamond as the search method. iprscan (interproscan) was used to provide functional annotations (Jones et al., 2014). Iprscan was run with default parameters except for disabling precomputation (the "-dp" flag) to generate functional annotations.

[0434] Funannotate predicted 75,295 protein-coding genes. Busco (version 5.4.2, Simulink) for predicted proteins using the Hymenoptera database (hymenoptera_odb10) JPEG2026500155000003.jpg32oetal.,2015) The analysis showed a basco score of 72.4% (15.8% fragmented). Identification of genes encoding honeybee nesting proteins

[0435] We identified nest material genes in Hylaeus spp. using a hidden Markov model constructed using mass spectrometry peptide sequence data from Hylaeus spp. nest material. Peptide sequences from the mass spectrometry sequence data were aligned using Muscle, and a hidden Markov model was generated using hmmbuild (version v3.8.1551; Edgar, 2004; version 3.3.2; Johnson et al., 2010) from the "Hmmer" software suite. Hmmsearch was used to search the Hylaeus Trinity Transcriptome protein database, using nest material genes as the highest-scoring matches. Using Salmon and Deseq2, we identified high expression sites for genes of interest. Genes encoding beehive nest material proteins were highly expressed in the salivary gland, accounting for approximately 10% of the genes expressed in this tissue. result

[0436] A combination of transcriptome, genome, and proteome sequencing allowed the identification of a highly unique nest material gene, which has an uncharacteristic gene structure consisting of 14 exons and unstructured introns, is highly repetitive, and has a high content of glutamine (30.5%) and serine (32.1%).

[0437] The longest contiguous sequence assembled from peptide identities assigned to the same sequence database entry was 86 amino acids in length: ...SSSGMSSQAQSQQQQAQLQQSQDQRQEQESQSAGSESAVKSSKSSSGMSSQAQSQQQQAQLQQSQAQSQEEESESAQSESEVHVSK... (SEQ ID NO: 33).

[0438] A search against the cDNA sequences (mRNA transcripts) of Hylaeus nubilosus revealed several highly significant hits in glutamine- and serine-rich sequences and other protein sequences.

[0439] A BLAST search against all protein sequences in nrNCBI revealed no significant homology between the glutamine-rich sequence and other known protein sequences.

[0440] Based on the above findings and those described elsewhere herein, we believe we have identified a novel nesting material polypeptide from Hylaeus nubilosus. This novel polypeptide consists of 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.

[0441] The alignment of the FUN module 7 sequence (derived from the long-read sequence) (SEQ ID NO: 14) with the assembled proteome sequence is shown in Figure 1. This is a promising alignment (it should be noted that proteome sequence analysis is less fidelity and more difficult than gene sequence analysis).

[0442] Mapping the predicted amino acid sequence of the long-read FUN_069765 sequence to the assembled proteome sequence generated from mass spectrometry of the nest material yielded an accuracy of 85%. Amino acid composition and sequences identified by proteome analysis

[0443] SEQ ID NO: 25 is the genomic sequence (20,870 bp) of the putative Hylaeus nubilosus gene encoding a nesting material polypeptide (FUN_069765-T1, also designated "FUN polypeptide"). Without wishing to be bound by theory, the inventors believe that the FUN polypeptide forms a protein biopolymer component of the nesting material of Hylaeus nubilosus. The genomic sequence is annotated with exons, introns, and untranslated regions (UTRs), and shows the predicted transcript (also designated FUN_069765-T1) and conceptual amino acid translation. FUN_069765cDNA

[0444] SEQ ID NO: 1 (16,353 bp) is derived from the FUN_069765-T1 transcript sequence (i.e., after intron splicing) and shows only the predicted coding region of the FUN cDNA, i.e., the FUN_069765-T1 polypeptide, excluding the 5' or 3' UTRs.

[0445] SEQ ID NO:2 is the predicted amino acid sequence of the FUN_069765-T1 polypeptide (5451 amino acid residues, theoretical MW = 578.4 kDa), which is proposed to form a protein biopolymer component of the nest material of Hylaeus nubilosus wasps. SEQ ID NO:2 is the amino acid sequence translation of SEQ ID NO:1, obtained by excising the intron from SEQ ID NO:25. Bioinformatics analysis of protein sequences

[0446] Translation of SEQ ID NO: 1 (FUN_069765 cDNA) and subsequent bioinformatics analysis of the polypeptide (FUN) was performed, leading to the following observations:

[0447] The FUN polypeptide consists of 5,451 amino acids and has a theoretical molecular weight of 578,350 Da.

[0448] The majority of the protein (amino acids 138–5451) is characterized by the presence of numerous repeat motifs, and this region of the protein is called the repeat region (see Figure 2). The repeat region is further subdivided into QS repeat regions (denoted as QS in Figure 2) that contain glutamine (Q) and serine (S)-rich repeat sequences. The QS repeat regions are separated by proline (P) and cysteine ​​(C)-rich spacer repeats. Details of these repeat sequences are discussed in more detail below.

[0449] In contrast, the first 137 amino acids of the protein lack repetitive sequence features. Using the SignalP5.0 server (see Almagro Armenteros et al., 2019), the first 17 residues are 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, amino acids 18–137) rich in serine (S, 16.67%), histidine (H, 15.83%), lysine (K, 11.67%), and glutamic acid (E, 10.00%). Predicted amino acid composition of FUN_069765-T1

[0450] The full-length mature protein is rich in serine (S, 32.14%) and glutamine (Q, 30.49%), and poor in tyrosine (Y, 0.04%), phenylalanine (F, 0.07%), tryptophan (W, 0.28%), cysteine ​​(C, 0.29%), and asparagine (N, 0.31%).

[0451] There is a clear bias for glutamine and asparagine, with glutamine occurring approximately 100 times more frequently than asparagine, which is completely absent in the QS repeat regions.

[0452] Amino acids with large aromatic side chains (Y, F, W) are all poorly represented and are virtually absent from QS repeat regions, occurring only in signal peptides, N-terminal domains, or spacer repeats. Similarly, cysteine ​​and proline residues are also absent from QS repeat regions, occurring only in signal peptides, N-terminal domains, or spacer repeats.

[0453] There are 16 cysteine ​​residues and one potential N-linked glycosylation site (Asn109-His-Thr) within the N-terminal domain. Example 3 - Recombinant expression and purification of FUN polypeptide

[0454] Our initial goal was to express a synthetic protein comparable in properties to those of nest material. To this end, we selected the insect cell line Sf9 (derived from the armyworm Podoptera frugiperda) for expression, since this insect belongs to the same phylum as honeybees. The secreted synthetic protein (SEQ No. 10) is a fusion of two modules (modules 1 and 7) that are predicted to provide the basic elements of nest material. Module 1 consists of a native signal peptide that can be recognized by the Sf9 cellular machinery and targeted for secretion into the culture medium. The N-terminal histidine-rich region of native module 1 was replaced with a hexahistidine tag for affinity purification.

[0455] The cDNA of SEQ ID NO:10 was cloned into Invitrogen's pFastBacDual entry vector. The plasmid grows best in E. coli DH5α cells or similar cells. Selection in liquid LB medium or LB agar medium supplemented with 75 μg ml-1 ampicillin is recommended. Transformation of E. coli DH5α cells with the entry vector was performed using a basic chemical heat shock protocol. Recovery of the propagated entry vector was performed using a chemical alkaline lysis protocol. Recombinant bacmids were generated using competent E. coli DH10 Multivac 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 Multivac cells were recovered from the cells by alkaline lysis. Insertion of the gene into the bacmid was confirmed by PCR.

[0456] Sf9 cells were transfected with recombinant bacmid DNA in six-well plates using Insect Gene Juice transfection reagent (Merck). Sf9 cells were grown at 28°C in Sf900-III serum-free insect cell culture medium (Thermofisher). Sf9 cells were cultured as monolayers on coverslips in six-well plates or in shaker flasks agitated at 130 rpm. Cells were incubated at 28°C for 4 hours, washed, and then incubated for an additional 72 hours. After 72 hours of incubation, the transfection mix was transferred to a sterile Falcon tube, the remaining Sf9 cells were pelleted by centrifugation (500g), and the clarified medium containing baculovirus was transferred to a new six-well plate. The primary amplification plate was incubated for an additional 72 hours at 28°C. The amplified virus was harvested and centrifuged at 500g to remove cells. The clarified virus stock was supplemented with 2% fetal bovine serum and stored at 4°C. For expression of the FUN polypeptide, the resulting recombinant virus was transfected into Sf9 cells (approximately 2.0-2.5 × 10 6 cells / ml).

[0457] Small-scale protein expression was performed in 200 ml volumes of Sf9 cells in serum-free medium. Cultures were infected with empirically derived virus ratios and grown at 28°C with shaking at 130 rpm. Cultures were periodically supplemented with sterile D-glucose, L-serine, and L-glutamine to 5 mM at times t48 and t72. Cultures were typically harvested 72–96 h postinfection and centrifuged at 1500 g before protein collection.

[0458] FUN polypeptide expression was scaled up to a total volume of 4.4 L in 250 ml shake flasks. The medium was harvested and stored frozen at -20°C for several weeks prior to downstream processing. 4.4 L of medium was processed using the NaCl-isopropanol method used for biomaterial production (B2.0).

[0459] 4.4 L of culture medium was stored frozen at -20°C in Schott bottles. The medium was then thawed at 4°C for 2 days before further processing. The medium was treated with the NaCl-isopropanol fractionation method (pH approximately 5.8) as follows: 500 ml of medium was measured using a polypropylene graduated cylinder and poured into a 1 L Schott bottle. Salt was gradually added to the mixture while stirring to titrate to a 2 M NaCl concentration. Once the NaCl was completely dissolved, 500 ml of 100% isopropanol (final NaCl concentration of 1 M) was added to the mixture and stirred well. The mixture was stirred for 30 minutes and centrifuged at 6750 g for 45 minutes. This procedure was repeated for the entire 4.4 L of medium, and the centrifugation pellet was collected and resuspended in 1x PBS containing 5% glycerol at pH 7.4. The pellet in PBS was collected in a Falcon tube and centrifuged at 4000 g for 30 minutes, after which the supernatant was discarded (PBS wash). The pellet was mixed with NuPAGE SDS loading buffer, boiled at 90°C for 10 minutes, and then loaded onto a precast 12% NuPAGE Bis-Tris gel (Thermo Fisher Scientific). Electrophoresis was performed at 160V for 40 minutes using 1X MOPS running buffer (Thermo Fisher Scientific). Protein bands were visualized by staining with Coomassie Brilliant Blue R-250.

[0460] The pellet was resuspended in PBS, and the slurry was then flash-frozen in liquid nitrogen and stored at -80°C. The pellet was thawed on ice and centrifuged at 4000g for 30 minutes, after which the supernatant was discarded. The product (approximately 11g wet weight) was transported via cold chain.

[0461] SDS-PAGE gel analysis of the NaCl-isopropanol precipitate confirmed that the purity of the expressed FUN polypeptide 2.0 was very low (estimated purity <30%) (not shown). Further purification of the FUN polypeptide was performed as follows.

[0462] The culture medium (harvested at t120) was centrifuged under reduced gravity to pellet cells and debris. To reduce the volume of work, we incorporated the NaCl-isopropanol fractionation as described above. The pellet containing the FUN polypeptide was then resuspended in PBS + 5% glycerol (1 / 5 of the starter culture medium volume) containing a minimum of 6 M urea (pH = 7.5 or 8). 番目 The mixture was resuspended in 1000kJ of HCl (pH 7.0). The mixture was then sonicated to disperse the protein and further release the His-tag. Sonication was performed at amplitude 10 (applied energy approximately 1600 J) for a total of 1 minute (1 second on, 1 second off). The medium was centrifuged at 10,000 x g for 20 minutes. The clarified supernatant was loaded onto a 5 mL Ni-HisTrap FF (FastFlow) column equilibrated with wash buffer (25 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, 5% glycerol, 6 M urea). The protein was eluted from the column with elution buffer (25 mM sodium phosphate, 300 mM NaCl, 300 mM imidazole, 5% glycerol, 6 M urea) and dialyzed overnight with stirring against a buffer containing 25 mM NaP, 150 mM NaCl, and 5% glycerol at pH 7.5 to remove the urea and imidazole. composition

[0463] To characterize this sample, we performed further characterization of the FUN polypeptide protein after NaCl-isopropanol precipitation, centrifugation, and filtration. In addition to monitoring the protein molecular weight using SDS-PAGE (Figure 4), we also performed chemical characterization using FT-IR (Figures 5 and 6). Preliminary Coating

[0464] Typically, before spinning fibers, coatings and films of the target protein are cast and their chemical and physical properties are evaluated. Using the drop-casting method, FUN polypeptide materials were cast onto various substrates for coating. The drop-casting method is suitable for small-area film formation. This method is performed by dropping 50–100 μL of FUN polypeptide sample (approximately 1 mg / mL) onto the substrate of interest and allowing the solution to evaporate for the required time. The FUN polypeptide coatings were characterized using various techniques. Fourier transform infrared spectrum (FTIR)

[0465] A 50 μL drop of FUN polypeptide sample (approximately 1 mg / mL) was placed on a 1 × 1 cm aluminum (Figure 5) or glass cover slip (Figure 6) and then immobilized on a solid support (whole glass slide). Fourier transform infrared (FTIR) spectra of these samples were measured using a Bruker Lumos FTIR spectrophotometer in attenuated total reflectance (ATR) mode. IR spectra were measured over the absorption range of 400–3900 cm-1 with a resolution of 2 cm-1 and a total of 128 scans. FTIR spectra of FUN polypeptide drop-cast films showed significant β-sheet formation. The presence of β-sheets is thought to render protein substrates insoluble in water, which is crucial for coating and textile applications. In comparison, FTIR spectra of silk fibroin cast films showed a predominant α-helix / coil structure. Ethanol treatment transformed the α-helix into a β-sheet structure (a peak shift was observed in the FTIR spectrum). As shown in this study, FUN polypeptide formed a β-sheet structure even before ethanol treatment. These results demonstrate structural characteristics of the FUN polypeptide that are predicted to behave in Hylaeus nubilosus nest material and indicate that the FUN polypeptides provided herein (and various compositions containing the FUN polypeptides) have great potential for use as functional coatings and fiber-forming materials. Characterization by scanning electron microscope (SEM)

[0466] SEM was used to observe the surface morphology of the FUN protein coating. For SEM analysis, a microfilm of FUN polypeptide (5 μl of 0.5 mg / ml) was cast onto a silicon wafer and analyzed by scanning electron microscopy using a Supra55-VP field emission scanning electron microscope (SEM, Zeiss, Germany) at an accelerating voltage of 3 keV. Images were taken at 10,000x and 100,000x magnifications with a working distance of 7–8 mm.

[0467] Figure 7 shows a typical SEM image of a FUN polypeptide coating, showing a smooth, clean surface with no fibrillar or network-like patterns. The results of the SEM study are consistent with what is currently known about the surface morphology of beehive materials. That is, the coatings and films constructed from the FUN polypeptides described here exhibit the characteristic patterns of beehive materials (as expected). Contact with water

[0468] Using the drop-casting method, a recombinant protein film of FUN polypeptide was deposited on a substrate with a surface area of ​​1 x 1 cm by dropping 100 μL of concentrated protein solution (approximately 2 mg / ml). 2The protein film was cast onto piranha-cleaned glass slides (glass slides from Westlab, Australia). Water contact angle measurements were performed to examine the hydrophobicity of the film surface. Measurements were performed on FUN polypeptide and silk coatings (Sigma) and ethanol-treated FUN polypeptide and silk coatings (Sigma) using the static sessile drop method with a Biolin Attention Theta Flow Tensiometer. A water drop was applied to the protein film-cast glass substrate. Images were taken immediately after contact and after 30 seconds of incubation at room temperature. Surfaces with measured contact angles less than 90° indicate hydrophilicity. The glass substrate was hydrophilic, with a constant angle of 14° remaining after 30 seconds of equilibration. The FUN polypeptide-coated surface exhibited a contact angle of approximately 74°, which decreased to approximately 62° after 30 seconds of equilibration, while the Sigma silk surface exhibited a contact angle of approximately 48°, which decreased to approximately 22° after 30 seconds of equilibration. The coating rendered the glass more hydrophobic, as indicated by better spreading of droplets on uncoated glass substrates than after protein coating. Ethanol-treated FUN polypeptides exhibited a hydrophobic angle of 96 degrees immediately upon contact, which decreased to 75 degrees after 30 seconds of equilibration. Thus, the results presented herein demonstrate that FUN polypeptides, or at least portions thereof, are relatively more hydrophobic than sigma silk when measured under the same conditions. Cleanability

[0469] A simple test was also performed to confirm the stability of the FUN polypeptide coating. 50 μL of FUN polypeptide sample (approximately 1 mg / mL) was drop-cast onto a 1 x 1 cm glass slide. The slide was immersed in pure ethanol solution for 2 hours and then dried under vacuum before use. A washability test was performed by immersing the FUN polypeptide-coated glass slide in water or 0.1 M PBS buffer (pH 7.4) for 24 hours. The immersed slide was rinsed with deionized water to remove excess salt. The dried "immersed" slide was stained with Coomassie Brilliant Blue stain for 3-4 hours and then destained overnight in deionized water. After 24 hours of immersion in water or PBS, the coating remained intact and completely covered by Coomassie stain (Figure 9). Example 4 - Proteomic or other analyses supporting conclusions from previous examples Proteomics

[0470] SDS-PAGE gels after protein purification were used to excise bands of interest for mass spectrometry fingerprinting. The highlighted boxes indicate the putative FUN polypeptide bands. The excised FUN polypeptide bands were pooled and sent to the University of Auckland Mass Spectrometry Facility for fingerprinting. Mass spectrometry fingerprinting

[0471] The goal was to achieve sequence coverage and confirm that the 100 kDa band on the denaturing gel was the expected protein and had the sequence as designed in the construct. GluC digestion of the FUN polypeptide protein band yielded approximately 83% sequence coverage (Figure 10). The underlined peptide indicates the unobserved region. The signaling peptide is thought to be cleaved upon secretion from Sf9 cells. Theoretically, the "SKSTHTAHKSSGGKSSQME" peptide (SEQ ID NO: 26) in the GluC digest would likely be picked up, but this peptide did not generate coverage. Due to the presence of two cysteines at the C-terminus, some degree of post-translational modification is expected, which may have led to the mismatch. To achieve complete sequence coverage, trypsin digestion combined with reduction and alkylation was performed, which was thought to improve coverage of the C-terminal region (Figures 11 and 12). Reduced and alkylated trypsin digestion captured this region, covering almost the entire C-terminal region except for the final cysteine ​​residue (Figure 11). The final cysteine ​​residue was excised by trypsin. The stretch preceding the C-terminus matched two high-quality peptides ("PTTTSSTPTVPSSEPR" (SEQ ID NO: 27) and "TGIPICSIWIR" (SEQ ID NO: 28)) and one medium-scoring peptide covering SSQWNEQPSSK (SEQ ID NO: 29). Therefore, the combined trypsin and GluC digestion yielded an overall coverage of approximately 96%. Trypsin digestion yielded: NLYFQGAK (SEQ ID NO: 30) KHHGHPNHHK (SEQ ID NO: 31) is likely to be detected, but the " TLK A suspect glycopeptide near "FHPHH" (SEQ ID NO: 32) may prevent cleavage.

[0472] Alkylated trypsin digestion (Figure 12) captured the entire region except for the last cysteine ​​residue, resulting in an overall coverage of 99.81%. Example 5 - HnM1M7-01 HnM1M7-01 construct

[0473] The secreted synthetic protein (SEQ ID NO: 10), based on the fusion of two modules (HnM1M7-01), begins with the proposed native signal peptide (SEQ ID NO: 47), which could be recognized by the Sf9 cellular machinery and targeted for secretion into the culture medium. In addition to the secretory signal peptide, this construct was designed to incorporate a hexahistidine tag for affinity purification and a 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. Generation of recombinant bacmids

[0474] The entry vector was used to transform competent E. coli DH10 Multibac cells (GenevaBiotech) containing the bacmid (baculovirus genome vector plasmid) and helper plasmids, generating recombinant bacmids, according to the manufacturer's instructions (Invitrogen). Briefly, 100 ng of the entry vector (HnM1M7-01 in pFastBac-DUAL) was transformed into chemically competent DH10 MultiBac cells and plated onto KGTIX plates (kanamycin, gentamicin, tetracycline, IPTG, and X-gal). Selection of successful recombinants (white colonies) was confirmed by replating several white colonies onto new KGTIX plates and ensuring a stable color phenotype (compared to control, non-recombinant, blue colonies plated on the same selective plate). The recombinant bacmid (HnM1M7-01 bacmid) was harvested from white DH10 Multibac cells by chemical alkaline lysis and used for transfection into logarithmically growing Sf9 cells. Sf9 transfection and virus amplification

[0475] Sf9 cells were transfected with the recombinant bacmid DNA in 6-well plates using Insect GeneJuice transfection reagent (Merck). Approximately 0.8 × 10 Sf9 cells in logarithmic growth phase were transfected. 6 The cells were seeded into wells of a 35mm 6-well cell culture plate at 1 ml / ml (approximately 80% confluence) and allowed to adhere to the plate for 60 minutes at 28°C. Four micrograms of recombinant bacmid was 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, and then the solution was brought to 1 ml with fresh Sf900-III medium. The medium was aspirated from the adherent cells from the 6-well plate, and 1 ml of the HnM1M7-01 bacmid / GeneJuice solution was layered on top of the cells. The cells were incubated for 4 hours at 28°C, washed, and an additional 1 ml of SF900-III medium was added. After 72 hours of incubation, the transfection mix was transferred to a sterile Falcon tube, and the remaining Sf9 cells were pelleted by centrifugation (500xg). The clarified medium containing the baculovirus was transferred to a new 6-well plate. Next, add 1.6 ml of Sf9 cells (1.5 x 10 cells) to each well. 6 The resulting mixture was then overlaid with 1000 μg of ... HnM1M7-01 expression

[0476] For expression of HnM1M7-01, the generated recombinant virus was incubated in a shaker flask with approximately 2.0-2.5 x 10 6Sf9 cells were infected at 1000 cells / ml. Small-scale protein expression was performed in 200 ml volumes 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 periodically supplemented with sterile D-glucose, L-serine, and L-glutamine to 5 mM at 48 and 72 hours post-infection and were typically harvested at 96 hours post-infection. Media was clarified by centrifugation at 1500g prior to protein harvest. Purification of HnM1M7-01

[0477] The clarified medium was adjusted to 50 mM Tris.Cl (pH 8.0) and 2 M NaCl. Once the NaCl was completely dissolved, an equal volume of 100% isopropanol was added to the mixture (final NaCl concentration 1 M) and mixed thoroughly. The mixture was stirred for 30 minutes and then centrifuged at 6750 x g for 45 minutes. The centrifuged pellet was resuspended in sonication buffer (25 mM Tris pH 8.0, 300 mM NaCl, 6 M urea, 5% glycerol) to 1 / 5 the volume of the starter medium. Sonication was performed on ice at 75% amplitude with 1-second pulses and 1-second pauses between pulses, until a minimum of 80,000 J was applied (Qsonix Midi Tip). The solution was clarified by centrifugation at 10,000 x g for 20 minutes. The clarified supernatant was adjusted to 15 mM imidazole and sequentially loaded onto a 5 mL Ni-HisTrap FF (Cytiva) column equilibrated with sonication buffer. After loading, the column was washed with 5 CV of wash buffer (25 mM Tris pH 8.0, 300 mM NaCl, 6 M urea, 5% glycerol), and the bound protein was eluted with elution buffer (25 mM Tris pH 8.0, 300 mM NaCl, 15 mM imidazole, 6 M urea, 5% glycerol). The eluted protein fractions were collected and analyzed on a 10% PAGE Tris-glycine gel to estimate protein purity. Protein fractions were pooled and dialyzed overnight at 4 °C against 2 L of dialysis buffer (25 mM Tris (pH 8.0), 150 mM NaCl, 5% glycerol). Emulsifying properties of HnM1M7-01

[0478] In this example, we aimed to demonstrate the emulsion-stabilizing effect of the HnM1M7-01 protein. To this end, we prepared oil / water emulsions using HnM1M7-01 as a self-emulsifier without adding any other surfactants. method :

[0479] HnM1M7-01 solution was prepared as described in Example 5.1. Oil-in-water (O / W) emulsions were prepared using MCT oil (caprylic / capric triglyceride, New Directions Australia) as the oil phase and buffer (control) or protein solution (treated) as the aqueous phase at a final ratio of 1:1. Samples were prepared by mixing the aqueous protein solution with MCT oil containing 5 μg / mL Nile Red (Merck). The mixture was emulsified using an Omi Sonic Ruptor 400 ultrasonic homogenizer equipped with a 3.8 mm ultra-high intensity treatment tip. Emulsification was performed using ten 1-second pulses at 50% power limit. Emulsions were photographed using a Nikon EclipseTi-S inverted microscope in fluorescence mode with a Texas Red filter. Emulsification was performed using ten 1-second pulses at 50% power limit. Emulsions were photographed using a Nikon EclipseTi-S inverted microscope in fluorescence mode with a Texas Red filter. To assess emulsion stability, samples were imaged at days 1, 2, and 7. Photographs of the tubes were also taken to assess bulk phase separation. result :

[0480] If the buffer layer contained oil droplets, spherules (Nile Red dissolved in oil, shown as white spherules in Figure 13) were observed, suggesting the formation of an emulsion. Confocal images of samples on days 1 and 2 showed the presence of spherules consisting of an emulsified oil-protein solution (Figure 13B), reflecting the surface activity of the HnM1M7-01-modified mixture. Even in the untreated buffer / oil mixture, the formation of oil droplets / spherules in the aqueous phase was observed during the first two days of the experiment (Figure 13A, days 1 and 2). However, these spherules were not stable and disappeared after day 7 (Figure 14A). In contrast, the spherical particles observed in the HnM1M7-01-stabilized mixture were found to be stable even after 7 days of equilibration.

[0481] Based on these observations, we demonstrated that HnM1M7-01 has surface activity. Without wishing to be bound by theory, we 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 thought to adsorb there to minimize interfacial tension. HnM1M7-01 at the interface rearranges to expose its hydrophilic chains toward the buffer phase and its hydrophobic chains toward the oil phase, thereby promoting the stabilization of the oil / buffer emulsion in this mixture. Example 6 - HnM1M7-03 - (SEQ ID NO: 50) Expression and purification of HnM1M7-03

[0482] This example describes the production of a polypeptide in E. coli BL21(DE3) using a kanamycin resistance plasmid, resulting in the production of purified polypeptide HnM1M7-03.

[0483] The nucleic acid sequence encoding the HnM1M7-03 protein was synthesized by non-template PCR. The virtual nucleic acid sequence was converted into an oligonucleotide sequence using the software suite LIMS (DNATwoPointO, Inc., Newark, CA, USA). The full-length nucleic acid sequence was synthesized by assembling oligonucleotides using template-free PCR. The amplicon was purified and cloned using standard cloning methods (Molecular Cloning. A Laboratory Manual. 2012. Green and Sambrook).

[0484] The gene encoding the synthesis of the HnM1M7-03 protein was cloned into the expression vector pD451-SR, which contains a T7-inducible promoter (DNA TwoPointO, Inc., Newark, CA, USA). The purified plasmid containing the gene was transformed into chemically competent E. coli BL21(DE3) cells by heat shock and plated on non-inducing agar medium containing 0.1 mg / L kanamycin. The plates were incubated overnight at 37°C. Glycerol stocks were prepared by selecting a single colony from the transformation plate, growing it in non-inducing medium, then suspending the cells in glycerol-containing medium and storing them at -80°C.

[0485] E. coli BL21(DE3) containing a plasmid capable of expressing the HnM1M7-03 protein was cultivated in a 100L fermenter. The medium was prepared and autoclaved in the fermenter. The medium components and concentrations were as follows: casein hydrolysate 12 g / L, yeast extract 24 g / L, NaCl 10 g / L, KHPO 48 g / L, and glycerol 30 g / L. After the medium cooled, 50 mg / L kanamycin was added. Preculture 1 flask was cultured at 37°C for approximately 6 hours. Preculture 2 flask was inoculated with preculture 1 and cultured at 28°C for approximately 12 hours. The fermenter was inoculated with two preculture flasks, and the temperature was controlled at 37°C during the initial growth phase. Dissolved oxygen was controlled at 30% air saturation, and the pH of the fermenter was maintained at 6.8. 5 g / L of Pluronic antifoam was added to suppress foaming.

[0486] The fermentor was cooled to 20°C immediately prior to induction, and HnM1M7-03 protein expression was induced with 0.2 mM IPTG at an OD of approximately 2. The biomass was concentrated by tangential flow filtration (TFF) approximately 22 hours post-induction and harvested by centrifugation. The biomass was frozen at -20°C until further processing.

[0487] Biomass was thawed overnight and resuspended in lysis buffer (25 mM Tris, 2 mM MgCl, 0.5% (w / v) Triton X-100 pH 8.0) using a Microcra D-9 rotor-stator. Lysing was performed at room temperature for 40 minutes using 2 mg of lysozyme per gram of biomass. DNA was digested with 25 units of benzonase per gram of biomass. Insoluble material was collected by centrifugation at 17,000 g for 20 minutes. The lysate pellet (i.e., the "insoluble" fraction) was washed with 25 mM Tris, 2 mM MgCl, 0.5% (w / v) Triton X-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 with 0.05 M sodium phosphate (pH 11.5). Insoluble material was recovered by centrifugation at 17,000 g for 40 minutes. The washed pellet was extracted with 10 mM Tris, 4 M guanidine, pH 8.0, for 40 minutes at room temperature. The extracted fraction containing HnM1M7-06 protein was centrifuged at 17,000 x g for 20 minutes to remove debris, and the supernatant was filtered. The extracted fraction was diluted to immobilized metal affinity chromatography (IMAC) loading conditions (10 mM Tris, 4 M guanidine, 500 mM NaCl, 20 mM imidazole, pH 8.0). The diluted material was loaded onto a HiScale column packed with nickel-charged IMAC Sepharose 6 FastFlow resin (Cytiva). 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).

[0488] The HnM1M7-03 protein was precipitated from the elution fraction with 2 M ammonium sulfate. The precipitated protein was collected by centrifugation at 12,000 g for 10 minutes. The precipitated protein pellet was resuspended and washed in ultrapure water. The HnM1M7-03 protein precipitate was collected by centrifugation at 12,000 g for 10 minutes. Physical properties of HmM1M7-03 Water contact angle of HnM1M7-03 protein-coated glass surface

[0489] A drop of protein solution (1 mg / mL HnM1M7-03 dissolved in 98% formic acid) was applied to a glass slide and allowed to dry overnight at room temperature. The coated glass slide was then mounted on a theta flow tensiometer (Biolin Scientific, UK). The water contact angle (WCA) was recorded continuously for 30 seconds.

[0490] The surface of untreated glass was hydrophilic, exhibiting a water contact angle of 22-24° that remained constant after 30 seconds of equilibration. Glass coated with HnM1M7-03 exhibited a contact angle of 60-65° that remained constant after 30 seconds of equilibration. We demonstrated that HnM1M7-03 is a useful material for modifying the relative hydrophobicity of materials. Example 6 - HnM7-06 (SEQ ID NO: 52) Expression and purification of HnM7-06

[0491] This example describes the production of a polypeptide in E. coli BL21(DE3) using a kanamycin resistance plasmid and the resulting purified polypeptide, HnM7-06.

[0492] The nucleic acid sequence encoding the HnM7-06 protein was synthesized by non-template PCR. The virtual nucleic acid sequence was converted into an oligonucleotide sequence using the software suite LIMS (DNA TwoPointO, Inc., Newark, CA, USA). The full-length nucleic acid sequence was synthesized by assembling oligonucleotides using template-free PCR. The amplicon was purified and cloned using standard cloning methods (Molecular Cloning. A Laboratory Manual. 2012. Green and Sambrook).

[0493] The gene encoding the HnM7-06 protein was cloned into the expression vector pD451-SR, which contains a T7-inducible promoter (DNA TwoPointO, Inc., Newark, CA, USA). The purified plasmid containing the gene was transformed into chemically competent E. coli BL21(DE3) cells by heat shock and plated on non-inducing agar medium containing 0.1 mg / L kanamycin. The plates were incubated overnight at 37°C. Glycerol stocks were prepared by selecting a single colony from the transformation plate, growing it in non-inducing medium, suspending the cells in glycerol-containing medium, and storing it at -80°C.

[0494] E. coli BL21(DE3) containing a plasmid capable of expressing the HnM7-06 protein was cultivated in a 100L fermenter. The medium was prepared and autoclaved in the fermenter. The medium components and concentrations were as follows: casein hydrolysate 12 g / L, yeast extract 24 g / L, NaCl 10 g / L, KHPO 48 g / L, and glycerol 30 g / L. After the medium cooled, 50 mg / L kanamycin was added. Preculture 1 flask was cultured at 37°C for approximately 7 hours. Preculture 2 flask was inoculated with preculture 1 and cultured at 28°C for 17 hours. Two preculture flasks were inoculated into the fermenter, and the temperature was controlled at 37°C during the initial growth phase. Dissolved oxygen was controlled at 30% air saturation, and the pH of the fermenter was maintained at 6.8. 5 g / L of Pluronic antifoam was added to suppress foaming.

[0495] The fermentor was cooled to 20°C immediately prior to induction, and HnM7-06 protein expression was induced with 0.2 mM IPTG at an OD of approximately 2. The biomass was concentrated by tangential flow filtration (TFF) and harvested by centrifugation approximately 22 hours post-induction. The biomass was frozen at -20°C until further processing.

[0496] Biomass was thawed overnight and resuspended in lysis buffer (25 mM Tris, 2 mM MgCl, 0.5% (w / v) Triton X-100 pH 8.0) using a Microcra D-9 rotor-stator. Lysis was performed at room temperature for 40 minutes using 2 mg of lysozyme per gram of biomass. DNA was digested with 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 with 25 mM Tris, 2 mM MgCl, 0.5% (w / v) Triton X-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 with 0.05 M NaOH. Insoluble material was collected by centrifugation at 17,500 g for 20 minutes. The washed pellet with 0.05 M NaOH was extracted with 10 mM Tris, 4 M guanidine, pH 8.0, for 40 minutes at room temperature. The extracted fraction containing HnM7-06 protein was centrifuged at 17,500 x g for 20 minutes to remove debris, and the supernatant was filtered. The extracted fraction was diluted to immobilized metal affinity chromatography (IMAC) loading conditions (10 mM Tris, 4 M guanidine, 500 mM NaCl, 20 mM imidazole, pH 8.0). The diluted material was loaded onto a HiScale column packed with nickel-charged IMAC Sepharose 6 FastFlow resin (Cytiva). 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).

[0497] The HnM7-06 protein was precipitated from the elution fraction using 2.5 M ammonium sulfate. The precipitated protein was collected by centrifugation at 12,000 g for 10 minutes. The precipitated protein pellet was resuspended in ultrapure water, and the washed HnM7-06 protein precipitate was collected by centrifugation at 12,000 g for 10 minutes. Physical properties of HnM7-06 Water contact angle of HnM7-06 protein-coated glass surface

[0498] A drop of protein solution (1 mg / mL HnM7-06 dissolved in 98% formic acid) was applied to a glass slide and allowed to dry overnight at room temperature. The coated glass slide was then mounted on a theta flow tensiometer (Biolin Scientific, UK). The water contact angle was recorded continuously for 30 seconds.

[0499] The surface of untreated glass was hydrophilic, exhibiting a water contact angle of 22–24° that remained constant after 30 seconds of equilibration. Glass coated with HnM7-06 exhibited a contact angle of 76–80° that remained constant after 30 seconds of equilibration. We demonstrate that HnM7-06 is a promising material for modifying the relative hydrophobicity of materials, which is desirable for use as films or coatings. Co-spinning of HnM7-06 protein and nylon

[0500] Following the same method as described in Example 8, HnM7-06 nylon fibers were wet-spun using a nylon dope solution in 98% formic acid (FA) with a nylon concentration of 15 wt %. HnM7-06 was dissolved in FA, and nylon was added with continuous mixing and shaking until a clear solution was obtained. Tensile strength tests were performed on the wet-spun fibers obtained from these solutions. Tensile strength of co-spun HnM7-06 nylon fiber

[0501] The mechanical properties of HnM7-06 nylon fibers were evaluated using a universal testing machine (UTM) (Agilent T150USA) equipped with a 0.5 N transducer. The fibers were mounted in a paper frame (with a 10 mm window) and subjected to a strain rate of 0.01 mm s. -1 The nominal gauge length was fixed at 10 mm. Two samples were measured from each fiber.

[0502] [Table 2]

[0503] As shown in Table 2, the inclusion of HnM7-06 protein in nylon co-spun fibers improved modulus, tensile strength, and toughness. When HnM7-06 protein was incorporated into nylon co-spun fibers at levels up to 10 wt.%, the elongation at break also increased. The inventors have demonstrated that HnM7-06 protein improves the strength of nylon fibers, imparting desirable properties to fibers used in many applications, including textiles, woven fabrics, and nonwoven fabrics. Example 8 - HnP1 (SEQ ID NO: 4)

[0504] In this example, the production of an oligopeptide by chemical solid-phase synthesis is described in the production of the peptide HnP1.

[0505] The peptide (SEQ ID NO: 4) was synthesized on a pre-loaded 2-Cl-Trt resin using the standard Fmoc synthesis protocol with DIC / HOBt coupling on an APEX396 automated synthesizer. The resin was swollen in DMF for 30 min, then treated with 20% piperidine-DMF at room temperature for 8 min to remove the Fmoc protecting group and washed three times with DMF. For the coupling reaction, the Fmoc-protected amino acid, HOBt, DIC, and NMP were added to the resin. The mixture was vortexed at room temperature for 20 min. The resin was then washed once with DMF. The deprotection and coupling cycle was repeated until the final amino acid residue was assembled. The resin was then washed with DMF and DCM and air-dried. The peptide was cleaved with a TFA cocktail (95% TFA, 2.5% water, 2.5% TIS) for 3 h. The crude peptide was precipitated by adding ice-cold anhydrous ethyl ether, washed three times with anhydrous ethyl ether, and then dried under vacuum. After synthesis, peptides were purified and salt converted (HCl or acetate) using conventional preparative HPLC. Co-spinning of HnP1 peptide and nylon

[0506] Nylon dope solutions were prepared by dissolving nylon 6 / 6 pellets (molecular weight 252.35 Daltons, Sigma-Aldrich, St. Louis, MO, USA) in 98% formic acid (FA) and stirring overnight to obtain a clear solution (15% w / w nylon). Nylon and HnP1 dopes were made by first dissolving HnP1 in FA (various concentrations) and then dissolving nylon in the HnP1 solution. The solutions were loaded into syringes and wet-spun in a water coagulation bath to produce single fibers for testing.

[0507] Scanning electron microscopy (SEM) was used to identify the morphology of the as-spun fibers, and pure nylon (0% HnP1) and HnP1 nylon fibers were coated with a 5 nm Pt conductive coating, followed by SEM imaging using a Zeiss Supra 55 VP field emission gun with an accelerating voltage of 5 KV, an aperture size of 20 μm, and a working distance of 7–8 mm. Co-spinning of HnP1 and silk

[0508] To produce regenerated silk, we used undegummed fibers reeled from bivoltine silkworms (SRR Silk Production Unit, Ramanagara, Karnataka, India). Lithium bromide (LiBr, 99%), sodium carbonate (NaCO, 99.5%), and ammonium sulfate ((NH)SO, 99%) (Sigma-Aldrich) were used to dissolve, degumm, and coagulate the silk fibers, respectively. Domesticated silkworm silk fibers were degummed in a 500 ml aluminum pot of an Ahiba IR Pro rotary dyeing machine (Datacolor, Lawrenceville, USA) using 0.2% sodium carbonate at a raw silk (g):liquid (ml) ratio of 1:50 at 98 °C for 30 min. The degummed silk was washed several times with deionized water and then dried in a fan-forced dryer at 60 °C. For regenerated silk fibroin (RSF), silk degummed with sodium carbonate was dissolved in 9.3 M lithium bromide solution at a raw silk (g):solution (ml) ratio of 1:7.5 at 60 °C for 40 min. The dissolved solution was dialyzed in a deionized water bath and concentrated to obtain a highly concentrated aqueous silk solution. The silk solution was freeze-dried for 48 h to obtain pure, solid regenerated silk crystals.

[0509] Using the same method as for co-spinning with nylon, 12 wt% silk fibers with different HnP1 concentrations were wet-spun. The morphology of the fibers immediately after spinning was observed by SEM. Discussion of SEM results:

[0510] The measured fiber diameters of wet-spun nylon, silk, HnP1 silk, and HnP1 nylon fibers were found to be constant at around 60-70 μm. For both nylon and silk, the fiber roughness gradually increased as the amount of HnP1 increased from 1 wt% to 10 wt%. As the surface roughness increased, the surface area-to-volume ratio also increased. While not wishing to be bound by theory, the inventors believe these properties are useful for applications such as functional fibers due to enhanced reactive sites, adsorption, surface adhesion, and heat transfer.

[0511] The cross-sectional morphology of nylon and HnP1 nylon and HnP1 silk fibers, as shown in Figures x and y, reveals the presence of a highly porous structure. Interestingly, increasing the amount of HnP1 in the HnP1-nylon and HnP1-silk mixed matrices from 1 wt% to 10 wt% resulted in the creation of smaller, more regular pores. Smaller pores enhance the adsorption rate of other chemicals, increase interfacial energy, and improve wettability. While not wishing to be bound by theory, we believe these properties are beneficial for applications requiring cell adhesion and aqueous coating formulations, for example, in the creation of functional fiber materials. Surface water contact angle measurements of fibers containing HnP1

[0512] The water contact angles (WCA) of individual fibers cospun with nylon or silk and HnP1 were measured using a theta flow tensiometer (Biolin Scientific, UK). WCAs were recorded continuously for 30 seconds. Discussion of WCA results

[0513] As shown in Figures 21 and 22, the WCA of nylon and silk fibers decreased when the fiber composition contained a large amount of HnP1, indicating that the surface hydrophilicity (wettability) changed in an HnP1 concentration-dependent manner.

[0514] These results indicate that the introduction of HnP1 acts to enhance the hydrophilicity of both nylon and silk fibers. Without wishing to be bound by theory, we believe that these properties provide useful advantages for applications requiring wettable fibers, such as the production of absorbent materials. Tensile strength of HnP1-nylon co-spun fibers

[0515] The mechanical properties of HnP1 nylon fibers were evaluated using a universal testing machine (UTM) (Agilent T150USA) equipped with a 0.5 N transducer. The fibers were mounted on a paper frame (with a 10 mm window) and subjected to a strain rate of 0.01 mm s. -1 The nominal gauge length was fixed at 10 mm. Two samples were measured from each fiber.

[0516] [Table 3] Discussion of tensile strength results

[0517] As shown in Table 3, the addition of HnP1 peptide to the cospun fibers improved the tensile strength of nylon.

[0518] The present inventors have demonstrated that the HnP1 peptide improves the strength of nylon fibers and confers properties to the fibers that are useful and desirable in a variety of applications, such as textiles, woven fabrics, and nonwoven fabrics.

[0519] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11] Table 4-12 Table 4-13 Table 4-14 Table 4-15 Table 4-16 Table 4-17 Table 4-18 Table 4-19 Table 4-20 Table 4-21 Table 4-22 Table 4-23 Table 4-24 Table 4-25 Table 4-26 Table 4-27 Table 4-28 [Table 4-29] [Table 4-30] [Table 4-31] [Table 4-32] [Table 4-33] [Industrial Applicability]

[0520] The present invention has industrial application in the production of polypeptides useful for providing amphiphilic, hydrophobic, hygroscopic and / or hydrophilic coatings to a wide variety of articles of manufacture, including, but not limited to, synthetic fibers, textiles, components thereof, biomedical devices, and components thereof.

Claims

1. An isolated polynucleotide encoding an isolated polypeptide comprising at least 70% amino acid sequence identity to SEQ ID NOs: 50, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 52.

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

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 NOs: 50, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 52.

4. A proteinaceous 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 of claim 1 or claim 2.

6. A vector comprising an isolated polynucleotide encoding the isolated polypeptide of claim 3 or the proteinaceous polymer of claim 4.

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

8. 8. The isolated host cell of claim 7, wherein the bacterial host cell is preferably Escherichia coli.

9. 8. The isolated host cell of claim 7, wherein the fungal host cell is preferably a Pichia or Aspergillus cell, preferably an Aspergillus niger cell.

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

11. A composition comprising an isolated polynucleotide according to claim 1 or claim 2, an isolated polypeptide according to claim 3, a proteinaceous polymer according to claim 4, and / or a vector according to claim 5 or claim 6, and / or an isolated host cell according to claim 7, 8, 9 or 10.

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

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

14. 12. Use of a FUN polypeptide or part thereof according to claim 3 or a composition according to claim 11 for coating on an article of manufacture.

15. 12. Use of a FUN polypeptide or part thereof according to claim 3 or a composition according to claim 11 to form a film on an article of manufacture.

16. 16. The use according to claim 14 or 15, wherein the article of manufacture is selected from the group consisting of a textile or a component or part thereof, and a biomedical device or a component or part thereof.

17. 16. The use according to claim 14 or 15, wherein the article of manufacture or a component or part thereof is a synthetic fiber.

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

19. 19. The use according to claim 17 or 18, wherein the synthetic fibres or polymers are selected from the group consisting of polyester, spandex, rayon, nylon, acrylic, microfiber, neoprene, polyamide, acetate, polyvinyl chloride (PVC), and synthetic or "faux" leather or fur fibres and polymers, preferably nylon.

20. 16. The use according to claim 14 or 15, wherein the article of manufacture or a component or part thereof is a natural fiber.

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

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

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

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

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

26. 16. The use of claim 14 or claim 15, wherein the article of manufacture is an air filtration device or a component or part thereof.

27. 27. The use according to claim 26, wherein the component or part thereof is an air filtration device.

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

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

Images