WO2013085540A2 - Cry crystals for the production of antimicrobial proteins - Google Patents

Abstract

Disclosed are fusion polypeptides comprising a Cry protein, or crystal-forming domain thereof, and an antimicrobial protein. Also provided are nucleic acid molecules encoding the fusion polypeptides, protein crystals comprising the fusion polypeptides, methods for producing and isolating the fusion polypeptides, methods for producing and isolating an antimicrobial polypeptides, compositions comprising the fusion polypeptides, and methods of using the fusion polypeptides.

Description

CRY CRYSTALS FOR THE PRODUCTION OF ANTIMICROBIAL PROTEINS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patent application number 12/795,485, filed June 7, 2010, which claims priority to U.S. Provisional Patent application numbers 61/313,525, filed March 12, 2010, and 61/184,637, filed June 5, 2009. All of these applications are expressly incorporated herein by reference in their entireties.

BACKGROUND

[0002] Microbes such as pathogenic and spoilage bacteria pose a serious challenge in a variety of fields such as the healthcare, food processing and manufacturing, and agriculture industries. Accordingly, there are continuous efforts to develop and identify new antimicrobial agents, as well as ensuring that a steady and adequate supply of existing antimicrobial agents are available for existing uses in various applications.

[0003] An environmentally friendly approach to controlling pests is the use of pesticidal crystal proteins derived from the soil bacterium Bacillus thuringiensis ("Bt"), commonly referred to as "Cry proteins." The Cry proteins are globular protein molecules which accumulate as protoxins in crystalline form during late stage of the sporulation of Bacillus thuringiensis ("Bt"). These crystalline protein structures exhibit pesticidal activity and have been used in a number of pest control applications, particularly in the agricultural setting. Mechanistically, the crystals are solubilized to release protoxins in the alkaline midgut environment of the pest (e.g., larvae) after ingestion. Protoxins (about 130 kDa) are converted into mature toxic fragments (about 66 kDa N-terminal region) by gut proteases. Many of these proteins are quite toxic to specific target insects, but harmless to plants and other non-targeted organisms.

[0004] Some Cry proteins have been recombinantly expressed in crop plants to provide pest- resistant transgenic plants. Among those, Bt-transgenic cotton and corn have been widely cultivated. A large number of Cry proteins have been isolated, characterized and classified based on amino acid sequence homology (Crickmore et al., 1998, Microbiol. Mol. Biol. Rev., 62: 807- 813). This classification scheme provides a systematic mechanism for naming and categorizing newly discovered Cry proteins. The Cryl classification is the best known and contains the highest number of cry genes which currently totals over 130. To date, the application and use of Cry proteins has been primarily limited to pest control related applications

[0005] Bacterial production of some proteins can pose a challenge, and in particular the production of a protein or enzyme that would typically exhibit antimicrobiral activity (e.g., kill) against the bacteria normally such as, for example, lysozyme.

SUMMARY

[0006] In an aspect, the disclosure relates to a fusion protein comprising a Cry protein, or a crystal-forming fragment thereof, fused to an antimicrobial polypeptide. In some embodiments, the Cry protein is selected from the group consisting of CrylAa, CrylAb, Cry2Aa, Cry3Aa, Cry4Aa, Cry4Ba, Cryl lAa, Cryl IBa, and Cryl9Aa, or a homolog or a crystal forming fragment thereof. In some embodiments the antimicrobial polypeptide comprises lysozyme.

[0007] In another aspect the disclosure relates to a protein crystal comprising a plurality of fusion proteins, wherein the fusion proteins comprise a Cry protein, or a crystal- forming fragment thereof, fused to an antimicrobial polypeptide.

[0008] In another aspect, the disclosure relates to a cultured bacterial cell comprising a protein crystal, wherein the protein crystal comprises a plurality of a fusion polypeptide comprising a Cry protein, or a crystal-forming fragment thereof, fused to an antimicrobial polypeptide.

[0009] In an aspect, the disclosure relates to a composition comprising a fusion polypeptide or a protein crystal comprising a Cry protein, or a crystal-forming fragment thereof, fused to an antimicrobial polypeptide, and a carrier, excipient, diluent, adjuvant, or vehicle.

[0010] In a further aspect, the disclosure relates to a method for producing a fusion protein comprising a Cry protein, or a crystal-forming fragment thereof, fused to an antimicrobial protein, wherein the method comprises: growing a recombinant bacterial cell in culture under conditions that allow for production of the fusion protein; and isolating the fusion protein from the recombinant bacterial cell, wherein the recombinant bacterial cell comprises a nucleic acid molecule or expression vector having a sequence that encodes a fusion polypeptide as described herein. [0011] Other aspects of the disclosure relate to isolated nucleic acid molecules comprising a sequence that encodes the fusion polypeptide described herein, expression vectors comprising the nucleic acid molecules, bacterial endospores and recombinant bacterial cells comprising a nucleic acid molecule having a sequence that encodes a fusion polypeptide as described herein.

[0012] The disclosure provides for other aspects and embodiments that will be apparent in light of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 depicts plasmid maps of pHT315 fusion expression vectors. (A) pHT315fusionCrylAb expression vector. (B) pHT315fusionCry3Aa-lysozyme expression vector. The Cry3Aa gene can be located at either the 5 '-end or the 3 '-end of the antimicrobial protein coding sequence.

[0014] Figure 2 depicts a flowchart outlining general steps of an illustrative method for the production and purification of fusion protein crystals produced in Bacillus thuringiensis .

[0015] Figure 3 depicts a flowchart outlining general steps of another illustrative method for the production, isolation, and purification of crystals of fusion proteins used to generate an immune response.

[0016] Figure 4 depicts the antimicrobial activity as determined by growth inhibition of bacteria on culture plates by an exemplary fusion protein comprising Cry3Aa and lysozyme (hen egg white lysozyme, (HEWL)). (A) Buffer only; (B) lysozyme (at 0.5 mg/mL); (C) soluble Cry3Aa-lysozyme fusion protein; (D) soluble Cry3Aa.

[0017] Figure 5 is a depiction of the phylogenic organization of a number of Cry proteins (Cryl-Cry25) having demonstrated insecticidal and pesticidal activity, and are illustrative of certain embodiments of the disclosure. Underlined proteins have three dimensional structures determined. See, e.g., Crickmore, et al., Microbiol. Mol. Biol. Rev. September 1998 vol. 62 no. 3 807-813, incoporated herein by reference. DETAILED DESCRIPTION

[0018] In a broad sense, the disclosure relates to fusion polypeptides that have antimicrobial activity and associated methods for use and production. The disclosure takes advantage of the unexpected finding that fusion polypeptides comprising a Cry protein (e.g., Cry crystal fusion polypeptide) and/or Cry protein crosslinking technology and an antimicrobial protein provides for the production of a fusion polypeptide, as well as crystals comprising the fusion polypeptide, that exhibit antimicrobial activity and can be effectively produced recombinantly in bacterial cells, such as Bacillus. Without being limited by any particular mechanism, it is proposed that by expressing these antimicrobial proteins as a fusion polypeptide with a crystal-forming Cry protein, the antimicrobial protein becomes encapsulated, preventing it from exerting an antimicrobial effect (e.g., killing) on the host cell. In addition to the fusion polypeptides in a crystal form, a linking domain comprising a cleavage site (e.g. intein self-cleaving domain or domain that can be activated for cleavage) can provide the antimicrobial protein separated from the crystal an isolated and active form. Such fusion proteins allow for the large-scale and cost- effective production of antimicrobial proteins that have been difficult to produce using recombinant technology.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments pertain. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of various embodiments, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0020] The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. It will be appreciated that there is an implied "about" prior to metrics such as temperatures, concentrations, and times discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings herein. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of "comprise", "comprises", "comprising", "contain", "contains", "containing", "include", "includes", and "including" are not intended to be limiting. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0021] Definitions

[0022] The term "Cry protein" or "Cry polypeptide" as used herein, refers to any one of the Cry polypeptides known in the art, such as those derived from Bacillus thuringiensis . A Cry protein, as used herein, can be a protein in the full length size, or can be in a truncated form as long as in vivo crystal forming activity is retained. The Cry protein can be a combination of different proteins in a hybrid or fusion protein. A "cry gene" or"cry DNA", as used herein, is a DNA sequence encoding a Cry protein. In the context of a fusion polypeptide comprising a Cry protein, each individual antimicrobial protein may possess unique folding characteristics during crystal formation. The size of the pocket generated within the crystal may vary not only in its size, but also in its shape. Accordingly, this platform technology is not limited by any particular size of a protein that may be incorporated into a fusion crystal.

[0023] The term "antimicrobial polypeptide," "antimicrobial peptide," or "antimicrobial protein" relates to an amino acid sequence that has antimicrobial activity. As used herein, antimicrobial activity of a protein relates to the inhibition of microbial growth (e.g., slowing or halting growth and/or proliferation, slowing or halting the rate of growth and/or proliferation, or stunning, inactivation, or killing of a microbe in response to exposure (e.g., contact) with the antimicrobial protein. Antimicrobial proteins can exhibit activity against bacteria, viruses, fungi, or molds, as well as parasites or pests. For purposes of the disclosure, antimicrobial activity may be determined according to any procedure that is described herein or that is otherwise known in the art

[0024] As used herein, a "crystal" refers to is a solid material, whose constituent atoms, molecules, or ions are arranged in an orderly repeating pattern extending in all three spatial dimensions. The determination of a crystal can be determined by any means including, inter alia, optical microscopy, electron microscopy, x-ray powder diffraction, solid state nuclear magnetic resonance (NMR) or polarizing microscopy. Microscopy can be used to determine the crystal length, diameter, width, size and shape, as well as whether the crystal exists as a single particle or is poly crystalline.

[0025] The term "endospore" used herein refers to any spore that is produced within a bacterium during periods of environmental stress.

[0026] A "fusion polypeptide" or "fusion protein" as used herein, is a polypeptide containing portions of amino acid sequence derived from two or more different proteins. Optionally, a fusion protein is expressed from a fusion gene in which a nucleotide sequence encoding a polypeptide sequence from one protein is appended in frame with, and optionally separated by a linker from, a nucleotide sequence encoding a polypeptide sequence from a different protein. The fusion gene can then be expressed by a recombinant host cell as a single protein.

[0027] As used herein, the term "nucleic acid sequence" refers to a polymer of deoxyribonucleotides or ribonucleotides in the form of a separate fragment or as a component of a larger construct. Nucleic acids expressing the products of interest can be assembled from cDNA fragments or from oligonucleotides that provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit. Polynucleotide or nucleic acid sequences include DNA, RNA, and cDNA sequences.

[0028] Fusion proteins

[0029] In an aspect, the disclosure provides a fusion polypeptide comprising a Cry protein and an antimicrobial protein or peptide. As described herein, the fusion polypeptide can be provided either as a crystal or in a non-crystal form (e.g., soluble or insoluble protein).

[0030] a. Cry protein

[0031] In some embodiments the fusion protein includes a Cry polypeptide. As noted above, the Cry protein (or polypeptide) can be any Cry protein or fragment, variant, or derivative thereof that has the ability to form a protein crystal. In some embodiments, the Cry polypeptide is derived from a Bacillus thuringiensis Cry polypeptide such as, for example, CrylAa, Cryl Ab, CrylAc, CrylAd, CrylAe, CrylAf, CrylAg, CrylBa, CrylBb, CrylBc, CrylBd, CrylBe, CrylCa, CrylCb, CrylDa, CrylDb, CrylEa, CrylEb, CrylFa, CrylFb, CrylGa, CrylGb, CrylHa, CrylHb, Crylla, Cryllb, Cryllc, CrylJa, CrylJb, CrylKa, Cry2Aa, Cry2Ab, Cry2Ac, Cry3Aa, Cry3Ca, Cry3Ba, Cry3Bb, Cry4Aa, Cry4Ba, Cry5Aa, Cry5Ab, Cry5Ac, Cry5Ba, Cry6Aa, Cry6Ba, Cry7Aa, Cry7Ab, Cry8Aa, Cry8Ba, Cry8Ca, Cry9Aa, Cry9Ba, Cry9Ca, Cry9Da, Cry9Ea, CrylOAa, Cryl lAa, Cryl lBa, Cryl lBb, Cryl2Aa, Cryl3Aa, Cry Aa, Cryl5Aa, Cryl6Aa, Cryl7Aa, Cryl 8Aa, Cryl9Aa, Cryl9Ba, Cry20Aa, Cry21Aa, Cry22Aa, Cry23Aa, Cry24Aa, and Cry25Aa including, but not limited to, the Cry-derived polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or as otherwise known and available in the art (FIG.5). In some embodiments, the Cry-derived polypeptides are encoded by nucleotide sequences comrpsing SEQ ID NOS: 1 , 3, 5, 7, 9, 1 1 , 13, 15, and 17. In some embodiments, the Cry polypeptide comprises CrylAb or Cry3Aa. In some embodiments the polypeptide sequence of Cry-derived polypeptides, can encompass variants thereof, including, but not limited to, any fragment, analog, homolog, naturally occurring allele, or mutant thereof. Polypeptides also encompass those polypeptides that are encoded by a Cry-derived nucleic acid. In various embodiments, shuffled polypeptides that form crystals in vivo and are at least 80%, 81 %, 82%, 83%, 84%, 88%, 90%, 95%, 98%, 99% or 99.5% identical to the polypeptide sequence of any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or variants thereof.

[0032] In some embodiments the Cry protein is encoded by a nucleic acid molecule of any of SEQ ID NOS: 1 , 3, 5, 7, 9, 1 1 , 13, 15, and 17. In embodiments that comprise a Cry protein fragment or analog that are at least partially functionally active, i.e., they are capable of forming biologically synthesized crystals, the nucleic acid molecule may be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99% or 99.5% identical to any of SEQ ID NOS: 1 , 3, 5, 7, 9, 1 1 , 13, 15, and 17, or a complimentary sequence thereof

[0033] b. Antimicrobial peptide

[0034] In some embodiments, the antimicrobial peptide comprises lysozyme. The lysozyme can be from any organism including mammals, birds, plants, and insects and can include naturally occurring or engineered variants thereof. In some embodiments, the lysozyme is selected from hen egg white lysozyme (UniProt accession no: P00698, mature peptide (amino acids 19-147) (SEQ ID NO:36) or human lysozyme (UniProt accession no: P61626 mature peptide (amino acids 19-147) (SEQ ID NO:38) or any functional variants thereof. The antimicrobial protein may also comprise a signal sequence that may be part of the native sequence (such as in embodiments comprising lysozyme), useful for expression or purification, or targeting of the protein to a cellular region. In embodiments comprising lysozyme, for example, the lysozyme can be expressed with a leader sequence which can be processed to form the mature lysozyme protein. [0035] In addition to lysozyme, a large number of antimicrobial peptides have been classified and structurally characterized. In some embodiments, the antimicrobial peptide comprises a peptide that exerts its antimicribial effect through interaction with the microbial cell membrane and/or cell wall. In some embodiments, the antimicrobial peptide can be selected from a host defense peptide. Host defense peptides are an evolutionarily conserved component of the innate immune response and are found among all classes of life. These peptides are potent, broad spectrum antibiotics and have been demonstrated antimicrobial activity against Gram positive and Gram negative bacteria, mycobacteria, viruses, and fungi.

[0036] Some non-limiting examples of antimicrobal peptides include alamethicin (from Trichoderma viride), cathelicidins such as human animicrobial peptide LL-37 (from lysosomes in macrophages and polymorphonuclear leukocytes (PMNs), defensins (from vertebrates, invertebrates, and plants), cecropins (from insects such as Hyalophora cecropia), dermcidins (encoded by the human DCD gene), histatins (from human saliva e.g., histatin 1 (Hstl) and histatin 2 (Hst2)), hydramacin-1 (from freshwater Hydra), melitten (from bee venom), magainins (from Xenopus laevis), MSI peptides (e.g., MSI-78, MSI-843 MSI-594), pexiganan (from African clawed frog), protegrins (from porcine), polyphemusin (from horseshoe crab), and thionins (from plants). A number of online databases listing antimicrobial peptides are available and can be consulted in selecting an appropriate antimicrobial peptide for use in accordance with the disclosure, including the database maintained by the Department of Pathology and Microbiology at the University of Nebraska Medical Center, Omaha, NE (http://aps.unmc.edu/AP/ main.php) .

[0037] Suitably, the antimicrobial peptide will preferentially interact with a microbial cell rather than the host cell (e.g., mammalian or plant cells), which provides the peptides with a selectivity for a microbial cell (e.g., bacterial cell) allowing for specific exertion of toxicity against the microbe, without being significantly toxic to the host cells.

[0038] The fusion polypeptide can comprise an optional "linker" group or moiety. When present, the chemical structure of the linker is not essential, as it can merely function as a spacer, or provide a convenient site for cleavage of the Cry protein from the antimicrobial protein. In some embodiments the linker can comprise a sequence of amino acids linked together by peptide bonds (peptidyl linker) as are known in the art. In some embodiments, a peptidyl can comprise from 1 up to about 40 amino acid residues, (e.g., 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, etc.) amino acid residues, including any number of amino acids falling within that range. In some embodiments, the linker is a chemical (i.e., non-peptide) linker such as those known in the art. In some embodiments the linker is robust and is not readily cleaved (e.g., by proteolytic enzymes). In some embodiments, the linker can provide for a convenient site for cleavage (e.g., proteolysis, autolysis, and/or self-cleavage), providing for the optional isolation and purification of the antimicrobial polypeptide, as are known in the art. In some embodiments the linker comprises a self-cleavage domain that, upon self-cleavage, can reform a peptide bond between the two domains (e.g., the Cry protein and the antimicrobial protein). In some embodiments, the linker comprises a sortase domain that comprises the sortase A catalytic core and the associated sortase A cleavage motif (See, e.g., Cossart, P. and Renaud, J., Proc Natl Acad Sci USA (2000 May 9) 97(10):5013-5015; Mazmanian SK., et al, Science 285 (5428): 760-3; Pallen MJ., et al, Curr Opin Microbiol 6 (5): 519-527, each incorporated by reference).

[0039] In some embodiments, the linker comprises an intein sequence that is able to excise itself and rejoin the remaining portions of an amino acid sequence (e.g., the Cry protein and antimicrobial protein) with a peptide bond. Inteins are known in the art (see, e.g., Gogarten, J Peter; et al, BMC Evol Biol (2006) 6:94; Anraku Y, et al. IUBMB Life (2005) 57(8):563-74; and de Grey, Aubrey D. N. J. Trends Biotechnol (2000) 18(9):394-399, each incorporated herein by reference). New England Biolabs maintains an online intein database (InBase) that allows for selection of an appropriate intein. In some embodiments, the intein is selected from a Mycobacterium intein. In some embodiments the intein comprises RecA intein or GyrA intein.

[0040] The arrangement and orientation of the Cry polypeptide and the antimicrobial protein of the fusion protein can vary, and will typically be determined by the arrangement of the nucleotide sequences in the expression systems. Some embodiments the arrangement provides for retaining, or substantially retaining the ability of the fusion polypeptide to form crystals. Suitably, the arrangement of the fusion polypeptide can be represented by one of the following schemes from N- to C-terminus:

[Cry polypeptide - optional linker domain - antimicrobial protein] or

[antimicrobial protein- optional linker domain - Cry polypeptide].

[0041] Some embodiments of this aspect relate to a fusion protein crystal comprising a Cry polypeptide and an antimicrobial protein. In some embodiments, the fusion protein crystal comprises a linker located between the Cry protein and the antimicrobial protein. In some further embodiments, at least one additional agent, polypeptide, nucleic acid, and or molecule may be bound and/or crosslinked to the crystal.

[0042] In an embodiment, the fusion polypeptide is expressed and forms a crystal in vivo in a cell. In further embodiments, the crystal of the fusion polypeptide is produced within a bacterial cell such as for example, a Bt or an E. coli cell. In some embodiments the crystal may be harvested directly from the bacterial cells, as the fusion polypeptide crystals are suitably stable. In exemplary embodiments, a simple purification strategy makes them relatively cheap to obtain.

[0043] In various embodiments, the biologically synthesized crystals are fairly consistent in size with about 150-500 protein molecules per crystal in them depending on the crystal size. In Bacillus, the crystals may be generated during the sporulation phase of the bacterium and are formed alongside the spore in a bacterium.

[0044] The fusion proteins and crystals comprising the fusion proteins described herein comprise Cry and antimicrobial proteins that can be from known amino acid sequences. In some embodiments the proteins can encompass functional fragments or conservative variants of the known amino acid sequences, such as those exemplified herein. Some specific examples of "conservative variants" include, but are not limited to, substitutions within the following groups: glycine and alanine; valine, alanine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine, and threonine; lysine, arginine, and histidine; and phenylalanine and tyrosine.

[0045] Also included in the embodiments are polypeptides carrying modifications such as substitutions, deletions, insertions, or inversions, and which substantially retain the crystal forming ability of the Cry polypeptide, as well as the antimicrobial activity of the antimicrobial protein. Consequently, included in the embodiments is a polypeptide, or a crystal forming fragment thereof, the amino acid sequence of which is at least 60% identical (e.g., at least 60%, 70%), 80%), or 95%) identical) the amino acid sequences of the proteins disclosed herein, including those set forth in the sequence listing. "Percent identity" is defined in accordance with the algorithm described herein.

[0046] As will be apparent form the disclosure, some embodiments provide an isolated polypeptide (e.g., either a fusion protein or an antimicrobial protein) encoded by a nucleic acid disclosed herein. An "isolated" polypeptide is a polypeptide that is substantially free from the proteins and other naturally occurring organic molecules with which it is naturally associated. Purity can be measured by any art-known method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC. An isolated polypeptide may be obtained, for example, by extraction from a natural source (e.g., a human cell); by expression of a recombinant nucleic acid encoding the polypeptide; or by chemical synthesis of the polypeptide. In the context of a polypeptide obtained by extraction from a natural source, "substantially free" means that the polypeptide constitutes at least 30% (e.g., at least 35%, 45%, 85%, etc.) of the dry weight of the preparation. A protein that is chemically synthesized or produced from a source different from the source from which the protein naturally originates, is by definition substantially free from its naturally associated components. Thus, an isolated polypeptide includes recombinant polypeptides synthesized, for example, in vivo, e.g., in Bt cells, or in vitro, e.g., in a mammalian cell line, E. coli or another single-celled microorganism, or in insect cells. In addition to the methods for purifying and isolating a polypeptide exemplified herein, methods for purifying and isolating a polypeptide are well known in the art.

[0047] The antimicrobial activity of the fusion proteins disclosed herein, in either soluble or crystal form, can be assessed by any method known in the art, including microscopy, fluorescence imaging, or any assay quantitative assays for for assessing bactericidal, virucidal or fungicidal activities. Some non-limiting examples of quantitative assays include tests provided by regulatory authorities such as detailed in European standards EN 1276 (against minimal concentrations of Pseudomonas aeruginosa, Escherichia coli, Enterococcus hirae, and Staphylococcus aureus) and EN 1650 (against fungi including Candida albicans and Aspergillus niger), or a time kill test, wherein upon exposure to an antibacterial agent the amount of bacteria killed over time is recorded. In a time kill test, the test composition is diluted so that after addition of inoculum the test composition is at use concentration. The test composition is then brought into contact with a known population of test bacteria for a specified time period at a specified temperature. The antimicrobial ingredients are neutralized at the end of the time period and the sample is plated to enumerate the surviving bacteria. The percent reduction from the original population is then calculated. Alternatively qualitative assays can be used to assess the effect of the fusion protein or, in some embodiments, the subsequently cleaved and isolated antimicrobial peptide.

[0048] Nucleic acid sequences [0049] In an aspect the disclosure provides a nucleic acid molecule comprising a sequence that encodes a fusion polypeptide comprising a Cry protein and an antimicrobial protein as described herein. In some embodiments the Cry nucleotide sequence is selected from a sequence comprising SEQ ID NOS: 1 , 3, 5, 7, 9, 1 1 , 13, 15, and 17. In some embodiments the antimicrobial nucleotide sequence is selected from a lysozyme coding sequence, or a codon- optimized sequence comprising SEQ ID NOS: 35, 37, or 39. Alternatively, the nucleotide sequences encoding the Cry and/or the antimicrobial protein can be obtained by several methods known in the art. For example, the DNA can be isolated using hybridization procedures that are well known in the art. These include, but are not limited to: (1 ) hybridization of probes to genomic or cDNA libraries to detect shared nucleotide sequences; (2) antibody screening of expression libraries to detect shared structural features; and (3) synthesis by the polymerase chain reaction (PCR). Sequences for specific genes and polypeptides can also be found in GenBank, National Institutes of Health computer database. (See the NCBI website).

[0050] The nucleic acid molecules are not limited strictly to the particular sequences specifically exemplified herein. Some embodiments provide for nucleic acid molecules that include one or more modifications such as substitutions, deletions, insertions, or inversions, but which nevertheless encode proteins that substantially retain the ability to form crystals. Some embodiments provide for nucleic acid sequences that can serve as hybridization probes for identifying a nucleic acid with one of the disclosed sequences. Included are nucleic acid molecules, the nucleotide sequence of which there is a portion that is at least 75% identical (e.g., at least 75%, 85%, 95%, or 99% identical) to the provided nucleotide sequences.

[0051] The determination of percent identity or homology between two sequences is accomplished using the algorithm of Karlin and Altschul (1990) Proc. Nat'l Acad. Sci. USA 87: 2264-2268, modified as in Karlin and Altschul (1993) Proc. Nat'l Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. MoT Biol. 215:403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See the NCBI website.

[0052] In some embodiments, a Cry-antimicrobial polypeptide fusion vector may be transformed into B. thuringeinsis cells where the fusion protein is expressed. Crystals comprising the fusion protein may be generated during the sporulation phase of the bacterium and are formed along with the spore in a bacterium, the macroscopic spore and crystal may be released allowing for their separation by centrifugation (e.g., in a renografin solution). In the final step, the spore and crystal particles may be separated by chromatography (e.g., CM-cellulose type).

[0053] In various embodiments, the endospores may be used as a convenient storage vehicle for transporting and packaging Bt cells transformed with nucleic acids encoding fusion polypeptides. Endospores are resistant to desiccation, temperature, starvation, and other environmental stresses. Accordingly, endospores containing nucleic acids encoding fusion polypeptides may be easily packaged and transported. When desired, the endospores of various embodiments may be reactivated through a process that includes the steps of activation, germination, and outgrowth to develop into a fully functional vegetative bacterial cell. These bacterial cells may then form crystals comprising fusion polypeptides.

[0054] Production of Fusion polypeptides

[0055] In an aspect, the disclosure provides a method for producing a fusion protein comprising a Cry protein, or a crystal-forming fragment thereof, fused to an antimicrobial protein, wherein the method comprises growing a recombinant cell in culture under conditions that allow for production of the fusion protein; and optionally isolating the fusion protein from the recombinant cell. In an embodiment the method produces a protein crystal comprising a plurality of the fusion proteins that comprise a Cry protein, or a crystal-forming fragment thereof, fused to an antimicrobial protein that is generated in vivo in a cell. In some embodiments, the protein crystals are generated in a bacterial cell. Any bacterial cell can be used in this embodiment, so long as it allows for the formation of the protein crytal. In some embodiments, the crystal proteins are produced in a bacterial cell selected from Bacillus such as, for example, B. thuringiensis or B. subtilis (See Agaisse, H and Lereclus, D. (1994) 176 (15) Journal of Bacteriology, p. 4734-4741 , incorporated herein by reference). Various other bacteria, including E. coli, may also be used to produce the fusion protein or crystals comprising a plurality of the fusion proteins in vivo. [0056] In alternative embodiments, the fusion protein and/or protein crystals may be generated in vivo in recombinant eukaryotic cells. For example, successful in vivo expression of Bt crystals in chloroplasts has been demonstrated in tobacco plants (Cosa et al. (2001) Nature Biotechnology 19:71 -74, herein incorporated by reference). Techniques for the isolation and purification of either microbially or eukaryotically expressed polypeptides may be by any conventional means known in the art, such as, for example, preparative chromatographic separations and immunological separations, such as those involving the use of monoclonal or polyclonal antibodies or antigen. In some embodiments, particularly embodiments relating to protein crystals, the expressed fusion proteins can be separated by gradient centrifugation.

[0057] Suitably, the fusion protein and/or protein crystal comprising the fusion protein can be produced by expression of nucleic acid encoding the protein in a bacterial cell such as, for example, a recombinant B thuringeinsis cell transformed with recombinant plasmid DNA, bacteriophage DNA, or cosmid DNA expression vectors encoding the fusion protein. Vector constructs can be expressed in B. thuringiensis in large scale.

[0058] In an aspect, the disclosure provides a method for the production of a protein crystal comprising a fusion polypeptide, wherein the method comprises growing a recombinant cell containing a nucleic acid encoding the fusion polypeptide under conditions that allow expression of the nucleic acid sequence, and recovering a crystal formed in the host cell. The nucleic acid sequences can optionally be operably linked to a promoter for expression in a particular prokaryotic or eukaryotic expression system. For example, a nucleic acid can be incorporated in an expression vector, such as described herein.

[0059] In various embodiments, purification of desired crystals is simple and cost effective. Initially, a shuttle vector is produced encoding an antimicrobial protein fused to a Cry polypeptide, or a crystal forming fragment thereof. In some embodiments, the expression vector is optimized for overexpression in B. thuringiensis. The vector (e.g., pHT315-fusionCryl Ab) may be transformed into a bacterium (e.g., a B. thuringeinsis cell) where the fusion protein is produced and forms crystals within the cell. In various embodiments, desired crystals may be generated during the sporulation phase of the bacterium and are formed alongside the spore in the bacterium (e.g., B. thuringiensis cells). Subsequently the spores/crystal mixture may be released from autolyzed Bt. Density gradient centrifugation may be performed using a Renograffin gradient. The bands containing the spores and crystals may then be isolated. In the final step, the spore and crystal particles may be separated by CM-cellulose chromatography to generate purified crystals comprising the desired fusion polypeptide.

[0060] In alternative embodiments, fusion protein crystal purification from bacteria may also be accomplished when the expression sequences include tags for one-step purification such as by nickel-chelate chromatography. The construct can also contain a tag to simplify isolation of the fusion polypeptide. For example, a polyhistidine tag of, e.g., six histidine residues, can be incorporated at the amino terminal end of the fluorescent protein. The polyhistidine tag allows convenient isolation of the protein in a single step by nickel-chelate chromatography. Other possible tags include CBP, CYD (covalent yet dissociable NorpD peptide), Strep II, FLAG, HPC (heavy chain of protein C) peptide tags, and the GST and MBP protein fusion tag systems. The fusion polypeptide can also be engineered to contain a cleavage site to aid in protein recovery. Alternatively, the fusion polypeptides of the embodiments can be expressed directly in a desired host cell for application in situ.

[0061] In other embodiments, the crystals can be used in an unpurified or partially purified state from which the activity associated with the crystal properties can still be utilized. Examples include the use of the crystal-containing cells or lysed proteins obtained after cell growth, or the crystal-containing fraction generated following centrifugation.

[0062] In another related aspect, the disclosure provides a method for producing an antimicrobial protein comprising: growing in culture a recombinant cell comprising a nucleic acid molecule (or an expression vector comprising the nucleic acid molecule) that encodes a fusion protein as described herein, under conditions that allow for production of the fusion protein and wherein the fusion protein comprises a cleavable linker; isolating the fusion protein from the recombinant cell; reacting the isolated fusion protein under conditions that allow for cleavage of the linker; and isolating the antimicrobial protein. In some embodiments, the fusion protein comprises a fusion protein crystal. In embodiments relating to production of a fusion protein crystal, the method step of isolating can comprise one or more centrifugation steps that employ a density gradient or an affinity media/method (e.g., a medium cross-linked with a moiety having binding specificity to the fusion protein crystal and/or a molecule attached to the fusion protein crystal). In some embodiments, the recombinant cell comprises a bacterial cell such as, for example B. thuringiensis, B. subtilis, or E. coli. In some embodiments, growth of the recombinant bacterial cell is continued until a spore/crystal mixture is released from the bacterium upon cell lysis (e.g., autolysis, mechanical, or chemical lysis). In some embodiments, the isolated antimicrobial protein comprises lysozyme.

[0063] Methods for purification and isolation of a protein can include any of the techniques routinely used and known in the art, including precipitation using salt solutions (e.g., ammonium sulfate), standard chromatographic methods using reverse-phase (separation by hydrophobicity), ion-exchange (separation by anionic/cationic charge), size-exclusion methods (separation by size and/or shape), or affinity chromatography (using amino acid/ligand tags and crosslinked column media) on any standard liquid chromatography system (e.g., HPLC or FPLC systems).

[0064] Expression Vectors

[0065] Delivery of a nucleic acid can be achieved by introducing the nucleic acid into a cell using a variety of methods known to those of skill in the art. For example, the construct can be delivered into a cell using a colloidal dispersion system. Alternatively, nucleic acid construct can be incorporated (i.e., cloned) into an appropriate vector. For purposes of expression, the nucleic acid sequences encoding the fusion polypeptide may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a plasmid, virus, or other vehicle known in the art that has been manipulated by insertion or incorporation of the nucleic acid sequences encoding the fusion polypeptides. The expression vector typically contains an origin of replication, a promoter, as well as specific genes that allow phenotypic selection of the transformed cells. Vectors suitable for use include, but are not limited to, the pSB6341 Ab expression vector for expression in Bacillus thuringiensis (Bt), the pHT315 expression vector for expression in Bacillus thuringiensis (Bt), the T7-based expression vector for expression in bacteria (Rosenberg et al., Gene, 56: 125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521 , 1988), baculovirus-derived vectors for expression in insect cells, cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV.

[0066] Depending on the vector utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see, e.g., Bitter et al., Methods in Enzymology, 153:516-544, 1987). These elements are well known to one of skill in the art. [0067] The term "operably linked" or "operably associated" refers to functional linkage between the regulatory sequence and the nucleic acid sequence regulated by the regulatory sequence. The operably linked regulatory sequence controls the expression of the product expressed by the nucleic acid sequence. Alternatively, the functional linkage also includes an enhancer element.

[0068] "Promoter" means the minimal nucleotide sequence sufficient to direct transcription. Also included are those promoter elements that are sufficient to render promoter-dependent nucleic acid sequence expression controllable for cell-type specific, tissue specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the native gene, or in the introns.

[0069] "Gene expression" or "nucleic acid sequence expression" means the process by which a nucleotide sequence undergoes successful transcription and translation such that detectable levels of the delivered nucleotide sequence are expressed in an amount and over a time period so that a functional biological effect is achieved.

[0070] Host Cells

[0071] An expression vector can be used to transform a target cell. By "transformation" is meant a permanent genetic change induced in a cell following incorporation of new DNA (i.e., DNA exogenous to the cell). Where the cell is a mammalian cell, the permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell. By "transformed cell" is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding a fusion protein comprising a Cry protein, or a fragment thereof. Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as bacterial cells (e.g., Bacillus, including B. thuringiensis, B. subtilis; E. coli, and the like) competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl₂ method by procedures well known in the art. Alternatively, MgCl₂ or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.

[0072] When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate co-precipitates, conventional mechanical procedures, such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransfected with DNA sequences encoding the fusion polypeptide, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein. (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). Preferably, a eukaryotic host is utilized as the host cell, as described herein.

[0073] Eukaryotic systems, such as mammalian expression systems, allow for proper post- translational modifications of expressed mammalian proteins to occur. Eukaryotic cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, phosphorylation, and advantageous secretion of the gene product should be used as host cells for the expression of the polypeptide. Such host cell lines may include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, and WI38.

[0074] Pharmaceutical Compositions

[0075] In another aspect, compositions, e.g., pharmaceutically acceptable compositions, are provided wherein the compositions comprise a crystals comprising a fusion polypeptide as described herein, formulated together with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, oral, nasal, or epidermal administration (e.g., by injection or infusion). For applications in animals, mucosal administration of a bacterial isolate comprising the crystalline fusion polypeptide, or a partially purified crystalline fraction is also acceptable.

[0076] The compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Useful compositions are in the form of injectable or infusible solutions. A useful mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). For example, the fusion protein and/or crystal can be administered by intravenous infusion or injection. In another embodiment, the fusion protein and/or crystal is administered by intramuscular or subcutaneous injection. These fusion protein and/or crystal compositions can also be administered via oral or nasal administration. For example, when delivered intranasally (i.n.), CrylAc is a potent mucosal immunogen and adjuvant. See Rodriguez-Monroy (2010) Scand. J. of Immunology 71 , pp: 159- 168.

[0077] Compositions for administration to animals and humans typically should be stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high crystal concentration, including the bacterial cell mass as isolated directly from cell culture or some lyophilized form. Sterile injectable solutions may be prepared by incorporating the fusion polypeptide in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. For administration in animals, feeding the animals the cell paste, or a partially purified composition, either directly, lyophilized, or in some dispersion may be advantageous.

[0078] The compositions can be administered by a variety of methods known in the art that are effective for any therapeutic and prophylactic applications. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.

[0079] In some embodiments, a composition comprising the fusion protein may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The fusion protein (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. Therapeutic compositions can be administered with medical devices known in the art.

[0080] Dosage regimens are adjusted to provide the optimum desired response (e.g., a prophylactic or therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms are dictated by and directly dependent on (a) the unique characteristics of the fusion protein and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding the fusion protein for the treatment of sensitivity in individuals.

[0081] In some embodiments, a non-limiting range for a therapeutically or prophylactically effective amount of a Cry-antimicrobial fusion polypeptide or fragment thereof is about 0.1 -100 mg/kg, e.g., 1 -10 mg/kg, and other ranges falling within the range. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. The exact dosage can vary depending on the route of administration. For intramuscular injection, the dose range can be 100 μg to 10 mg per injection. Multiple injections can be administered as needed.

[0082] A therapeutically effective amount or a prophylactically effective amount of the fusion polypeptide can vary according to factors that are specific to each subject such as, for example, disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the pharmaceutical composition is outweighed by the therapeutically beneficial effects. The ability of a fusion polypeptide to inhibit a measurable parameter can be evaluated in an animal model system predictive of efficacy in the target subject (e.g., a human subject). Alternatively, this property of a composition can be evaluated by examining the ability of the compound to modulate, such modulation in vitro by assays known to the skilled practitioner. As used herein a "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, e.g., protective immunity against a subsequent challenge by a pathogen. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0083] Accordingly, the fusion polypeptides described herein have in vitro and in vivo diagnostic, therapeutic, and prophylactic utilities. As used herein, the term "subject" is intended to include humans and non-human animals. The term "non-human animals" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, pigs, chickens and other birds, mice, dogs, cats, cows, horses, and fish.

[0084] Methods of administering a crystal comprising a fusion polypeptide and dosage determination are described above. In some embodiments, vaccines can be used to prevent various disease conditions by inducing a protective immunity in the inoculated subject, or to treat an existing disease state if improved immune responses can be useful in controlling the relevant pathogen. For example, the Cry crystals comprising fusion proteins can fused with specific antigens can be used to prevent, reduce, or alleviate bacterial and or an acute influenza infection.

[0085] In other embodiments, immunogenic compositions and vaccines that contain an immunogenically effective amount of an antigenic polypeptide, or antigenic fragments thereof, fused or crosslinked to a crystal comprising the fusion polypeptide described herein, are provided. Immunogenic epitopes in a polypeptide sequence can be identified according to methods known in the art, and proteins or fragments containing those epitopes can be delivered by various means, in a vaccine composition. Suitable compositions can include, for example, lipopeptides (e.g., Vitiello et al., J. Clin. Invest., 95:341 , 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) ("PLG") microspheres (see, e.g., Eldridge et al., Molec. Immunol, 28:287-94, 1991 ; Alonso et al, Vaccine, 12:299-306, 1994; Jones et al, Vaccine, 13:675-81 , 1995), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al, Nature, 344:873-75, 1990; Hu et al, Clin. Exp. Immunol, 1 13:235-43, 1998), and multiple antigen peptide systems (MAPs) (see, e.g., Tarn, Proc. Natl. Acad. Sci. U.S.A., 85:5409-13, 1988; Tarn, J. Immunol. Methods, 196: 17-32, 1996). Toxin- targeted delivery technologies, also known as receptor-mediated targeting, such as those of Avant Immunotherapeutics, Inc. ( eedham, Mass.) can also be used.

[0086] Useful carriers that can be used with immunogenic compositions and vaccines are well known, and include, for example, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like. The compositions and vaccines can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, typically phosphate buffered saline. Besides the crystal itself, the compositions and vaccines may also include an additional adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, CTL responses can be primed by conjugating influenza or other viral polypeptides (or fragments, derivatives or analogs thereof) to lipids, such as tripalmitoyl-S-glycerylcysteinyl-seryl-serine.

[0087] Immunization with a composition or vaccine containing a protein composition, e.g., via injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, induces the immune system of the host to respond to the composition or vaccine by producing large amounts of CTLs, and/or antibodies specific for the desired antigen. Consequently, the host typically becomes at least partially immune to later infection (e.g., M. tuberculosis), or at least partially resistant to developing an ongoing chronic infection, or derives at least some therapeutic benefit. For example, the subject is protected against subsequent infection by the target virus or bacteria.

[0088] Also provided herein are kits including one or more of a nucleic acid vector encoding a fusion polypeptide as described herein, or a crystal-forming component thereof; bacteria for in vivo expression of a fusion protein crystal, and or a composition comprising a crystal comprising a fusion polypeptide. The kit can include one or more other elements including: instructions for use; other reagents, e.g., a label, a therapeutic agent, or an agent useful for crosslinking, recombinantly engineering or otherwise coupling or fusing a Cry protein to a an antimicrobial protein, and/or diagnostic agent; devices or other materials for preparing the composition for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject. [0089] The following examples merely serve to illustrate some of the aspects and embodiments described herein, and should not be taken to limit the scope of the disclosure. One of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention.

[0090] Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used, for example, for nucleic acid purification and preparation, chemical analysis, recombinant nucleic acid, and oligonucleotide synthesis. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the instant specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. 2000). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of described herein are those well known and commonly used in the art.

EXAMPLES

[0091] Example 1: Cry-antimicrobial fusion protein

[0092] In order to assess whether the Cry protein platform would be effective to produce and form crystal fusion proteins in vivo, a fusion polypeptide is constructed by inserting a codon- optimized nucleotide sequence encoding lysozyme (SEQ ID NO: 39) in- frame with a nucleotide sequence encoding Cry3Aa polypeptide. The resulting fusion protein is predicted to have a sequence of Cry3Aa- lysozyme.

[0093] Fig. 1 is a general shuttle vector for expressing Cry fusion crystals. The fusion protein expression vector in Fig. 1 includes a pHT315 shuttle vector as a platform for expressing Cry fusion protein crystals. The cloning vector pHT315 is vector previously developed in order to carry out recombinant expression of crystal proteins of Bacillus thuringiensis . The vector bears an origin of replication (ori) for replicating in E.coli and another for Bt. In addition, the vector has two antibiotic resistance genes one for ampicillin (Amp ) and second for erythromycin (Ery^R). Additional detailed information about the construction of the pHT315 vector and for information about obtaining the vector is available in Arantes, O. and Lereclus, D. "Construction of cloning vectors for Bacillus thuringiensis", Gene (1991) 108; pp: 1 15-1 19 (incorporated herein by reference).

[0094] As described in related international application PCT/US2010/037650 (incorporated herein by reference) the pHT315 vector depicted in Fig. 1 as including a CrylAb coding sequence (SEQ ID NO: 3), can be modified to express a wide variety of Cry fusion crystals in vivo. In this example, the Cry fusion protein vector modified so that it is driven by a cry3A promoter (SEQ ID NO: 32) operably linked to a 1950 bp Cry3Aa coding sequence (SEQ ID NO: 7) from Bacillus thuringienis var tenebrionis. Once the Cry3Aa sequences are incorporated into the expression vector, a codon-optimized nucleotide sequence (SEQ ID NO: 39) encoding hen egg white lysozyme (SEQ ID NO: 36) is positioned downstream of the cry3Aa gene followed by a stop codon.

[0095] Once the sequence of the expression vector was confirmed, the pHT315-fusion Cry3Aa-lysozyme vector is transformed into bacterial cells (e.g., Bt or E.coli cells) and the cells cultured under conditions that allow for expression of the Cry3Aa-lysozyme fusion protein. Interestingly, the production of the fusion protein does not appear to have any adverse effect on the bacterial cells during production. Without being limited by any particular theory, it may be that the fusion protein is sequestered upon forming a crystal such that the antimicrobial effect of lysozyme is inhibited (e.g., prevents interaction between lysozyme and bacterial cell membrane).

[0096] Crystals bearing the following constructs are also prepared using the Bacillus thuringiensis crystal expression system described above. Cry3Aa-RecAintein-LyzC in pHT315 vector; Cry3A-Sortase-LyzC in pHT315 vector (where "sortase" relates to a domain containing the catalytic core of sortase A as well as the associated sortase A cleavage motif (e.g., at the T-G bond of an LPXTG domain (for S. aureus)); LyzC-GyrAintein-Cry3Aa in pHT315 vector; and LyzC-GyrAintein-CrylAb in pHT315 vector. The various intein sequences (e.g., RecA intein, GyrA intein) can be found on InBase, the Intein Database and Registry (Perler, F. B. (2002). InBase, the Intein Database. Nucleic Acids Res. 30, 383-384) and are incoporated herein by reference. In light of the robust nature of the Cry-based expression platform, it is expected that each of these constructs will provide fusion proteins having antimicrobial activity. [0097] General protocols and methods for isolating and purifying the crystal fusion protein are illustrated in Figures 2-3. The Cry3 Aa- lysozyme fusion protein crystals are isolated from the bacterial culture and the lysozyme can be cleaved from the Cry protein using the following protocol:

1. Harvest grown crystals by centrifuging at 8k rpm for 10 min

2. Resuspend in sterile distilled water

3. Layer 4 ml on each density gradient of 60% iodixanol

4. Centrifuge at 5000rpm for 70 min

5. Separate the visual bands using hypodermic needle

6. Wash each layer with 50ml water and centrifuge at 8000rpm for 10 min

7. Repeat step 6 for 6-1 Ox

8. Resuspend 200μί of pellet in sterile water and add to autoclaved 1.5mL tubes

9. Centrifuge at 10k rpm for 2 min

10. To pellet add desired buffer (40 mm bis-tris in x pbs ph 6.2)

11. Incubate the mixture for 16-18 hrs @ 25oc/4oc with shaking on a nutator mixer.

12. Centrifuge at 10k rpm for 2 min

13. Collect 40μL· supernatant and boil samples for SDS-PAGE analysis

14. Run gel

15. Stain with coomasie blue and identify for bands of 15-20 kDa (expected 17 kDa band) for lysozyme (cleaved) against a lysozyme standard on one lane.

[0098] Example 2: Antimicrobial activity of Cry-antimicrobial fusion protein

[0099] We investigated whether the fusion protein retained any of the antimicrobial activity expected to be provided by lysozyme, by performing a straightforward semiquantitative plate disc assays using growth inhibition of Micrococcus luteus. The exemplary Cry3Aa-RecAintein- LyzC fusion construct detailed above was used in this assay.

[00100] Upon purification of the protein fusion crystals from the Bt growth media, the crystals were treated with 40mM Bis-Tris, pH 6.2, in IX Phosphate Buffered Saline (400μί for every 100 μL· of crystals). The crystals are incubated at room temperature, or at 4°C overnight, with gentle shaking. The samples are centrifuged and the supernatant is collected and put into a fresh tube. The protein concentration was measured and was adjusted to a concentration of 0.5mg/mL to allow for normalization.

[00101] Filter paper discs (0.2cm each) were autoclaved. Following sterilization, aliquots (100 μΐ,) of the supernatant protein solution were applied to each disc, one disk per sample or control. Filter discs were prepared with the following samples:

[00102] The samples were air-dried on the filter paper discs.

[00103] BHIG (Beef Heart Infusion -Glucose) media was prepared and plated for the growth of the target microbe M. luteus. M. luteus cells (100 μΐ, of lxl 0⁷ cells/mL) were spread on each BHIG plate. One of the prepared filter paper discs were placed at the center of each culture plate, and the plates were incubated at 37°C overnight. Lysozyme activity is measured by the diameter of clearance (translucent area) around the filter paper disc and compared to the positive control (purified lysozyme)

[00104] Representative results are depicted in Figure 4. The plate containing the soluble Cry3Aa-lysozyme fusion protein exhibited an observable zone of inhibition (Figure 4C) relative to the control plates (Figure 4A, B, and D).

[00105] The ability of Cry3Aa-lysozyme fusion proteins to be able to be produced in bacterial cells and form crystals in vivo that retain antimicrobial activity is a significant discovery as it provides a platform by which any host of antimicrobial proteins can be generated on large scale and purified using low-cost and straightforward techniques.

[00106] This data indicates that the fusion proteins disclosed herein can provide a useful platform for topical, mucousal, or oral delivery of antimicrobial proteins based on regularly shaped micrometer-sized protein crystals produced within the bacterium Bacillus thuringiensis and may encapsulate the antimicrobial proteins within a protective crystalline framework. In this regard, some desirable features of these biologically-generated Cry protein crystals include (1) relatively uniform size and degree of purity, and (2) their stability under standard physiological conditions.

[00107] As discussed above, various embodiments incorporate a target antimicrobialprotein into the Cry crystal platform by fusing the gene of the target protein or enzyme to either the 5'- or 3'-end of a cry gene, with an optional linker such as an intein or sortase domain. In the exemplary embodiments described above, the fusion cassette may be cloned into an expression vector, such as the pHT315 E. coli-B. thuringiensis shuttle vector. This vector can be transfected into a bacterium and used to produce a Cry-antimicrobial fusion protein crystals within the live bacterium (e.g., B. thuringiensis or E. coli). Notably, and according to the protocol described above, because B. thuringiensis autolyses following sporulation and given that the crystals have a distinct density, the crystals can be readily purified by centrifugation. The direct synthesis of these crystals in bacteria combined with their ease of purification makes the production of these Cry-antimicrobial fusion protein crystals an economical alternative to other methods of antimicrobial protein production. [00108] Our work that demonstrates the ability of Cry crystals to form encapsulated proteins comprising lysozyme (this work) and speaks to its ability to encapsulate similar enzymes in general and allow for their simple and cost-effective production in bacteria..

Claims

CLAIMS We Claim:

1. A fusion protein, comprising a Cry protein, or a crystal-forming fragment thereof, fused to an antimicrobial polypeptide.

2. The fusion protein of claim 1 , wherein the Cry protein is selected from the group

consisting of Cryl Aa, CrylAb, Cry2Aa, Cry3Aa, Cry4Aa, Cry4Ba, Cryl 1 Aa, Cryl IBa, and Cryl9Aa, or a homolog or a crystal forming fragment thereof.

3. The fusion protein of claim 2, wherein the Cry protein is selected from Cry3Aa and

CrylAb.

4. The fusion protein of claim 1 , wherein the antimicrobial polypeptide comprises lysozyme.

5. The fusion protein of claim 4, wherein the lysozyme comprises a sequence selected from the group SEQ ID NO:36 and SEQ ID NO:38.

6. The fusion protein according to any of claims 1 or 4, further comprising a linker group between the Cry protein and the antimicrobial protein.

7. The fusion protein according to claim 6, wherein the linker group comprises a protein domain, peptide, or amino acid having a cleavable site.

8. The fusion protein according to claim 6, wherein the linker group comprises a self- cleaving domain.

9. The fusion protein according to claim 8, wherein the self-cleaving domain comprises an intein.

10. The fusion protein according to claim 7, wherein the cleavable site comprises a sortase domain.

11. A protein crystal comprising a plurality of fusion proteins, wherein the fusion proteins comprise a Cry protein, or a crystal-forming fragment thereof, fused to an antimicrobial polypeptide.

12. The protein crystal of claim 1 1 , wherein the Cry protein is selected from the group

consisting of Cryl Aa, CrylAb Cry2Aa, Cry3Aa, Cry4Aa, Cry4Ba, Cryl 1 Aa, Cryl IBa, and Cryl9Aa, or a homolog or a crystal forming fragment thereof.

13. The protein crystal of claim 12, wherein the Cry protein is selected from the group

consisting of CrylAb and Cry3Aa.

14. The protein crystal of claim 1 1, wherein the antimicrobial polypeptide comprises lysozyme.

15. The fusion protein of claim 14, wherein the lysozyme comprises a sequence selected from the group SEQ ID NO:36 and SEQ ID NO:38.

16. The protein crystal of any of claims 11-15, wherein the protein crystal is isolated or

purified.

17. The protein crystal of any of claims 11-15, wherein the fusion protein comprises a linker group between the Cry protein and the antimicrobial protein.

18. The fusion protein according to claim 17, wherein the linker group comprises a protein domain, peptide, or amino acid having a cleavable site.

19. The fusion protein according to claim 17, wherein the linker group comprises a self- cleaving domain.

20. The fusion protein according to claim 19, wherein the self-cleaving domain comprises an intein.

21. The fusion protein according to claim 18, wherein the cleavable site comprises a sortase domain.

22. A cultured bacterial cell comprising a protein crystal, wherein the protein crystal

comprises a plurality of a fusion polypeptide comprising a Cry protein, or a crystal- forming fragment thereof, fused to an antimicrobial polypeptide.

23. A composition comprising the fusion polypeptide according to any of claims 1-8, or the protein crystal of any of claims 11-15, and a carrier, excipient, diluent, adjuvant, or vehicle.

24. The composition according to claim 23, wherein the carrier, diluent, adjuvant, or vehicle comprises a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, or vehicle.

25. An isolated nucleic acid molecule having a sequence that encodes the fusion polypeptide according to any of claims 1-10.

26. An expression vector comprising the nucleic acid of claim 25.

27. A bacterial endospore comprising a nucleic acid molecule having a sequence that encodes a fusion polypeptide according to any of claims 1-10.

28. A recombinant bacterial cell comprising the nucleic acid molecule of claim 25.

29. A recombinant bacterial cell comprising the expression vector of claim 26.

30. A method for producing a fusion protein comprising a Cry protein, or a crystal-forming fragment thereof, fused to an antimicrobial protein, wherein the method comprises growing a recombinant bacterial cell in culture under conditions that allow for

production of the fusion protein; and

isolating the fusion protein from the recombinant bacterial cell,

wherein the recombinant bacterial cell comprises the nucleic acid molecule of claim 25, or the expression vector of claim 26.

31. The method of claim 30, wherein the fusion protein comprises a protein crystal

comprising a plurality of fusion proteins that comprise a Cry protein, or a crystal-forming fragment thereof, fused to an antimicrobial polypeptide.

32. The method of claim 31 , wherein the isolating comprises centrifuging the protein crystal using a density gradient or affinity method; and isolating the protein crystal.

33. The method of claim 30, wherein the growing of the recombinant bacterial cell is

performed until a spore/crystal mixture is released from the bacterium upon autolysis.

34. The method of claim 33 comprising centrifuging the spore/crystal mixture using a density gradient or affinity method; and isolating the protein crystal.

35. The method of claim 30, wherein the bacterial cell is selected from B. thuringiensis, B. subtilis, or E. coli.

36. A method for producing an antimicrobial protein comprising:

growing a recombinant bacterial cell comprising the nucleic acid molecule of claim 25, or the expression vector of claim 26 in culture and under conditions that allow for production of a fusion protein encoded by the nucleic acid molecule or the expression vector, wherein the fusion protein comprises a cleavable linker;

isolating the fusion protein from the recombinant bacterial cell;

reacting the isolated fusion protein under conditions that allow for cleavage of the

linker; and

isolating the antimicrobial protein.

37. The method of claim 36, wherein the fusion protein comprises a fusion protein crystal.

38. The method of claim 37, wherein the isolating of the fusion protein crystal comprises centrifuging the fusion protein crystal using a density gradient or affinity method.

39. The method of claim 37, wherein the growing of the recombinant bacterial cell is performed until a spore/crystal mixture is released from the bacterium upon autolysis.

40. The method of claim 39 comprising centrifuging the spore/crystal mixture using a density gradient or affinity method.

41. The method of claim 36, wherein the bacterial cell is selected from B. thuringiensis, B. subtilis, or E. coli.

42. The method of any of claims 36-41 , wherein the antimicrobial protein comprises

lysozyme.