Compositions and methods for industrial scale production and recovery of lectins

Abstract

Certain aspects of the present disclosure relate to industrial scale production and recovery of lectin proteins. Certain aspects relate to recombinant gram-positive bacterial cells that produce heterologous lectin proteins. Certain other aspects relate to compositions and methods for recovering and / or purifying one or more lectins and lectin formulations with improved purity thereof.

Description

Technical Field

[0001] The present disclosure generally relates to the fields of microbial cells, molecular biology, fermentation, protein production, protein recovery, protein purification, protein preparation, etc. Certain aspects of the present disclosure relate to industrial-scale production and recovery of lectin proteins, recombinant Gram-positive bacterial cells producing heterologous lectins, and compositions and methods for recovering and / or purifying one or more lectins, lectin preparations with improved purity, etc.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 323,256, filed on March 24, 2022, which is hereby incorporated by reference in its entirety.

[0004] References to sequence listings

[0005] The electronically submitted content of the text file sequence listing named "NB41857-WO-PCT_SequenceListing.txt" was created on March 21, 2023 and is 41KB in size, which is hereby incorporated by reference in its entirety. Background Art

[0006] Lectins are generally defined as carbohydrate-binding proteins that can recognize and bind simple or complex carbohydrates in a reversible and highly specific manner while not showing catalytic activity (Lagarda-Diaz et al., 2017). Lectin proteins were originally named hemagglutinins due to their ability to agglutinate red blood cells (and other cells). Recently, lectins such as the red algae (Griffithsia sp.) griffithsin (GRFT) protein, the red algae (Kappaphycus alvarezii) KAA-2 protein, the concanavalin A (ConA) protein from jack-bean, the jackfruit lectin protein from A. heterophyllus, the cyanobacterial antiviral protein-N (CV-N) protein from N. ellipsosporum, etc. have been evaluated for their antiviral activity (Whitley et al., 2013).

[0007] For example, PCT publications WO 2005 / 118627 and WO 2007 / 064844 describe methods for isolating natural griffithsin (GRFT) lectin from red algae (Trichoderma species), cloning its wild-type (grft) gene, generating its recombinant polynucleotide, fermenting and producing it in Escherichia coli (E. coli) host cells, then isolating recombinant His-tagged GRFT protein from the E. coli host and characterizing its antiviral activity. PCT publication WO 2010 / 01424 generally describes methods for inhibiting hepatitis C virus infection in a host, which methods include administering to the host an effective amount of a glycosylation-resistant GRFT (variant) protein (or a polypeptide conjugate thereof) in combination with another antiviral protein. For example, as described in this disclosure, the inventors noted that the antiviral protein combination of scytovirin (SVN) and griffithsin (GRFT) has (nanomolar) activity against hepatitis C virus (HCV).

[0008] U.S. Patent Publication No. US20110263485 describes methods for inhibiting human immunodeficiency virus (HIV) infection in a host, which methods include administering to the host an effective amount of gp120Griffithsin and a peptide selected from gp41 binding protein, CCR5 binding protein, gp120 binding protein, or another griffithsin, which are potent inhibitors of HIV infection. PCT Publication No. WO 2016 / 130628 discloses variant griffithsin proteins with mutations that change the isoelectric point of GRFT protein, which reportedly change their solubility in various pH ranges, thereby allowing improved product release. PCT Publication No. WO 2019 / 108656 generally describes a microbicidal composition comprising an endosperm extract and an anti-HIV lectin, an anti-HIV antibody, or an antigen-binding antibody fragment thereof.

[0009] Recombinant production of GRFT in tobacco plants (Nicotiana benthamiana) has been described by O'Keefe et al. (2009), where GRFT accumulated to levels of about 1 gram of recombinant GRFT per kilogram of Nicotiana benthamiana leaf material when expressed via an infectious tobacco mosaic virus (TMV)-based vector. For example, as contemplated in the O'Keefe et al. disclosure, while it is hoped that biologics such as griffithsin will act as HIV prophylactics, their practical use as topical microbicides is hampered by high production costs, with any manufacturing system that relies on growth under sterile conditions unlikely to be price competitive with male condoms, which are necessary if the product is to be used by persons at risk for sexual transmission of HIV.

[0010] Hirayama et al. (2016) have described the elucidated primary structure of the KAA-2 lectin using peptide mapping and complementary DNA (cDNA) cloning, and prepared its active recombinant using an E. coli expression system. Gengenbach et al. (2019) described the transient expression of a mistletoe lectin named "viscumin" (Viscum album) in intact Nicotiana benthamiana plants, and the purification of the recombinant mistletoe lectin from crude plant extracts by affinity chromatography, where the performance and economics of the tobacco plant-based method were compared with the corresponding method based on E. coli expression. As summarized by Gengenbach et al., the E. coli method has low recovery, requires a lot of dilution and is complicated, while the plant-based method includes only half the number of steps. According to a direct cost comparison between the two methods conducted in the study of Gengenbach et al., the plant expression system is 50% cheaper than heterologous expression in the natural host mistletoe or E. coli host cells.

[0011] Petrova et al. (2016) described a probiotic Lactobacillus rhamnosus strain (GR-1) that was documented to survive and interfere with urogenital pathogens when implanted on vaginal epithelium. As described in this publication, a genomic region encoding a protein with homology to lectin-like proteins (i.e., the llp1 gene encoding lectin-like protein 1 (Llp1)) was identified, wherein phenotypic analysis of a knockout mutant strain (GR-1_Δllp1) of the Lactobacillus rhamnosus (GR-1) parent strain revealed a two-fold reduction in adhesion to vaginal and ectocervical epithelial cell lines compared to the wild type.

[0012] Recently, Petrova et al. (2018) described probiotic Lactobacillus rhamnosus strains expressing (HIV inhibitory lectin) actinohivin (AH) or griffithsin (GRFT) for in situ delivery. As generally summarized in this disclosure, Lactobacillus rhamnosus strains are unable to produce intracellular or extracellular AH, and it is assumed that the observed results may be that AH is toxic during export outside the cell wall of Lactobacillus rhamnosus strains, which may be through binding to essential glycosylated cell wall molecules, such as peptidoglycans of the Sec pathway, exopolysaccharides or glycosylated proteins. Petrova et al. (2018) further described Lactobacillus rhamnosus strains constructed for extracellular expression of GRFT, where the results obtained "suggest that Lactobacillus rhamnosus strains can synthesize GRFT, but do not secrete it outside the cell under the conditions tested." For example, as described in this publication, for the recombinant strains CMPG10731, CMPG10734, CMPG10767, and CMPG10768, a band corresponding to GRFT was detected in the cell wall fraction, indicating that the recombinant protein may be trapped in the cell wall.

[0013] Based on the foregoing, it is apparent that there remains a continuing and unmet need in the art for improved host organisms capable of producing heterologous lectins for various antimicrobial uses, and for novel methods and compositions capable of cost-effectively producing, recovering and / or purifying lectins on an industrial scale. Summary of the invention

[0014] As described and exemplified below, the present disclosure provides, inter alia, novel recombinant (modified) Gram-positive bacterial cells (strains) expressing heterologous lectin proteins, polynucleotides (e.g., vectors, expression cassettes) comprising nucleic acids encoding heterologous lectins, methods and compositions for producing heterologous lectins in recombinant Gram-positive host cells, methods and compositions for recovering and / or purifying lectins, and the like.

[0015] Therefore, some one or more embodiments or aspects of the present disclosure relate to methods for expressing / producing heterologous lectins in Gram-positive bacterial (host) cells. In certain embodiments, the present disclosure provides methods for producing heterologous lectins in Gram-positive bacterial cells, which are performed by introducing an expression cassette encoding the lectin into the cell, wherein the cassette comprises an upstream (5') promoter sequence operably linked to a downstream (3') open reading frame (ORF) encoding the lectin, and fermenting the cell under suitable conditions for producing the lectin. In certain embodiments of these methods, at least two cassettes encoding the lectin are introduced into the cell, wherein the cassettes comprise an upstream promoter sequence operably linked to a downstream ORF encoding the lectin. In certain other embodiments of these methods, the introduced cassettes are integrated into the genome of the cell. In certain other embodiments of these methods, at least two introduced cassettes are integrated into the genome of the cell. In other one or more embodiments of these methods, at least two introduced cassettes comprise the same ORF encoding the same lectin, or comprise different open reading frames (ORFs) encoding different lectins.

[0016] Certain other one or more embodiments or aspects of the present disclosure relate to methods for producing a heterologous lectin in a Gram-positive bacterial cell, the methods comprising introducing an expression cassette encoding the lectin into the cell, wherein the cassette comprises an upstream (5') promoter sequence operably linked to a downstream nucleic acid encoding a signal (secretion) sequence, the signal (secretion) sequence being operably linked to a downstream (3') open reading frame (ORF) encoding the lectin, and fermenting the cell under suitable conditions for producing the lectin. In certain embodiments of these methods, the introduced cassette is integrated into the genome of the cell. In yet other embodiments of these methods, the cell comprises at least two introduced cassettes encoding the lectin, wherein the cassettes comprise an upstream promoter sequence operably linked to a downstream nucleic acid encoding a signal sequence, the signal sequence being operably linked to a downstream ORF encoding the lectin. In other one or more embodiments of these methods, at least two introduced cassettes are integrated into the genome of the cell. In certain other embodiments of these methods, at least two introduced cassettes comprise the same ORF encoding the same lectin, or comprise different ORFs encoding different lectins.

[0017] Certain other one or more embodiments or aspects of the present disclosure relate to methods for producing a heterologous lectin in a Gram-positive bacterial cell, the methods comprising introducing a first and a second expression cassette encoding the lectin into the cell, wherein the first cassette comprises an upstream promoter sequence operably linked to a downstream open reading frame (ORF) encoding the lectin, and the second cassette comprises an upstream promoter sequence operably linked to a downstream nucleic acid encoding a signal (secretion) sequence, the signal (secretion) sequence being operably linked to the downstream ORF encoding the lectin, and fermenting the cell under suitable conditions for producing the lectin. In certain embodiments of these methods, the introduced first cassette or the introduced second cassette is integrated into the genome of the cell. In certain other embodiments of these methods, the introduced first and second cassettes are integrated into the genome of the cell. In yet other embodiments of these methods, at least two introduced cassettes comprise the same ORF encoding the same lectin, or comprise different ORFs encoding different lectins.

[0018] In any of the preceding embodiments of the methods, the lectin is expressed intracellularly, and / or the lectin is expressed and secreted extracellularly.

[0019] In yet other embodiments of these methods, the ORF encoding is derived from a natural lectin or variant lectin of a cyanobacterial cell, a eukaryotic cell or a bacterial cell. In certain other embodiments of these methods, the natural or variant lectin is derived from a eukaryotic cell selected from the group consisting of: a plant cell, a fungal cell, an insect cell and an animal cell.

[0020] In one or more other embodiments of these methods, the ORF encodes a lectin selected from the group consisting of: a natural griffithsin (GRFT) lectin (or a variant GRFT lectin derived therefrom), a natural scytovirin (SVN) lectin (or a variant SVN lectin derived therefrom), a natural cyanobacterial antiviral protein-N (CVN) lectin (or a variant CVN lectin derived therefrom), a natural Kappaphycus longiflorus KAA-1 lectin (or a variant KAA-1 lectin derived therefrom), a natural Kappaphycus longiflorus KAA-2 lectin (or a variant KAA-2 lectin derived therefrom), a natural Microcystis viridis (MVL) lectin (or a variant MVL lectin derived therefrom), a natural Boodlea coacta lectin (BCA) lectin (or a variant BCA lectin derived therefrom), a natural Artocarpus heterophyllus (jackfruit lectin) lectin (or variant jackfruit lectin derived therefrom), natural Musa acuminata (banana) lectin (or variant banana lectin derived therefrom), natural Aaptos papilleta (sponge) lectin (or variant sponge lectin derived therefrom), natural Abrusprecatorius (abrus bean) lectin (or variant abrac bean lectin derived therefrom), natural Aegapodium podagraria (abrus podagraria) (abrus podagraria) lectin (or variant abrac podagraria lectin derived therefrom), Agaricus bisporus (common mushroom) lectin (or variant common mushroom lectin derived therefrom), natural Albizzia julibrissin (mimosa seed) lectin (or variant mimosa seed lectin derived therefrom), natural Allomyrina dichotoma) (Japanese beetle) lectin (or variant Japanese beetle lectin derived therefrom), natural Aloe arborescens (aloe plant) lectin (or variant aloe plant lectin derived therefrom), natural Amphicarpaea bracteata (isomorphic bean) lectin (or variant isomorphic bean lectin derived therefrom), natural Anguilla (eel) lectin (or variant eel lectin derived therefrom), natural Aplysia depilans (mollusk) lectin (or variant mollusk lectin derived therefrom), natural Arachis hypogaea (peanut) lectin (or variant peanut lectin derived therefrom), natural Bauhinia (Bauhiniapurpurea) (Camel's hoof tree) lectin (or variant Camel's hoof tree lectin derived therefrom), natural Bryonia diocia (White Bryonia) lectin (or variant White Bryonia lectin derived therefrom), natural Caragana Arborescens (Siberian Pea Tree) lectin (or variant Siberian Pea Tree lectin derived therefrom), natural Carcinoscorpius rotundacauda (Horseshoe Crab) lectin (or variant Horseshoe Crab lectin derived therefrom), natural Microcystis aeruginosa (Microcystis cerevisiae) lectin (or variant aeruginosa) (cyanobacteria) microvirin (MVN) lectin (or variant MVN lectin derived therefrom), natural Eucheumaserra (red algae) ESA-2 lectin (or variant ESA-2 lectin derived therefrom), natural Musa acuminate (banana) BanLec lectin (or variant BanLec lectin derived therefrom), natural Aspidistra elatior AEL lectin (or variant AEL lectin derived therefrom), natural Chaetopterus variopedatus (marine worm) CVL lectin (or variant CVL lectin derived therefrom), Vicia faba (broad bean) lectin (or variant broad bean lectin derived therefrom), natural Lensculinaris (lentil) (or variant Lensculinaris lectin derived therefrom), natural Pisum sativum (Pisum gracilis) (Lentil) (or variant Lensculinaris lectin derived therefrom), natural sativum) (pea) lectin (or variant pea lectin derived therefrom), natural jacaranda lectin-like lectin (or variant jacaranda lectin-like lectin derived therefrom), including but not limited to the jacaranda lectin-like lectin listed in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 39, natural CVN-like lectin (or variant CVN-like lectin derived therefrom), including but not limited to the CVN-like lectin listed in SEQ ID NO: 10 and SEQ ID NO: 11, natural OAA-like lectin (or variant OAA-like lectin derived therefrom), including but not limited to the OAA-like lectin listed in one of SEQ ID NO: 14-SEQ ID NO: 23 or SEQ ID NO: 29-SEQ ID NO: 38, natural galectin-1-like lectin (or variant galectin-1-like lectin derived therefrom), including but not limited to SEQ ID NO: 25-SEQ ID NO: 39.Galectin-1-like lectins listed in NO:28, and natural ricin-like lectins (or variant ricin-like lectins derived therefrom), including but not limited to the ricin-like lectin listed in SEQ ID NO:24.

[0021] In certain other embodiments of these methods, the Gram-positive bacterial cell is selected from members of the phylum Firmicutes.

[0022] Certain other one or more embodiments or aspects of the present disclosure relate to methods for recovering (and optionally purifying) lectins produced by Gram-positive bacterial cells. Thus, certain one or more embodiments of the present disclosure provide methods for recovering heterologous lectins expressed and retained within cells and / or provide methods for recovering heterologous lectins expressed and secreted outside cells.

[0023] Thus, certain embodiments relate to methods for recovering an intracellular lectin, the methods comprising fermenting recombinant cells expressing an intracellular lectin and lysing the cells at the end of the fermentation to obtain a lysed cell broth, heat treating the lysed broth at a pH between 1.5 and 8.5, then cooling the broth and harvesting the cooled broth, subjecting the harvested cooled broth to a clarification process, and subjecting the clarified broth to a concentration process, wherein the lectin is recovered in the clarified concentrated broth. In certain aspects of these methods, the pH between 1.5 and 8.5 is about pH 1.5, about pH 2.0, about pH 2.5, about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.5 to about pH 9.0.

[0024] Accordingly, certain other embodiments relate to methods for recovering secreted lectins, the methods comprising fermenting recombinant cells expressing and secreting a lectin and harvesting the broth, subjecting the harvested broth to a clarification process, and subjecting the clarified broth to a concentration process, wherein the lectin is recovered in the clarified, concentrated broth.

[0025] Certain other embodiments relate to methods for improving the purity of a lectin concentrate, the methods comprising obtaining a lectin concentrate recovered according to any of the foregoing methods, (a) adjusting the pH of the concentrate to between 1.5 and 8.5, (b) incubating the concentrate of step (a) at a temperature between about 5° C. and 55° C. for a sufficient amount of time, and (c) centrifuging the concentrate and collecting the supernatant, or filtering the concentrate and collecting the filtrate, wherein the collected supernatant or the collected filtrate comprises substantially purified lectin relative to the purity of the lectin concentrate obtained in step (a). In certain embodiments or aspects of step (a), the pH between 1.5 and 8.5 is about pH 1.5, about pH 2.0, about pH 2.5, about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.5 to about pH 9.0.

[0026] Certain other embodiments provide methods for increasing the purity of a lectin concentrate, the methods comprising obtaining a lectin concentrate recovered according to any of the foregoing methods, (a) adjusting the pH of the concentrate to about pH 2, incubating the concentrate at pH 2 at a temperature between about 55°C and 65°C for a sufficient amount of time, and (b) centrifuging the incubated concentrate and collecting the supernatant, or filtering the concentrate and collecting the filtrate, wherein the collected supernatant or the collected filtrate comprises substantially purified lectin relative to the purity of the lectin concentrate obtained in step (a). In certain embodiments or aspects of step (a), the temperature between about 55°C-65°C is about 54.5°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C to about 65.5°C.

[0027] In one or more other embodiments, the present disclosure provides methods for crystallizing a lectin, the methods comprising (a) obtaining a lectin concentrate recovered according to one of the foregoing methods, and (b) adding a salt or a mixture of salts to the concentrate, the salt or mixture of salts comprising sodium, calcium, ammonium, sulfate or chloride ions, adjusting the pH of the salted concentrate to about pH 2 to 5, and mixing and incubating the concentrate at about 5° C. to 50° C. for an appropriate amount of time, wherein after mixing and incubating for an appropriate amount of time in step (b), the lectin crystallizes from the concentrate. In certain aspects, the pH of the salted concentrate is adjusted to about pH 2, about pH 2.5, about pH 3, about pH 3.5, about pH 4, about pH 4.5, or about pH 5.

[0028] In certain other one or more embodiments, the present disclosure provides methods for crystallizing a lectin, the methods comprising (a) obtaining a lectin concentrate recovered according to one of the foregoing methods, and (b) adding about 0.5% to about 10% of a salt or a mixture of salts to the concentrate, the salt or mixture of salts comprising sodium, calcium, ammonium, sulfate or chloride ions, adjusting the pH of the salted concentrate to about pH 2 to 5, and mixing and incubating the concentrate at about 5° C. to 50° C. for an appropriate amount of time, wherein after mixing and incubating for an appropriate amount of time in step (b), the lectin crystallizes from the concentrate. In certain related embodiments of the method, the pH is about 2.5 to about 3.5, the salt is about 1% to about 5% sodium sulfate, and the incubation temperature is about 5° C. to about 25° C. In other embodiments of the method, the pH is about 2.8 to about 3.2, the salt is about 1.8% to about 2.5% sodium sulfate, and the incubation temperature is about 15° C. to about 25° C.

[0029] One or more other embodiments of the present disclosure are directed to methods for improving the purity of a lectin (protein) preparation, the methods comprising (a) obtaining a lectin crystal slurry according to one of the aforementioned methods, optionally washing the crystal slurry with water and mixing, and centrifuging the slurry for a sufficient amount of time, decanting and collecting the supernatant after centrifugation, adding an appropriate pH buffer to maintain a pH of about 4.8 to about 8.6 while mixing at room temperature for a sufficient amount of time, and (b) filtering the supernatant and collecting the filtered supernatant, wherein the collected supernatant comprises a high purity lectin preparation.

[0030] In certain other one or more embodiments, the present disclosure provides methods for recovering (and optionally purifying) lectins produced by other recombinant cells expressing native or heterologous lectins, such as recombinant tobacco cells / plants expressing heterologous lectins, recombinant E. coli cells expressing heterologous lectins, etc. Thus, certain embodiments or aspects of the present disclosure provide methods for recovering secreted lectins from fermentation broths in which recombinant cells have been fermented to express lectins. In certain embodiments, such methods include (a) obtaining and harvesting a whole fermentation broth containing secreted lectins, (b) subjecting the harvested broth to a clarification process, and (c) subjecting the clarified broth to a concentration process, wherein the lectin is recovered in the clarified, concentrated broth. Certain other one or more embodiments of the present disclosure relate to methods for recovering an intracellular lectin, which methods generally include (a) obtaining and lysing a whole fermentation comprising recombinant cells that have been fermented to express the lectin, heat treating the lysed broth at a pH between about 1.5 and about 8.5, then cooling the broth and harvesting the cooled broth, (b) subjecting the harvested cooled broth to a clarification process, and (c) subjecting the clarified broth to a concentration process, wherein the lectin is recovered in the clarified, concentrated broth. In a related embodiment, the present disclosure provides methods for improving the purity of a lectin concentrate, the methods comprising (a) obtaining a lectin concentrate recovered according to the foregoing method of this paragraph, adjusting the pH of the concentrate to between about 1.5 and about 8.5, (b) incubating the concentrate of step (a) at a temperature between about 5° C. and about 55° C. for a sufficient amount of time, and (c) centrifuging the concentrate and collecting the supernatant, or filtering the concentrate and collecting the filtrate, wherein the collected supernatant or the collected filtrate comprises substantially purified lectin relative to the purity of the lectin concentrate obtained in step (a). In certain other related embodiments, the present disclosure provides methods for improving the purity of a lectin concentrate, the methods comprising (a) obtaining a lectin concentrate recovered according to the foregoing method of this paragraph, adjusting the pH of the concentrate to about pH 2, incubating the concentrate at about pH 2 at a temperature between about 55° C. and about 65° C. for a sufficient amount of time, and (b) filtering the incubated concentrate and collecting the supernatant, or filtering the concentrate and collecting the filtrate, wherein the collected supernatant or the collected filtrate comprises substantially purified lectin relative to the purity of the lectin concentrate obtained in step (a).

[0031] In one or more other related embodiments, the present disclosure provides methods for crystallizing a lectin, the methods comprising (a) obtaining a lectin concentrate recovered according to the method described above, (b) adding a salt or a mixture of salts to the concentrate, the salt or mixture of salts comprising sodium, calcium, ammonium, sulfate or chloride ions, adjusting the pH of the salted concentrate to about 2 to about 5, and mixing and incubating the concentrate at between about 5° C. and about 50° C. for an appropriate amount of time, wherein after mixing and incubating for an appropriate amount of time in step (b), the lectin crystallizes from the concentrate.

[0032] In certain other one or more related embodiments, the present disclosure provides methods for crystallizing lectins, the methods comprising (a) obtaining a lectin concentrate recovered according to the methods described above, (b) adding about 0.5% to about 10% of a salt or a mixture of salts to the concentrate, the salt or mixture of salts comprising sodium, calcium, ammonium, sulfate or chloride ions, adjusting the pH of the salted concentrate to a pH of about 2 to about 5, and mixing and incubating the concentrate at about 5° C. to about 50° C. for an appropriate amount of time, wherein after mixing and incubating for an appropriate amount of time in step (b), the lectin crystallizes from the concentrate. In certain aspects, the pH in the foregoing method is about 2.5 to about 3.5, the salt is about 1% to about 5% sodium sulfate, and the incubation temperature is about 5° C. to about 25° C. In other embodiments or aspects, the pH is about 2.8 to about 3.2, the salt is about 1.8% to about 2.5% sodium sulfate, and the incubation temperature is about 15° C. to about 25° C.

[0033] Therefore, certain other one or more embodiments provide methods for improving the purity of a lectin preparation, the methods comprising (a) obtaining a lectin crystal slurry according to the method described above, optionally washing the crystal slurry with water and mixing, and centrifuging the slurry for a sufficient amount of time, decanting and collecting the supernatant after centrifugation, adding an appropriate pH buffer to maintain a pH of about 4.8 to about 8.6, while mixing at room temperature for a sufficient amount of time, and (b) filtering the supernatant and collecting the filtered supernatant, wherein the collected supernatant contains a high purity lectin preparation.

[0034] Accordingly, certain other embodiments relate to highly pure lectin preparations produced and obtained according to one or more methods of the present disclosure.

[0035] In certain embodiments, the present disclosure provides methods for producing and recovering a high-purity griffithsin (GRFT) protein preparation, which methods include (a) constructing a recombinant Gram-positive bacterial cell expressing a polynucleotide encoding the GRFT protein, (b) fermenting the cell under conditions suitable for producing the GRFT protein, lysing the cell at the end of the fermentation to obtain a lysed cell broth, and treating the lysed broth by maintaining the broth at a pH of about 4.8 to about 5.2 and a temperature of about 50° C. to about 80° C. for about 1 to about 4 hours, (c) clarifying the broth of step (b) by a filtration or microfiltration process, and concentrating the clarified broth by an ultrafiltration process, (d) subjecting the concentrated broth of step (c) to a crystallization process, the crystallization process comprising adding about 2% sodium sulfate to the concentrate, adjusting the pH to about 3 and incubating at about 22° C. for a sufficient amount of time, (e) centrifuging the incubated concentrate of step (d), decanting the supernatant to harvest GRFT crystals, and precipitating the GRFT crystals at a pH of about 1.5 to about 2.0. 4.5 to about 5.5 in about 100 mM sodium acetate buffer to obtain an aqueous GRFT protein preparation, and centrifuging the GRFT preparation to remove any insoluble impurities therein and collecting the supernatant, or filtering the GRFT preparation to remove any insoluble impurities therein and collecting the filtrate, wherein the supernatant collected in step (e) or the filtrate collected in step (e) contains a high-purity GRFT protein preparation.

[0036] In yet other embodiments, the present disclosure relates to methods for recovering a high-purity GRFT preparation, which methods include (a) obtaining a whole fermentation broth comprising recombinant cells expressing a GRFT protein, lysing the cells in the cell broth, and treating the lysed broth by maintaining the broth at a pH of about 4.8 to 5.2 and a temperature of about 50° C. to 80° C. for about 1 to 4 hours, (b) clarifying the broth of step (b) by a filtration or microfiltration process, and concentrating the clarified broth by an ultrafiltration process, (c) subjecting the concentrated broth of step (b) to a crystallization process, the crystallization process comprising adding about 2% sodium sulfate to the concentrate, adjusting the pH to about 3 and incubating at about 22° C. for a sufficient amount of time, and (d) centrifuging the incubated concentrate of step (c), decanting the supernatant to harvest GRFT crystals, and concentrating the GRFT crystals at a pH of about 1 to 4 hours. 4.5 to about 5.5 in about 100 mM sodium acetate buffer to obtain an aqueous GRFT protein preparation, and centrifuging the GRFT preparation to remove any insoluble impurities therein and collecting the supernatant, or filtering the GRFT preparation to remove any insoluble impurities therein and collecting the filtrate, wherein the supernatant collected in step (d) or the filtrate collected in step (d) contains a high-purity GRFT protein preparation.

[0037] In certain embodiments of these methods, the high purity GRFT preparation comprises a native GRFT protein or a variant GRFT protein preparation. In certain other embodiments of these methods, the high purity GRFT preparation is at least 2.0 times higher than the recovered GRFT concentrate, such as by 280 In some other embodiments of these methods, when visualized by SDS-PAGE, the GRFT is the main band in the high-purity GRFT preparation, or the only band of about 12.7 kDa. In one or more other embodiments, when measured / screened for one or more animal red blood cells (red blood cell / erythrocyte), the high-purity GRFT has hemagglutination activity.

[0038] In certain other embodiments of these methods, the recombinant cells of the present disclosure are made deficient in the production of one or more endogenous genes encoding one or more highly expressed background (native) proteins, including but not limited to amylases, xylanases, pullulanases, phytases, pectate lyases, glucanases, mannosidases, lipases, esterases, and the like.

[0039] Therefore, certain other embodiments of the present disclosure relate to one or more recombinant Gram-positive bacterial cells (strains) expressing heterologous lectins. In certain embodiments, the present disclosure relates to a recombinant Gram-positive bacterial cell expressing a polynucleotide encoding a heterologous lectin. In certain embodiments, the recombinant cell comprises an introduced polynucleotide box encoding the lectin, wherein the box comprises an upstream promoter operably connected to a downstream nucleic acid encoding the lectin, or wherein the box comprises an upstream promoter operably connected to a downstream nucleic acid encoding a signal sequence, and the signal sequence is operably connected to a downstream nucleic acid encoding the lectin. In other embodiments, the recombinant cell comprises at least two introduced boxes encoding the lectin, wherein the at least two introduced boxes comprise an upstream promoter operably connected to a downstream nucleic acid encoding the lectin. In yet other embodiments, the recombinant cell comprises at least two introduced boxes encoding the lectin, wherein the introduced boxes comprise an upstream promoter operably connected to a downstream nucleic acid encoding a signal sequence, and the signal sequence is operably connected to a downstream nucleic acid encoding the lectin. In certain other embodiments, the recombinant cell comprises at least two introduced cassettes encoding the lectin, wherein one introduced cassette comprises an upstream promoter operably linked to a downstream nucleic acid encoding the lectin, and a second introduced cassette comprises an upstream promoter operably linked to a downstream nucleic acid encoding a signal sequence operably linked to the downstream nucleic acid encoding the lectin.

[0040] In certain embodiments of these recombinant cells, the lectin is expressed intracellularly. In certain other embodiments of these recombinant cells, the lectin is expressed and secreted extracellularly. In yet other embodiments of these recombinant cells, the lectin is expressed intracellularly and the lectin is expressed and secreted extracellularly.

[0041] In certain other embodiments of the recombinant cells, the cassette is integrated into the genome of the cell. In other embodiments of the recombinant cells, at least one of the at least two introduced cassettes is integrated into the genome of the cell. In still other embodiments of the recombinant cells, at least two introduced cassettes are integrated into the genome of the cell.

[0042] In other embodiments of the recombinant cells, the at least two introduced cassettes encode the same lectin or encode different lectins.

[0043] In other embodiments of the recombinant cells, the Gram-positive bacterial cell is selected from members of the phylum Firmicutes. In other embodiments, the Gram-positive bacterial cell is selected from members of the family Bacillaceae. In certain other embodiments, the Gram-positive bacterial cell is selected from the group consisting of: Alkalibacillus sp. cells, Amphibacillus sp. cells, Anoxybacillus sp. cells, Bacillus sp. cells, Caldalkalibacillus sp. cells, Cerasilbacillus sp. cells, Exiguobacterium sp. cells, Filobacillus sp. cells, Geobacillus sp. cells, Gracilibacillus sp. cells, Halobacillus sp. cells, Halolactibacillus sp. cells. sp. cells, Jeotgalibacillus sp. cells, Lentibacillus sp. cells, Marinibacillus sp. cells, Oceanobacillus sp. cells, Ornithinibacillus sp. cells, Paraliobacillus sp. cells, Paucisalibacillus sp. cells, Pontibacillus sp. cells, Sea Bacillus sp. cells, Saccharococcus sp. cells, Salibacillus sp. cells, Salinibacillus sp. cells, Tenuibacillus sp. cells sp. cells, Thalassobacillus sp. cells, Ureibacillus sp. cells, and Virgibacillus sp. cells.

[0044] In one or more other embodiments of the recombinant cells, the heterologous lectin is derived from a bacterial cell, a eukaryotic cell or a cyanobacterial cell. In another embodiment of the recombinant cells, the heterologous lectin is derived from a eukaryotic cell selected from the group consisting of a plant cell, a fungal cell, an insect cell, and an animal cell. In certain other one or more embodiments of the recombinant cells, the heterologous lectin is selected from the group consisting of a natural griffithsin (GRFT) lectin (or a variant GRFT lectin derived therefrom), a natural scytovirin (SVN) lectin (or a variant SVN lectin derived therefrom), a natural cyanobacterial antiviral protein-N (CVN) lectin (or a variant CVN lectin derived therefrom), a natural Kappaphycus longicornis KAA-1 lectin (or a variant KAA ... scytovirin (SVN) lectin (or a variant -1 lectin), natural Kappaphycus longifolia KAA-2 lectin (or variant KAA-2 lectin derived therefrom), natural Microcystis viridis (MVL) lectin (or variant MVL lectin derived therefrom), natural B. hemispherica lectin (BCA) lectin (or variant BCA lectin derived therefrom), natural Jackfruit (Jacaranda lectin) lectin (or variant jacaranda lectin derived therefrom), natural Banana (Banana) lectin (or variant banana lectin derived therefrom), natural Aaptospapilleta (sponge) lectin (or variant sponge lectin derived therefrom), natural Abrus precatorius (abrus bean) lectin (or variant Abrus bean lectin derived therefrom), natural Cercidiphyllum serrata (Cercidiphyllum serrata) lectin (or variant Cercidiphyllum serrata lectin derived therefrom), Agaricus bisporus (common mushroom) lectin (or variant common mushroom lectin derived therefrom), natural Albizia julibrissin (mimosa tree seed) lectin (or variant mimosa tree seed lectin derived therefrom), natural Rhinoceros beetle (Japanese beetle) lectin (or variant Japanese beetle lectin derived therefrom), natural Wood Aloe vera (aloe plant) lectin (or variant aloe plant lectin derived therefrom), natural euphorbia pulex (isotype bean) lectin (or variant isotype bean lectin derived therefrom), natural anguilla (eel) lectin (or variant eel lectin derived therefrom), natural common sea hare (mollusk) lectin (or variant mollusk lectin derived therefrom), natural arachis (peanut) lectin (or variant peanut lectin derived therefrom), natural Bauhinia oleracea (camel hoof tree) lectin (or variant camel hoof tree lectin derived therefrom), natural bryony radix (white bryony radix) lectin lectin (or variants thereof derived from Bryonia suffruticosa lectin), natural Caragana oleracea (Siberian pea tree) lectin (or variants thereof derived from Siberian pea tree lectin), natural Horseshoe Crab (Horseshoe Crab) lectin (or variants thereof derived from Horseshoe Crab lectin), natural Microcystis aeruginosa (cyanobacteria) microvirin (MVN) lectin (or variants thereof derived from MVN lectin), natural Eucheuma dentatum (red algae) ESA-2 lectin (or variants thereof derived from ESA-2 lectin), natural Banana (Banana) BanLec lectin (or variants thereof derived from The present invention relates to a variant BanLec agglutinin, a variant BanLec agglutinin derived from a natural Aegilops punctatus AEL agglutinin (or a variant AEL agglutinin derived therefrom), a natural Trichoptera (marine worm) CVL agglutinin (or a variant CVL agglutinin derived therefrom), a Vicia faba (broad bean) lectin (or a variant Vicia faba lectin derived therefrom), a natural Lentil (lentil) lectin (or a variant Lentil agglutinin derived therefrom), a natural Pisum sativum (pea) lectin (or a variant Pisum sativum lectin derived therefrom), a natural Artocarpus lectin-like lectin (or a variant Artocarpus lectin-like lectin derived therefrom), including but not limited to SEQ ID NO: 113. SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 39, natural CVN-like lectins (or variant CVN-like lectins derived therefrom), including but not limited to SEQ ID NO: 10 and SEQ ID NO: 11, natural OAA-like lectins (or variant OAA-like lectins derived therefrom), including but not limited to SEQ ID NO: 14-SEQ ID NO: 23 or SEQ ID NO: 24.OAA-like lectins listed in one of SEQ ID NO:29 to SEQ ID NO:38, natural galectin-1-like lectins (or variant galectin-1-like lectins derived therefrom), including but not limited to galectin-1-like lectins listed in SEQ ID NO:25 to SEQ ID NO:28, and natural ricin-like lectins (or variant ricin-like lectins derived therefrom), including but not limited to ricin-like lectins listed in SEQ ID NO:24.

[0045] In certain other embodiments, the recombinant cells of the present disclosure are made deficient in the production of one or more endogenous genes encoding one or more highly expressed background (native) proteins, including but not limited to amylases, xylanases, pullulanases, phytases, pectate lyases, glucanases, mannosidases, lipases, esterases, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The mature amino acid sequences of exemplary lectins GRFT (SEQ ID NO: 1), Q-GRFT (SEQ ID NO: 2) and KAA-2 (SEQ ID NO: 4) are shown. More specifically, the native griffithsin (GRFT) protein comprises 121 amino acid residues, wherein the amino acid "X" at position 31 of the native GRFT is an unknown non-naturally occurring amino acid residue ( Figure 1 A, SEQ ID NO: 1). Variants of the natural griffithsin protein Figure 1 B (Q-GRFT; SEQ ID NO: 2). For example, variant Q-GRFT comprises 121 amino acid residues, wherein in the native GRFT ( Figure 1 A; SEQ ID NO: 1) at position 31 of the non-natural residue X in Q-GRFT ( Figure 1 B; SEQ ID NO: 2) was substituted with alanine (A) at position 31, and the native GRFT ( Figure 1 A; SEQ ID NO: 1) at position 78 of methionine (M) in Q-GRFT ( Figure 1 B; SEQ ID NO: 2) is substituted with glutamine (Q) at position 78. The native KAA-2 lectin protein contains 269 amino acid residues ( Figure 1 C; SEQ ID NO:4).

[0047] Figure 2 The mature amino acid sequence of an exemplary japanese lectin-like lectin is shown ( Figure 2 A-2E).

[0048] Figure 3 The mature amino acid sequence of an exemplary CVN-like lectin is presented ( Figure 3 A-3B).

[0049] Figure 4 The mature amino acid sequence of an exemplary OAA-like lectin is presented ( Figure 4 A-4T).

[0050] Figure 5 The mature amino acid sequences of exemplary ricin-like and galectin-like lectins are presented ( Figure 5 A-5E).

[0051] Figure 6 A schematic diagram of an exemplary lectin polynucleotide expression cassette of the present disclosure is presented. More particularly, Figure 6 As shown, an expression cassette can be constructed for intracellular expression of lectin proteins in Gram-positive (host) cells ( Figure 6 A, Figure 6 B) or extracellular expression / secretion ( Figure 6 C, Figure 6 D) Figure 6 As shown, the promoter sequence (abbreviated as "pro"), signal sequence (abbreviated as "ss"), and optional terminator sequence (abbreviated as "term") of the expression cassette are generally selected to function in the desired Gram-positive host. Figure 6 C and Figure 6 As shown in D, the DNA sequence encoding the mature lectin protein (lectin) is located downstream (3') and operably linked to the nucleic acid sequence (ss) encoding the secretion signal sequence (5'). In order to enhance the expression of the cassette in a specific Gram-positive (host) cell, the DNA sequence encoding the mature lectin protein (lectin) can be codon optimized using techniques and methods known to those skilled in the art.

[0052] Figure 7 Shown is an SDS-PAGE gel of broth supernatants obtained from B. subtilis strains CB447 (secreted Q-GRFT) and CB476 (intracellular Q-GRFT) evaluated in a 10 L bioreactor. More specifically, Figure 7 As presented in FIG. 1 , ten (10) μL of 10-fold diluted samples (lanes 1-6) were evaluated along with See Blue Plus 2 molecular weight standards (labeled, "kDa") and T4 lysozyme protein standards (McLab), followed by staining and retention of the gel using standard molecular biology procedures. Figure 7As shown, lanes 1, 2 and 3 are the broth supernatants from strain CB447 at eighteen (18) hours, twenty-four (24) hours and thirty (30) hours of fermentation, respectively, and lanes 4, 5 and 6 are the broth supernatants from strain CB476 at eighteen (18) hours, twenty-four (24) hours and thirty (30) hours of fermentation, respectively, in which the Q-GRFT protein appears as a single band with a molecular weight of 12.7 kDa.

[0053] Figure 8 The Q-GRFT protein formulation described in Example 11 is presented ( Figure 8 ) of SDS-PAGE gel. Figure 8 , the lanes labeled "MW" are molecular weight ladders, lane 1 is crystal slurry at 47 hours, lane 2 is the non-washed crystal slurry supernatant, lanes 3, 4 and 5 are non-washed crystal precipitates plus buffer, lanes 6, 7 and 8 are non-washed crystal precipitates plus buffer, filtered, lane 9 is 1x washed crystal slurry supernatant, lanes 10, 11 and 12 are washed crystal precipitates plus buffer, lanes 13, 14 and 15 are washed crystal precipitates plus buffer, filtered, as described in Example 11. For example, the buffer in lanes 3, 6, 10, 13 is 100 mM Tris (pH 8.6), the buffer in lanes 4, 7, 11, 14 is 100 mM Bis-Tris (pH 6.5), and the buffer in lanes 5, 8, 12, 15 is 100 mM sodium acetate (pH 5.5).

[0054] Fig. 9 A chromatogram indicating the total protein composition of the purified lectin (Q-GRFT) described in Example 14 is shown. Fig. 9 As presented in , Q-GRFT purity was approximately 90% of the total protein composition.

[0055] Fig.10 A schematic diagram of the plasmid p3JM used to express the lectin gene (ORF) coding sequence is presented. Fig.10 As shown, p3JM comprises an upstream (5′) Bacillus subtilis aprE promoter region, which is operably linked to a downstream (3′) nucleic acid encoding a target lectin protein, which is operably linked to a downstream terminator (Tlat) sequence. In addition, plasmid p3JM ( Fig.10 ) contains the β-lactamase gene coding sequence (Bla) and the chloramphenicol acetyltransferase coding gene (CAT).

[0056] Fig.11 SDS-PAGE analysis of purified jacaranda lectin-like, CVN-like, OAA-like, ricin-like and galectin-1-like lectins expressed in Gram-positive bacterial cells is shown. More particularly, Fig.11 The amino acid sequence expressed in the supernatant of Bacillus subtilis strain CBS12 is shown (SEQ ID NO, see e.g. Figure 1-5 ) SDS-PAGE results of various lectins. These expressed lectins show a variety of different molecular weights, structural folds and source species.

[0057] Fig.12 The hemagglutination activity of the lectin on different erythrocytes is shown. More specifically, Fig.12 A shows the hemagglutination activity of different doses of lectin on 1% mouse erythrocytes, showing that the hemagglutination of lectin is dose-dependent. Fig.12 B shows the hemagglutination activity of different lectins (100 μg / ml) on 1% animal erythrocytes. The lane labeled PB indicates phosphate buffer used as a control.

[0058] Fig.13 The hemagglutination activity of the jacaranda-like, CVN-like, OAA-like, and galectin-like lectins produced in Gram-positive bacterial cells of the present disclosure is shown. The lane labeled PB indicates phosphate buffer used as a control. The various lectins annotated with sequence identification numbers (SEQ ID No.) at the top of the gel were tested against different animal red blood cells. The hemagglutination activity indicates that the produced lectins have functional activity.

[0059] Biological sequence description

[0060] SEQ ID NO: 1 is the amino acid sequence of native mature griffithsin (GRFT) protein isolated from Chaetodon species.

[0061] SEQ ID NO: 2 is the amino acid sequence of a variant grifithsin (Q-GRFT) protein having a methionine (M) to glutamine (Q) substitution at amino acid position 78 (M78Q).

[0062] SEQ ID NO: 3 is a DNA sequence encoding the Q-GRFT protein (SEQ ID NO: 2), which has been codon-optimized for expression in Bacillus subtilis cells.

[0063] SEQ ID NO: 4 is the amino acid sequence of the natural high mannose binding lectin KAA 2 isolated from Kappaphysonae.

[0064] SEQ ID NO: 5 is the amino acid sequence of Banana (Banlec) lectin.

[0065] SEQ ID NO: 6 is the amino acid sequence of Helianthus annuus lectin.

[0066] SEQ ID NO: 7 is the amino acid sequence of Renouxia sp. lectin.

[0067] SEQ ID NO: 8 is the amino acid sequence of Artocarpus integrifolia (Artocarpus integrifolia lectin).

[0068] SEQ ID NO: 9 is the amino acid sequence of Nostoc ellipsosporum (CV-N) lectin.

[0069] SEQ ID NO: 10 is the amino acid sequence of Microcystis viridis (MVL) lectin.

[0070] SEQ ID NO: 11 is the amino acid sequence of Microcystis aeruginosa PCC7806 (MVN) lectin.

[0071] SEQ ID NO: 12 is the amino acid sequence of Homo sapiens (DCSIGN) lectin.

[0072] SEQ ID NO: 13 is the amino acid sequence of Kappaphysonii longiflorum (KAA-1) lectin.

[0073] SEQ ID NO: 14 is the amino acid sequence of Eucheuma denticulatum (EDA2) lectin.

[0074] SEQ ID NO: 15 is the amino acid sequence of Meristotheca papulosa (MPA-2) lectin.

[0075] SEQ ID NO: 16 is the amino acid sequence of Oscillatoria agardhii NIES-204 (OAA) lectin.

[0076] SEQ ID NO: 17 is the amino acid sequence of Eucheuma serra (ESA-2) lectin.

[0077] SEQ ID NO: 18 is the amino acid sequence of the Herpetosiphon aurantiacus DSM 785 lectin.

[0078] SEQ ID NO: 19 is the amino acid sequence of Roseofilum reptotaenium AO1-C lectin.

[0079] SEQ ID NO: 20 is the amino acid sequence of Pseudomonas baetica lectin.

[0080] SEQ ID NO: 21 is the amino acid sequence of a lectin from the family Verrucomicrobiaceae bacterium.

[0081] SEQ ID NO: 22 is the amino acid sequence of Melittangium boletus lectin.

[0082] SEQ ID NO: 23 is the amino acid sequence of a lectin from Rhodocyclaceae bacterium.

[0083] SEQ ID NO:24 is the amino acid sequence of Longispora albida (Actinohivin) lectin.

[0084] SEQ ID NO:25 is the amino acid sequence of Mus musculus (Galectin-1) lectin.

[0085] SEQ ID NO: 26 is the amino acid sequence of the vampire bat (Desmodus rotundus) (Galectin-1) lectin.

[0086] SEQ ID NO: 27 is the amino acid sequence of Scleropages formosus (Galectin-1) lectin.

[0087] SEQ ID NO: 28 is the amino acid sequence of Callorhinchus milii lectin.

[0088] SEQ ID NO: 29 is the amino acid sequence of Trichodesmium sp. ALOHA_ZT_67 lectin.

[0089] SEQ ID NO: 30 is the amino acid sequence of Burkholderia ubonensis lectin.

[0090] SEQ ID NO: 31 is the amino acid sequence of Aquimarina longa lectin.

[0091] SEQ ID NO: 32 is the amino acid sequence of Microcystis aeruginosa lectin.

[0092] SEQ ID NO: 33 is the amino acid sequence of Corallococcus sp. Z5C101001 lectin.

[0093] SEQ ID NO:34 is the amino acid sequence of Agardhiella subulata lectin.

[0094] SEQ ID NO: 35 is the amino acid sequence of Sphingomonas sp. TDK1 lectin.

[0095] SEQ ID NO: 36 is the amino acid sequence of a lectin from Proteobacteria bacteria.

[0096] SEQ ID NO: 37 is the amino acid sequence of Sinobacterium caligoides lectin.

[0097] SEQ ID NO: 38 is the amino acid sequence of Aquimarina sp. TRL1 lectin.

[0098] SEQ ID NO: 39 is the amino acid sequence of Musa troglodytarum lectin. DETAILED DESCRIPTION

[0099] I. Overview

[0100] As briefly described above and described below, certain embodiments of the present disclosure provide, inter alia, novel recombinant Gram-positive bacterial cells expressing heterologous lectin proteins, wherein these lectin proteins can be the same lectin or a combination of different lectin proteins, recombinant polynucleotides encoding heterologous lectins (e.g., vectors, expression cassettes) for introduction (e.g., transformation) into Gram-positive host cells to express the heterologous lectins, fermentation broths containing lectin proteins (and lectin preparations obtained therefrom), lectin proteins recovered from fermentation broths (and lectin preparations obtained therefrom), purified lectin preparations, etc. Therefore, certain embodiments of the present disclosure provide novel methods for recovering and / or purifying lectins derived from any naturally occurring lectin source (e.g., plant lectins, algal lectins, cyanobacterial lectins, etc.). Thus, certain other embodiments of the present disclosure provide novel methods for recovering and / or purifying lectins obtained from recombinant cells expressing one or more lectins (e.g., recombinant Gram-negative bacterial cells, recombinant Gram-positive bacterial cells, recombinant plant cells (e.g., tobacco), etc.).

[0101] II. Definitions

[0102] The following terms and phrases are defined in view of recombinant cells expressing heterologous lectins, polynucleotides comprising nucleic acids encoding heterologous lectins, one or more recombinant lectins (e.g., natural lectins and / or functional variants thereof) encoded by such recombinant host cells, fermentation methods for producing such recombinant lectins, methods for their recovery and / or purification, etc. Terms not defined herein shall be given their ordinary meaning as used in the art.

[0103] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the compositions and methods of the present invention are applied. Although any methods and materials similar or equivalent to the methods and materials described herein can also be used in the practice or testing of the compositions and methods of the present invention, representative illustrative methods and materials are now described. All publications and patents cited herein are incorporated by reference in their entirety.

[0104] It is further noted that the claims may be drafted to exclude any optional element. Therefore, this statement is intended to serve as a prerequisite basis (or condition thereof) for the use of exclusive terminology such as "solely," "only," "excluding," "not including," etc., in connection with the recitation of claim elements, or the use of a "negative" limitation. In certain embodiments, an example of a condition used herein is that the "recombinant lectin protein" produced and / or recovered and / or purified according to the present disclosure is not a "His-tagged lectin."

[0105] As will be apparent to those skilled in the art after reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the inventive compositions and methods described herein. Any recited method may be performed in the order of events recited or in any other order that is logically possible.

[0106] As used herein, the term "recombinant" or "non-natural" refers to an organism, microorganism, cell, nucleic acid molecule, vector, etc. that has at least one engineered genetic change or has been modified by the introduction of a heterologous nucleic acid molecule; or refers to a cell (e.g., Gram-positive cell) that has been changed so that the expression of a heterologous nucleic acid molecule or an endogenous nucleic acid molecule or gene can be controlled. Recombination also refers to a cell derived from a non-natural cell, or a progeny of a non-natural cell with one or more such modifications. Genetic changes include, for example, the introduction of modifications of expressible nucleic acid molecules encoding proteins, or the addition, deletion, substitution or other functional changes of other nucleic acid molecules of the genetic material of the cell. For example, a recombinant cell can express genes or other nucleic acid molecules (e.g., polynucleotide constructs) that are not found in a natural (wild-type) cell in the same or homologous form, or can provide an altered endogenous gene expression pattern, such as overexpression, underexpression, minimal expression or no expression at all. "Recombination", "recombining" or the generation of a "recombinant" nucleic acid is usually an assembly of two or more nucleic acid fragments, wherein the assembly produces a chimeric DNA sequence that would otherwise not be found in the genome.

[0107] The term "derived" encompasses the terms "originating from", "obtained", "obtainable", and "produced", and generally indicates that a specified material or composition finds its origin in another specified material or composition, or has characteristics that can be described with reference to another specified material or composition. For example, the recombinant Gram-positive bacterial cells of the present disclosure can be derived from / obtained from any known Gram-positive bacterial strain (e.g., Bacillus subtilis 168 strain, etc.). Natural lectin proteins (and / or functional lectin variants thereof) and DNA sequences encoding the same can be derived from / obtained from known lectin proteins and / or functional variants thereof.

[0108] As used herein, "endogenous gene" refers to a gene that is located in its natural location in the genome of an organism.

[0109] As used herein, a "heterologous" gene, "non-endogenous" gene, or "foreign" gene refers to a gene (or gene coding sequence (CDS) or (ORF)) that is not normally found in the host organism, but is introduced into the host organism by gene transfer. The term "foreign" gene(s) includes a native gene (or ORF) inserted into a non-native organism and / or a chimeric gene inserted into a native or non-native organism.

[0110] As used herein, "heterologous control sequences" refer to gene expression control sequences (e.g., promoters, enhancers, terminators, etc.) that do not essentially function to regulate (control) the expression of a gene of interest. Typically, heterologous nucleic acids are not endogenous (natural) to the cell or part of the genome in which they are present, and have been added to the cell by infection, transfection, transduction, transformation, microinjection, electroporation, etc. "Heterologous" nucleic acid constructs may contain control sequence / DNA coding (ORF) sequence combinations that are the same or different from those found in the natural host cell.

[0111] As used herein, the terms "signal sequence" and "signal peptide" refer to a sequence of amino acid residues that can participate in the secretion or directional transport of a mature protein or a precursor form of a protein. Typically, the signal sequence is located at the N-terminus of the precursor or mature protein sequence. The signal sequence can be endogenous or exogenous. There is generally no signal sequence in the mature protein. Typically, the signal sequence is cut from the protein by a signal peptidase during the translocation process.

[0112] As used herein, the term "expression" refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid molecules of the present disclosure. Expression can also refer to the translation of mRNA into polypeptides. Therefore, the term "expression" includes any steps involved in the production of polypeptides, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, secretion, etc.

[0113] As used herein, "nucleic acid" refers to nucleotide or polynucleotide sequences and fragments or portions thereof, as well as DNA, cDNA and RNA of genomic or synthetic origin, which may be double-stranded or single-stranded, whether representing a sense strand or an antisense strand. It should be understood that due to the degeneracy of the genetic code, a variety of nucleotide sequences may encode a given protein.

[0114] It should be understood that the polynucleotides (or nucleic acid molecules) described herein include "genes", "vectors" and "plasmids".

[0115] Accordingly, the term "gene" refers to a polynucleotide encoding a specific sequence of amino acids, which contains all or part of a protein coding sequence and may include regulatory (non-transcribed) DNA sequences, such as a promoter sequence, which determines, for example, the conditions under which a gene is expressed. The transcribed region of a gene may include an untranslated region (UTR) (including introns, 5'-untranslated region (UTR) and 3'-UTR) as well as a coding sequence.

[0116] As used herein, the term "coding sequence" refers to a nucleotide sequence that directly specifies the amino acid sequence of its (encoded) protein product. The boundaries of a coding sequence are generally determined by an open reading frame (hereinafter, "ORF") that usually begins with the ATG start codon. Coding sequences typically include DNA, cDNA, and recombinant nucleotide sequences.

[0117] As used herein, the term "promoter" refers to a nucleic acid sequence that is capable of controlling the expression of a coding sequence or functional RNA. Typically, the coding sequence is located (3') downstream of the promoter sequence. The promoter may be derived entirely from a natural gene, or may be composed of different elements derived from different promoters found in nature, or may even comprise a synthetic nucleic acid segment.

[0118] As used herein, the term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence (e.g., ORF) when expression of the coding sequence can be achieved (i.e., the coding sequence is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in the sense or antisense direction.

[0119] Therefore, when a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "effectively connected" to another nucleic acid sequence. For example, if the DNA encoding a secretory leader sequence (i.e., a secretory signal sequence) is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA encoding the secretory leader sequence is operably connected to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably connected to the sequence; if a ribosome binding site is positioned to promote translation, the ribosome binding site is operably connected to the coding sequence. Generally, "operably connected" means that the DNA sequence being connected is continuous, and in the case of a secretory leader sequence, it is continuous and in the reading phase. However, enhancers do not have to be continuous. Connection is achieved by connecting at a convenient restriction site. If such sites do not exist, synthetic oligonucleotide adapters or joints are used according to conventional practice.

[0120] As used herein, "a functional promoter sequence that controls the expression of a target gene (or its open reading frame) linked to a protein coding sequence of the target gene" refers to a promoter sequence that controls the transcription and translation of a coding sequence in a desired Gram-positive host cell. For example, in certain embodiments, the present disclosure relates to a polynucleotide comprising an upstream (5') promoter (or a 5' promoter region, or a tandem 5' promoter, etc.) that is functional in Gram-positive cells, wherein the promoter region is operably linked to a nucleic acid sequence (e.g., ORF) encoding a lectin protein.

[0121] As used herein, "suitable regulatory sequence" refers to a nucleotide sequence that is located upstream (5' non-coding sequence), internal or downstream (3' non-coding sequence) of a coding sequence and affects transcription, RNA processing or stability or translation of the relevant coding sequence. Regulatory sequences can include promoters, transcription leader sequences, RNA processing sites, effector binding sites and stem-loop structures.

[0122] As used herein, a "functional gene" is a gene that can be used by cellular components to produce an active gene product (typically a protein). Functional genes are the opposite of mutagenized genes that have been modified so that they cannot be used by cellular components to produce an active gene product, or have a reduced ability to be used by cellular components to produce an active gene product.

[0123] As used herein, a "functional protein" is a protein that has an activity (such as enzymatic activity, binding activity, surface active properties, etc.) and which has not been mutagenized, truncated, or otherwise modified to eliminate or reduce such activity.

[0124] As used herein, the terms "modification" and "genetic modification" are used interchangeably and include, but are not limited to: (a) introduction, substitution or removal of one or more nucleotides in a gene (or its ORF), or introduction, substitution or removal of one or more nucleotides in a regulatory element required for transcription or translation of a gene or its ORF, (b) gene disruption, (c) gene conversion, (d) gene deletion, (e) gene down-regulation, (f) specific mutagenesis of any one or more genes disclosed herein and / or (g) random mutagenesis.

[0125] As used herein, "disruption of a gene" or "gene disruption" are used interchangeably and broadly refer to any genetic modification that substantially prevents a host cell from producing a functional gene product (e.g., protein). Therefore, as used herein, gene disruption includes, but is not limited to, frameshift mutations, premature termination codons (i.e., so that no functional protein is produced), substitutions that eliminate or reduce protein activity (so that no functional protein is produced), internal deletions, insertions that destroy the coding sequence, mutations that remove the operable connection between the natural promoter required for transcription and the open reading frame, etc.

[0126] As used herein, the term "introduced", as used in phrases such as "introducing into a bacterial cell" or "introducing into a bacterial cell" at least one polynucleotide open reading frame (ORF), or a gene thereof, or a vector thereof, includes methods known in the art for introducing a polynucleotide into a cell, including but not limited to protoplast fusion, natural or artificial transformation (e.g., calcium chloride, electroporation), transduction, transfection, conjugation, and the like.

[0127] As used herein, "transformed" or "transformation" means a cell transformed by using recombinant DNA technology. Transformation typically occurs by inserting one or more nucleotide sequences (e.g., polynucleotides, ORFs, or genes) into a cell. The inserted nucleotide sequence may be a heterologous nucleotide sequence (i.e., a sequence that does not naturally occur in the cell to be transformed). Thus, transformation generally refers to the introduction of exogenous DNA into a host cell such that the DNA remains as a chromosomal integrant or a self-replicating extrachromosomal vector.

[0128] As used herein, "transforming DNA", "transforming sequence" and "DNA construct" refer to DNA for introducing a sequence into a host cell or an organism. Transforming DNA is DNA for introducing a sequence into a host cell or an organism. DNA can be generated in vitro by PCR or any other suitable technique. In certain embodiments, the transforming DNA comprises an input sequence, and in other embodiments, it further comprises an input sequence flanked by homology boxes. In yet other embodiments, the transforming DNA comprises other non-homologous sequences (i.e., stuffing sequences or flanking) added to the ends. The ends can be closed so that the transforming DNA forms a closed loop, such as, for example, as inserted into a vector.

[0129] As used herein, "input sequence" refers to a DNA sequence introduced into a Gram-positive host cell chromosome. In certain embodiments, the input sequence is a part of a DNA construct. In other embodiments, the input sequence encodes one or more target proteins. In certain embodiments, the input sequence comprises a sequence that may or may not be present in the genome of the cell to be transformed (that is, it may be a homologous or heterologous sequence). In certain embodiments, the input sequence encodes one or more target proteins, genes and / or mutations or modified genes. In alternative embodiments, the input sequence encodes functional wild-type genes or operons, functional mutant genes or operons or non-functional genes or operons. In certain embodiments, non-functional sequences can be inserted into genes to destroy the function of genes. In another embodiment, the input sequence includes a selective marker. In a further embodiment, the input sequence includes two homology boxes.

[0130] As used herein, "homologous box" or "homologous arm" refers to a nucleic acid sequence homologous to a sequence in a gram-positive bacterial cell chromosome. More particularly, according to the present invention, a homologous box is an upstream or downstream region that has a sequence identity between about 80% and 100%, a sequence identity between about 90% and 100%, or a sequence identity between about 95% and 100% with a gene or a part of a gene to be deleted, destroyed, inactivated, downregulated, etc. These sequences guide the integration position of the DNA construct in the gram-positive bacterial cell chromosome, and guide which part of the gram-positive bacterial cell chromosome is replaced by the input sequence. Although it is not intended to limit the present disclosure, the homology box can include between about 1 base pair (bp) and 200 kilobases (kb). Preferably, the homology box includes between about 1 bp and 10.0 kb; between 1 bp and 5.0 kb; between 1 bp and 2.5 kb; between 1 bp and 1.0 kb; and between 0.25 kb and 2.5 kb. The homology box may also include about 10.0 kb, 5.0 kb, 2.5 kb, 2.0 kb, 1.5 kb, 1.0 kb, 0.5 kb, 0.25 kb, and 0.1 kb. In some embodiments, the 5' and 3' ends of the selectable marker are flanked by homology boxes, wherein the homology boxes contain nucleic acid sequences that are closely flanked by the coding region of the gene.

[0131] As used herein, the term "nucleotide sequence encoding a selectable marker" refers to a nucleotide sequence that is capable of being expressed in a host cell and wherein expression of the selectable marker confers upon the cells containing the expressed gene the ability to grow in the presence of the corresponding selective agent or in the absence of essential nutrients.

[0132] As used herein, the terms "selectable marker" and "selective marker" refer to a nucleic acid (e.g., a gene) that can be expressed in a host cell that allows easy selection of those hosts containing the vector. Examples of such selectable markers include, but are not limited to, antimicrobial agents. Thus, the term "selectable marker" refers to a gene that provides an indication that the host cell has taken up the input DNA of interest or that some other reaction has occurred. Typically, a selectable marker is a gene that confers antimicrobial resistance or a metabolic advantage to a host cell to allow cells containing exogenous DNA to be distinguished from cells that have not received any exogenous sequences during transformation.

[0133] A "residing selectable marker" is a marker located on the chromosome of the microorganism to be transformed. The residing selectable marker encodes a gene that is different from the selectable marker on the transforming DNA construct. Selectable markers are well known to those skilled in the art. As indicated above, the marker can be an antimicrobial resistance marker (e.g., amp R , phleoR 、spec R ,kan R ery R , tet R 、cmp R , and neo R ). Other markers useful according to the present invention include, but are not limited to, auxotrophic markers, such as serine, lysine, tryptophan; and detection markers, such as β-galactosidase.

[0134] As defined herein, a host cell "genome", a Gram-positive bacterial (host) cell "genome", a Bacillus sp. (host) cell "genome", etc. includes chromosomal genes and extrachromosomal genes.

[0135] As used herein, the terms "plasmid", "vector" and "cassette" refer to an extrachromosomal element that usually carries genes that are typically not part of the central metabolism of the cell and is usually in the form of a circular double-stranded DNA molecule. Such elements can be linear or circular self-replicating sequences derived from single-stranded or double-stranded DNA or RNA from any source, genome integrating sequences, bacteriophages, or nucleotide sequences in which multiple nucleotide sequences have been linked or recombined into a single construct that is capable of introducing a promoter fragment and DNA sequence for a selected gene product, along with appropriate 3' untranslated sequences, into a cell.

[0136] As used herein, the term "plasmid" refers to a circular double-stranded (ds) DNA construct that is used as a cloning vector and forms an extrachromosomal self-replicating genetic element in many bacteria and some eukaryotic organisms. In some embodiments, the plasmid is incorporated into the genome of the host cell. In some embodiments, the plasmid is present in the parent cell and is lost in the daughter cell.

[0137] As used herein, "transformation cassette" refers to a specific vector that contains a gene (or its ORF) and has elements in addition to the foreign gene that facilitate transformation of a specific host cell.

[0138] As used herein, the term "vector" refers to any nucleic acid that can replicate (propagate) in a cell and can carry a new gene or DNA segment to a cell. Therefore, the term refers to a nucleic acid construct designed to be transferred between different host cells. Vectors include viruses, phages, proviruses, plasmids, phagemids, transposons, and artificial chromosomes such as YAC (yeast artificial chromosome), BAC (bacterial artificial chromosome), PLAC (plant artificial chromosome), etc. that are "episomes" (i.e., they replicate autonomously) or can be integrated into the chromosome of a host organism.

[0139] As used herein, the terms "expression cassette" and "expression vector" refer to a nucleic acid construct that is recombinantly or synthetically generated with a series of specified nucleic acid elements (i.e., these are vectors or vector elements, as described above) that allow a specific nucleic acid to be transcribed in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes (in addition to other sequences) a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the DNA construct also includes a series of specified nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. In certain embodiments, the DNA construct of the present disclosure includes a selectable marker and an inactivated chromosome, or a gene, or a DNA segment as defined herein.

[0140] As used herein, "targeting vector" is a vector comprising a polynucleotide sequence that is homologous to the region in the chromosome of the host cell to which the targeting vector is transformed and that can drive homologous recombination in the region. For example, the targeting vector can be used to introduce mutations into the chromosome of the host cell by homologous recombination. In certain embodiments, the targeting vector comprises other non-homologous sequences (i.e., stuffing sequences or flanking sequences) that are, for example, added to the end. The end can be closed so that the targeting vector forms a closed loop, such as, for example, as inserted into a vector. For example, in certain embodiments, the cell is modified (e.g., transformed) by introducing one or more "targeting vectors" into a Bacillus species (host) cell.

[0141] As used herein, "flanking sequence" refers to any sequence upstream or downstream of the sequence in question (for example, for gene ABC, gene B is flanked by A and C gene sequences). In certain embodiments, the flank of the input sequence on each side is a homology box. In another embodiment, the input sequence and the homology box are included in the unit of the flank on each side as a stuffing sequence. In certain embodiments, the flanking sequence is only present on one side (3' or 5'), but in a preferred embodiment, it is on each side of the sequence flanked. The sequence of each homology box is homologous to the sequence in the Bacillus chromosome. These sequences guide the integration position of the new construct in the Bacillus chromosome and which part of the Bacillus chromosome will be replaced by the input sequence. In other embodiments, the flanks of the 5' and 3' ends of the selectable marker are polynucleotide sequences comprising the part of the inactivated chromosome segment. In certain embodiments, the flanking sequence is only present on one side (3' or 5'), and in other embodiments, it is present on each side of the sequence flanked.

[0142] As used herein, "host cell" refers to a cell that has the ability to serve as a host or expression vehicle for newly introduced DNA sequences.

[0143] In certain aspects, the host cells of the present disclosure are Gram-positive bacterial cells / strains. As will be appreciated by those skilled in the art, many Gram-positive host strains are generally recognized as safe (GRAS) according to US FDA guidelines, and therefore, Gram-positive host cells are particularly useful protein production hosts relative to Gram-negative hosts (e.g., E. coli expression systems) that require additional expensive processing steps to remove endotoxins (e.g., LPS).

[0144] As used herein, Bacillus subtilis strains designated herein as "CB447", "CB476", "CB460", "CB462", "CB488", "CBS6", "CZ438", "CBS6" and "CBS12" were constructed for expression of mature Q-GRFT variants (SEQ ID NO: 2). More specifically, as described in the Examples section, Bacillus subtilis strains CB447, CB476, CB460 and CZ438 were constructed for extracellular secretion of mature Q-GRFT variants, Bacillus subtilis strain CB476 was constructed for intracellular expression of mature Q-GRFT variants, Bacillus subtilis strains CBS6 and CBS12 were constructed for intracellular or secreted expression of mature Q-GRFT variants, and Bacillus subtilis strain CB488 was constructed for both intracellular and secreted expression of mature Q-GRFT variants. In certain embodiments, the recombinant Gram-positive bacterial cell expressing a heterologous lectin comprises at least one introduced cassette encoding a heterologous lectin, or at least two introduced cassettes encoding the same heterologous lectin or different heterologous lectins. For example, in the case of one or more modified Bacillus subtilis strains exemplified herein, the CZ438 strain comprises two introduced expression cassettes encoding a mature Q-GRFT variant (SEQ ID NO: 2) for secretory expression (Example 6). As further described in Example 15, cassettes encoding various other classes of lectin proteins were introduced into Bacillus subtilis strain CBS12 and screened for lectin expression / production (Table 3).

[0145] As used herein, the terms "purified," "isolated," or "enriched" mean that a biomolecule (e.g., a polypeptide or polynucleotide) is altered from its native state by separating it from some or all of the naturally occurring components with which it is associated in nature. Such separation or purification can be accomplished by art-recognized separation techniques, such as ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, heat treatment, ammonium sulfate precipitation or other protein salt precipitation, crystallization, centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis, or gradient separation to remove undesirable whole cells, cell debris, impurities, foreign proteins, or enzymes in the final composition. Components that provide additional benefits, such as activators, anti-inhibitors, desired ions, compounds that control pH, or other enzymes or chemicals, can then be further added to the purified or isolated biomolecule composition.

[0146] As used herein, a "protein preparation" is any material, typically a solution, usually aqueous, containing one or more proteins.

[0147] As used herein, the terms "broth", "culture broth", "fermentation broth" and / or "whole fermentation broth" are used interchangeably and refer to a preparation produced by cell fermentation that does not undergo processing steps after fermentation is complete. For example, whole fermentation broth is typically produced when a microbial culture is grown to saturation, incubated under carbon-limited conditions to allow protein synthesis (e.g., expression of proteins by host cells; and optionally secretion of proteins into the cell culture medium). Typically, whole fermentation broth is unfractionated and contains spent cell culture medium, metabolites, extracellular polypeptides, and microbial cells.

[0148] As used herein, the phrase "treated broth" refers to broth that has been conditioned by changing the chemical composition and / or physical properties of the broth. Broth "conditioning" can include one or more treatments such as cell lysis, pH modification, heating, cooling, addition of chemicals (e.g., calcium, one or more salts, one or more flocculants, one or more reducing agents, one or more enzyme activators, one or more enzyme inhibitors, and / or one or more surfactants), mixing, and / or timed holding of the broth (e.g., 0.5 to 200 hours) without further processing.

[0149] As used herein, the process of "cell lysis" includes any cell lysis technique known in the art, including but not limited to enzymatic treatment (e.g., lysozyme, proteinase K treatment), chemical means (e.g., ionic liquids), physical means (e.g., French press, ultrasound), simply maintaining the culture without feed, etc.

[0150] As used herein, the terms "recovery," "recovered," and "recovering" refer to at least partially separating a protein from one or more components of a microbial broth, and / or at least partially separating a protein from one or more solvents (e.g., water or ethanol) in the broth.

[0151] In certain aspects, the broth in which the host cells have been fermented for the production of the lectin protein is clarified, with or without broth treatment. As used herein, "clarified" broth means a broth that has been subjected to at least one clarification process to remove cell debris and / or other insoluble components. As understood in the art, clarification processes include, but are not limited to, centrifugation techniques, cross-flow membrane filtration techniques, solid / liquid filtration techniques, and the like.

[0152] "Cellular debris" refers to cell wall and other insoluble components released or formed after disruption of the cell membrane (eg, after undergoing a cell lysis procedure).

[0153] In certain aspects, separation of the solvent includes, but is not limited to, ultrafiltration, evaporation, spray drying, freeze drying, as understood in the art. The solution obtained is referred to as "clarified broth concentrate", "UF concentrate" or "ultrafiltration concentrate".

[0154] As used herein, the terms "one or more lectins" and "lectin protein" are used interchangeably and refer to carbohydrate binding proteins (or glycoproteins) that can recognize and bind simple or complex carbohydrates in a reversible and highly specific manner while not exhibiting catalytic activity. Therefore, the lectin protein described herein has the same meaning as the lectin described in the art (e.g., see Lagarda-Diaz et al., 2017).

[0155] As used herein, the term "one or more functional lectin variants" and when used in phrases such as "natural lectin and its functional (lectin) variants", "natural griffithsin (GRFT) and its functional GRFT variants", etc., refers to variant (mutant) lectins derived from natural (parent) lectin proteins, wherein these functional (lectin) variants have carbohydrate binding activity. In certain aspects, the functional lectin variants may have reduced carbohydrate binding activity relative to the parent (natural) lectin, the same carbohydrate binding activity relative to the parent (natural) lectin, or increased carbohydrate binding activity relative to the parent (natural) lectin.

[0156] III. Lectin Protein

[0157] As briefly described above, lectins are proteins (or glycoproteins) with non-catalytic carbohydrate binding sites. As generally understood in the art, lectins are different from enzymes because their carbohydrate binding properties never change, and they are different from antibodies because they are not induced as immune responses. For example, some of the most well-known lectins are found in leguminous seeds, which are believed to be responsible for innate immunity and defense mechanisms in plants (Peumans and Van Damme, 1998). Recently, the use of lectins in mitigating viral infections (e.g., HIV, MERS-CoV, SARS-CoV-2, HCV, Ebola, etc.) has been significantly concerned (PCT Publication Nos. WO 2005 / 118627, WO 2007 / 064844, WO 2010 / 01424, WO2016 / 130628, WO 2019 / 108656, and US Publication No. US20110263485). However, as described above in the Background, the economics of recombinant lectin production (eg, using currently available host expression systems and their downstream recovery processes) significantly limits the acceptance and / or use of lectins as antimicrobial compositions.

[0158] For example, PCT publications WO 2005 / 118627 and WO 2007 / 064844 describe methods for isolating a natural griffithsin (GRFT) lectin from a red alga (Trichoderma sp.), cloning its wild-type (grft) gene, generating its recombinant polynucleotide, fermenting and producing it in an E. coli (E. coli) host cell, followed by isolating a recombinant His-tagged GRFT protein from the E. coli host, and characterizing its antiviral activity. However, as described in WO 2005 / 118627 and WO 2007 / 064844, the recombinant GRFT protein encoded by the nucleic acid of Example 2 (and its C-terminal His-tagged GRFT protein) was not efficiently translocated to the periplasmic fraction of E. coli after GRFT protein expression, wherein most of the produced GRFT protein accumulated in the inclusion bodies of E. coli without cleavage of the pelB signal sequence located at the N-terminus of the griffithsin protein. Therefore, as described above, steps were taken to express griffithsin in the cytosolic fraction of E. coli using N-terminally (His) tagged GRFT or a His-tagged variant of GRFT.

[0159] Similarly, PCT Publication No. WO 2010 / 01424 generally describes methods for inhibiting hepatitis C virus infection in a host, which methods include administering to the host an effective amount of a glycosylation-resistant GRFT protein (or its polypeptide conjugate) in combination with another antiviral protein. For example, as described in this disclosure, the inventors noted that the antiviral protein combination of scytovirin (SVN) and griffithsin (GRFT) has (nanomolar) activity against hepatitis C virus (HCV). U.S. Patent Publication No. US20110263485 further describes methods for inhibiting human immunodeficiency virus (HIV) viral infection in a host, which methods include administering to the host an effective amount of gp120 Griffithsin and a peptide selected from gp41 binding protein, CCR5 binding protein, gp120 binding protein or another griffithsin, which combinations are potent inhibitors of HIV infection.

[0160] PCT Publication No. WO 2016 / 130628 discloses variant griffithsin proteins having mutations that alter the isoelectric point of the GRFT protein, which reportedly alter its solubility in various pH ranges, thereby allowing for improved product release.

[0161] PCT Publication No. WO 2019 / 108656 generally describes a microbicidal composition comprising an endosperm extract and an anti-HIV lectin, an anti-HIV antibody or an antigen-binding antibody fragment thereof. More particularly, the inventors utilized transgenic plants expressing two or more cyanobacterial antiviral proteins-N (CVN) proteins, griffithsin (GRFT) proteins, scytovirin (SVN) proteins, and other anti-HIV lectin proteins. However, as described in WO 2019 / 108656, the production of such microbicidal components is expensive because a fermenter-based expression platform is required, and downstream processing facilities must comply with good manufacturing practices (GMP) to ensure the removal of viruses or endotoxins, wherein when each microbicide requires two or three separate products with separate manufacturing processes, the capacity, scalability, and cost issues affecting the fermenter are exacerbated.

[0162] Recombinant production of GRFT in tobacco plants (Nicotiana benthamiana) has been described by O'Keefe et al. (2009), where GRFT accumulated to levels of approximately 1 gram of recombinant GRFT per kilogram of N. benthamiana leaf material when expressed via an infectious Tobacco Mosaic Virus (TMV) based vector.

[0163] Hirayama et al. (2016) have described the elucidated primary structure of KAA-2 lectin using peptide mapping and complementary DNA (cDNA) cloning and prepared its active recombinant using an E. coli expression system.

[0164] Gengenbach et al. (2019) describe the transient expression of a mistletoe lectin named "viscumin" (Viscum album) in intact Nicotiana benthamiana plants, and the purification of the recombinant mistletoe lectin from crude plant extracts by affinity chromatography, where the performance and economics of the tobacco plant-based method were compared to the corresponding method based on E. coli expression. For example, as described in Gengenbach et al., full-length mistletoe lectin was produced in Nicotiana benthamiana leaves at levels up to 7 mg / kg purified product, where the yield of full-length mistletoe lectin was comparable to that of plant lectins expressed in yeast, but about 6 times lower than that of refolded mistletoe lectin A and B chains expressed in E. coli. As summarized by Gengenbach et al., the E. coli method has low recovery, requires extensive dilutions and is complex, while the plant-based method includes only half the number of steps. According to a direct cost comparison between the two approaches performed in a study by Gengenbach et al., the plant expression system is 50% cheaper compared to heterologous expression in the natural host mistletoe or in E. coli host cells.

[0165] As described herein, the present disclosure addresses many ongoing and unmet needs in the art, particularly those associated with industrial-scale production, recovery and / or purification of lectin proteins. More particularly, as described below and in the following examples, applicants disclose novel end-to-end methods for large-scale production, recovery and purification of any recombinant lectin protein (or proteins) in Gram-positive bacterial (host) cells. As described in Example 1, exemplary Gram-positive bacterial cells were designed, constructed, and evaluated for their ability to express heterologous (exogenous) lectins, wherein applicants have demonstrated that Gram-positive bacterial cells can express and produce significant amounts of a heterologous (i.e., eukaryotic) lectin called griffithsin (GRFT), which was surprising and unexpected based on the prior art.

[0166] In other aspects, it is surprising and unexpected that Gram-positive bacterial cells can express and produce significant amounts of such heterologous (i.e., eukaryotic plant) lectins, which are known to agglutinate various Gram-positive bacterial cells (e.g., Bacillus species cells; Cole et al., 1984). For example, the sword bean (Canavalia ensiformis) lectin concanavalin A (ConA) agglutinates Bacillus species cells (e.g., Bacillus subtilis 168, Bacillus sphaericus, Bacillus amyloliquefaciens) by binding to cell wall sugars (e.g., α-D-mannopyranosyl, α-D-glucopyranosyl, and spatially related residues).

[0167] Studies of leguminous plant (Leguminosiae) lectins (Ayouba et al., 1991) characterized other monosaccharides (e.g., Glc / Man-specific lectins, Gal / GalNAc-specific lectins, MurAc-specific lectins, and MurNAc-specific lectins) as being widely distributed on bacterial cell walls. As described in Ayouba et al. (1991), interactions of these sugar residues may explain the previously reported agglutination of various bacteria by leguminous plant lectins. Gram-positive cells are generally not considered for use in large-scale production of recombinant (eukaryotic) lectin proteins.

[0168] Therefore, as contemplated and described herein, certain embodiments of the present disclosure relate in particular to nucleic acids encoding lectin proteins, recombinant cells expressing / producing one or more lectin proteins, recovery of lectin proteins, purification of lectin proteins, lectin (protein) preparations, and the like. More particularly, in certain embodiments, natural and / or variant lectin proteins and / or DNA (nucleic acid) sequences encoding them can be derived from / obtained from known lectin proteins. In certain aspects, lectin proteins are derived from host organisms that naturally produce lectin proteins. Therefore, in certain embodiments, lectin proteins of the present disclosure are derived from eukaryotic cells, bacterial cells, or cyanobacterial cells. In certain other one or more embodiments, the eukaryotic cell is a photosynthetic plant cell, a red algae cell, an animal cell, or an insect cell.

[0169] In certain embodiments, the lectin protein is derived from one or more antiviral lectins described in U.S. Patent Publication Nos. US 20040204365, US20020127675, US20110189105, and US20110263485 and / or PCT Publication Nos. WO 2005 / 118627, WO2008 / 022303, WO 2010 / 014248, WO 2014 / 197650, WO 2016 / 130628, and WO 2019 / 108656 (each of which is incorporated herein by reference in its entirety). Thus, in certain aspects, the lectin protein is scytovirin (SVN), grifithsin (GRFT), cyanobacterial antiviral protein-N (CVN), functional fragments thereof, and / or functional variants (mutants) thereof.

[0170] In other embodiments, the lectin protein is one or more antiviral lectins described in PCT Publication No. WO 2019 / 108656, such as jackfruit (Jacaranda lectin) lectin, banana (banana) lectin, hemispheric algae lectin, green Microcystis lectin, etc., and / or functional fragments thereof that retain the ability to bind to carbohydrates on the viral envelope described therein, and / or functional variants thereof.

[0171] In other embodiments, the lectin protein is derived from a eukaryotic lectin source as described in Singh and Sarathi (2012), including but not limited to Aaptos as described in Singh and Sarathi (2012). papilleta (sponge) lectin, Abrus abrinus (abbean) lectin, Apium graveolens (sheep's horn celery) lectin, Agaricus bisporus (common mushroom) lectin, Albizia julibrissin (mimosa tree seed) lectin, Diplocoriaceae (Japanese beetle) lectin, Aloe arborescens (aloe plant) lectin, Dimorphotheca bryophylli (isomorph bean) lectin, Anguilla anguilla (eel) lectin, Aplysia vulgaris (mollusc; Mediterranean) lectin, Arachis aconitifolia (peanut) lectin, Jackfruit (jackfruit lectin) lectin, Bauhinia oleracea (camel hoof tree) lectin, Bryonia ovata (white bryonia) lectin, Caragana oleracea (Siberian pea tree) lectin, Scolopendra subspinipes (horseshoe crab) lectin, and / or functional fragments thereof that retain the ability to bind to a specified carbohydrate (moiety), and / or functional variants thereof.

[0172] In certain aspects, the lectin proteins of the present disclosure can be divided into multiple groups, including but not limited to "galactose (Gal)" specific lectin, "glucose (Glu)" specific lectin, "fucose (Fuc)" specific lectin, "mannose (Man)" specific lectin, "N-acetylgalactosamine (GalNAc)" specific lectin, "N-acetylglucosamine (GluNAc)" specific lectin, "sialic acid" specific lectin, etc.

[0173] In certain embodiments, the lectin proteins of the present disclosure can be divided into multiple groups based on the classification of their source species, including but not limited to lectins derived from "red algae", lectins derived from "plants", lectins derived from "cyanobacteria", lectins derived from "mammals", lectins derived from "bacteria", and lectins derived from "fish".

[0174] In other embodiments, one or more lectin proteins of the present disclosure can be grouped or characterized based on protein fold, such as β-barrel, β-prism, β-trefoil, etc. (eg, see Table 3).

[0175] In certain other embodiments, one or more lectin proteins of the present disclosure may be grouped or classified as "japanese lectin-like" lectins ( Figure 2 ), "CVN-like" lectins ( Figure 3 ), "OAA-like" lectins ( Figure 4 ), "ricin-like" lectin ( Figure 5 ) and galectin-1-like lectin ( Figure 5 ).

[0176] Thus, in certain aspects, lectins suitable for use in accordance with the present disclosure can be derived / isolated from a eukaryotic lectin source organism. For example, in certain embodiments, the lectin (protein) can be isolated from a eukaryotic (source) organism using affinity chromatography techniques known to those skilled in the art (i.e., one of the above carbohydrate moieties (Gal, Man, GalNAc, etc.) is attached to an inert (chromatographic) matrix such that the lectin protein having binding specificity for the carbohydrate moiety will be retained).

[0177] In certain other embodiments, the lectin protein is a microvirin (MVN) lectin derived from the cyanobacterium Microcystis aeruginosa (PCC7806), which has a mannose-specific affinity. For example, as described in Breitenbach Barroso Coelho et al. 2018 (incorporated herein by reference), the MVN lectin can inhibit HIV-1 infection, syncytium formation between infected and uninfected CD4 T cells, and HIV-1 transmission. In other embodiments, the lectin protein is scytovirin (SVN) derived from the cyanobacterium Scytonema varium, which binds to mannose residues on the envelope glycoprotein of the virus with high affinity and inhibits viral replication, as observed by Zaire Ebola virus (e.g., see Breitenbach Barroso Coelho et al., 2018). In yet other aspects, the lectin protein is an ESA-2 lectin derived from the red alga Eucheuma dentatum, which exhibits anti-HIV activity and potent inhibition of influenza A virus (H1N1) infection (e.g., see Breitenbach Barroso Coelho et al., 2018). In another embodiment, the lectin protein is a BanLec (Jalapenopsis lectin-related) lectin derived from the fruit of the banana (Bananas brasiliensis), which recognizes high mannose glycans found on viral envelopes (such as HIV-1) (e.g., see Breitenbach Barroso Coelho et al., 2018).

[0178] In certain other embodiments, the lectin protein is a mannose-binding lectin derived from the rhizome of Aspidistra oleracea (AEL), which has been shown to have significant in vitro inhibitory activity against vesicular stomatitis virus, coxsackievirus B4, and respiratory syncytial virus (see, e.g., Breitenbach Barroso Coelho et al., 2018).

[0179] In certain other embodiments, the lectin protein is the CVL lectin (β-galactose specific) derived from the marine worm Trichoptera with anti-HIV-1 activity (see, e.g., Breitenbach Barroso Coelho et al., 2018).

[0180] In certain other embodiments, the lectin proteins of the present disclosure can be derived from seeds of broad bean (fava bean), lentil (lentil), and pea (pisa) as generally described in El-Araby et al., 2020 (incorporated herein by reference). As generally described in the El-Araby et al. (2020) reference, crude extracts of the three legumes were purified by affinity chromatography using mannose agarose, wherein the purified broad bean, lentil, and pea lectins had molecular weights of 18 kDa, 14 kDa, and 17 kDa, respectively, as determined by amino acid sequence analysis. For example, the minimum inhibitory concentration (MIC) values ​​of these purified lectins ranged from 1.95 μg / ml to 250 μg / ml when tested against bacteria (Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumonia) and fungi (Candida albicans).

[0181] In another embodiment, the lectin protein of the present disclosure is an anti-tumor (anti-cancer) lectin derived from mistletoe (Viscum), such as the mistletoe lectin I (MLI) and mistletoe lectin III (MLIII) B-subunit described in Pevzner et al., 2004 and Gengenbach et al., 2019 (incorporated herein by reference).

[0182] As contemplated herein, native lectins and / or one or more variant lectins derived therefrom can be evaluated for function or activity by means including, but not limited to, hemagglutination activity assays, carbohydrate / glycan binding affinity assays, antimicrobial inhibition assays, combinations thereof, and the like, as described and illustrated in El-Araby et al. (2020). Thus, in certain aspects, the native lectins and / or variant lectins of the present disclosure have antimicrobial activity (e.g., antiviral activity, antifungal activity, antibacterial activity).

[0183] In one or more other embodiments, the variant lectin has at least about 40% to about 99.9% sequence identity with a natural (wild-type) lectin amino acid sequence. In certain other embodiments, the variant lectin has at least about 40% sequence identity with a natural (wild-type) lectin amino acid sequence and has carbohydrate binding activity. In certain other embodiments, the variant lectin has at least about 40% sequence identity with a natural (wild-type) lectin amino acid sequence and has antimicrobial binding activity.

[0184] In other embodiments, the variant lectin has at least about 40%, 41%, 42%, 43%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135 7%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity.

[0185] In yet other embodiments, the variant lectin has at least about 40%, 41%, 42%, 43%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity.

[0186] In certain embodiments, the functional lectin protein comprises one or more non-catalytic carbohydrate binding sites. Thus, in certain one or more embodiments or aspects, the functional lectin protein can be characterized or evaluated based on its carbohydrate binding activity. In certain embodiments, the natural, variant and / or functional lectin can be described or defined by antimicrobial function / activity (e.g., antiviral activity, antifungal activity, antibacterial activity). In other one or more embodiments or aspects, the natural, variant and / or functional lectin can be screened or described by hemagglutination activity assays, etc.

[0187] IV. Gram-positive cells producing heterolectins

[0188] As briefly described above and further described in the Examples section below, Applicants have designed, constructed, and evaluated exemplary Gram-positive bacterial cells for their ability to express heterologous lectins. For example, in certain embodiments, a polynucleotide encoding a variant Q-GRFT protein (Q-GRFT; SEQ ID NO: 2) (expression cassette; Figure 6 ) were introduced into Bacillus cells and evaluated in large-scale bioreactors. More specifically, expression cassettes encoding Q-GRFT (SEQ ID NO: 2) variants were constructed for intracellular expression and / or secreted expression of Q-GRFT in Bacillus cells and evaluated using large-scale (about 10 L) bioreactors, as presented in Example 1. As described in that example, exemplary Bacillus strains containing an introduction cassette for secreted Q-GRFT expression or an introduction cassette for intracellular Q-GRFT expression were evaluated in Figure 6 As shown in , these strains produced comparable amounts of Q-GRFT under the large-scale conditions tested, demonstrating that Gram-positive bacterial cells / strains are particularly useful host strains for large-scale fermentation and production of lectin proteins.

[0189] In certain one or more embodiments, applicants contemplate that eliminating or reducing one or more (several) background enzymes (activities) will aid / facilitate the lectin downstream recovery and purification processes described herein (e.g., by reducing undesirable host cell background (native) protein contaminants). For example, the use of Bacillus species deficient in endogenous enzyme activity allows one to simplify the fermentation medium by eliminating complex protein sources, thereby producing a cleaner fermentation broth, faster and more efficient recovery of lectin proteins. Thus, in certain embodiments, making the recombinant Gram-positive host cell deficient in the production of one or more endogenous genes encoding one or more highly expressed background proteins (e.g., amylases, xylanases, pullulanases, phytases, pectate lyases, β-glucanases, mannosidases, lipases, esterases, etc.), applicants contemplate that the one or more lectin downstream recovery and purification processes described herein will be facilitated and / or the one or more lectin downstream recovery and purification processes described herein will be reduced in cost (e.g., see Section VII).

[0190] For example, in the case of certain Bacillus subtilis strains exemplified herein, these strains can be modified to be deficient in the production of one or more background enzymes (activities) such as α-amylase (e.g., amyE), protease (e.g., aprE), β-glucanase (e.g., bglS), etc. In particular, as described in the Examples below, certain Bacillus subtilis strains of the present disclosure have been modified to be deficient in the production of native α-amylase (amyE). In other embodiments, the Bacillus subtilis strains of the present disclosure are modified to be deficient in the production of native proteases.

[0191] In one or more specific embodiments, Bacillus subtilis strain CB462 comprises a deletion of the endogenous genes aprE, nprE (encoding the background proteases aprE and nprE) and alrA (used as a selection marker), Bacillus subtilis strain CB460 comprises a deletion of the endogenous genes aprE, nprE, epr, isp, bpr, wprA (encoding the background proteases aprE, nprE, epr, isp, bpr, wprA) and alrA (used as a selection marker), and Bacillus subtilis strain CB447 comprises a deletion of the endogenous genes aprE, nprE, epr, isp, bpr, wprA, vpr, m pr, nprB (encoding background proteases aprE, nprE, epr, isp, bpr, wprA, vpr, mpr, nprB) and alrA (used as a selection marker), Bacillus subtilis strain CB476 contains a deletion of the endogenous genes aprE, nprE, epr, isp, bpr, wprA, vpr, mpr, nprB (encoding background proteases aprE, nprE, epr, isp, bpr, wprA, vpr, mpr, nprB) and alrA (used as a selection marker), Bacillus subtilis strain CBS6 contains a deletion of the endogenous genes aprE, nprE, epr, isp, bpr, wprA, vpr, mpr, nprB subtilis strain CZ438 contains the deletion of the endogenous genes aprE, nprE, epr, isp, bpr, wprA, vpr, mpr, nprB (encoding the background proteases aprE, nprE, epr, isp, bpr, wprA, vpr, mpr, nprB) and alrA (used as a selection marker), and Bacillus subtilis strain CB488 contains the deletion of the endogenous genes aprE, nprE, epr, isp, bpr, wprA, vpr, mpr, nprB (encoding the background proteases aprE, nprE, epr, isp, bpr, wprA, vpr, mpr, nprB) and alrA (used as a selection marker). subtilis strain CBS12 contains a deletion of the endogenous genes aprE, nprE, epr, isp, bpr, wprA, vpr, mpr, nprB (encoding the background proteases aprE, nprE, epr, isp, bpr, wprA, vpr, mpr, nprB), amyE (encoding the background amylase AmyE), and scoC.

[0192] As further described in the Examples below (Example 15; Table 3), different classes of lectin proteins (e.g., jacaranda-like lectins, CVN-like lectins, OAA-like lectins, galectin-1-like lectins, ricin-like lectins, etc.) representing different protein folds (e.g., β-barrel, β-sandwich, β-prism, etc.) were screened for expression / production in recombinant (modified) Gram-positive cells of the present disclosure. More particularly, as demonstrated in the Examples, it was surprisingly observed that Gram-positive bacterial cells / strains (e.g., Bacillus sp. cells) are particularly suitable host strains for large-scale fermentation, production, and recovery of different families of lectin proteins (Table 3).

[0193] Therefore, certain embodiments of the present disclosure provide recombinant Gram-positive bacterial cells expressing one or more heterologous nucleic acids (polynucleotides) encoding lectin proteins. In certain embodiments, the recombinant Gram-positive bacterial cells express heterologous polynucleotides encoding natural lectin proteins or functional variants derived from natural lectin proteins. In certain aspects, the heterologous polynucleotides encoding lectin proteins are described as expression cassettes introduced into recombinant cells. In certain embodiments, at least one expression cassette is introduced into the Gram-positive bacterial cell. In other embodiments, at least two expression cassettes are introduced into the Gram-positive bacterial cell. For example, Figure 6 and Fig.10 Presented are suitable for intracellular expression of lectin proteins (e.g., Figure 6 A-6B) and / or extracellular expression / secretion (e.g., Figure 6 C-6D) is a schematic diagram of an exemplary lectin polynucleotide cassette.

[0194] Thus, in certain one or more embodiments or aspects, the Gram-positive host cells of the present disclosure comprise one or more lectin expression cassettes introduced therein, wherein when cultured under suitable conditions, the host cells express the lectin. In certain other one or more embodiments, the recombinant cells of the present disclosure comprise at least two introduced expression cassettes encoding the same heterologous lectin, or at least two introduced expression cassettes encoding different (heterologous) lectins. In certain other one or more embodiments or aspects of the present disclosure, the recombinant cells comprise one or more introduced expression cassettes encoding lectins for intracellular expression and / or one or more introduced expression cassettes encoding lectins for secretory (extracellular) expression and combinations thereof. For example, the recombinant cells of the present disclosure can be constructed to express both intracellular lectins and secretory lectins, which can be recovered and purified according to one or more methods / processes described herein.

[0195] In certain related embodiments, the gram-positive bacteria include Bacilli, Clostridium, and Mollicutes (e.g., including Lactobacillales with Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, Oscillospiraceae, Streptococcaceae, and Lactobacillales with Alicyclobacillus family). llaceae, Bacillaceae, Caryophanaceae, Listeriaceae, Paenibacillaceae, Planococcaceae, Sporolactobacillaceae, Staphylococcaceae, Thermoactinomycetaceae, Turicibacteraceae, Bacillales).

[0196] In certain embodiments, species of the Bacillaceae family include alkali-tolerant Bacillus, bibacillus, anaerobic Bacillus, Bacillus, thermoalkali Bacillus, cherry-like Bacillus, microbacterium, line Bacillus, geobacillus, tenuis, halobacillus, halolactobacillus, marine Bacillus, oceanic Bacillus, ornithonic Bacillus, marine Bacillus, halogen Bacillus, marine Bacillus, marine Bacillus, saccharococcus, halobacillus, halogen Bacillus, tenuis, deep-sea Bacillus, urea Bacillus, and branched Bacillus.

[0197] In other embodiments, Bacillus species cells include, but are not limited to, B. acidiceler, B. acidicola, B. acidocaldarius, B. acidoterrestris, B. aeolus, B. aerius, B. aerophilus, B. agaradhaerens, B. agri, B. aidingensis, B. acidoterrestris, B. aerolius, B. aerius, B. aerophilus, B. agaradhaerens, B. agri, B. aidingensis, B. acidoterrestris, B. aerolius ... akibai), Bacillus alcalophilus, Bacillus algicola, Bacillus alginolyticus, Bacillus alkalidiazotrophicus, Bacillus alkalinitrilicus, Bacillus alkalitelluris, Bacillus altitudinis, Bacillus alveayuensis, Bacillus alvei, Bacillus amylolyticus ), Bacillus aneurinilyticus, Bacillus aneurinolyticus, Bacillus anthracis, Bacillus aquimaris, Bacillus arenosi, Bacillus arseniciselenatis, Bacillus arsenicoselenatis, Bacillus arsenicus, Bacillus arvi, Bacillus asahii, Bacillus atrophaeus, Bacillus aurantii, Bacillus arsenicus, Bacillus arvi, Bacillus asahii, Bacillus atrophaeus, Bacillus aurantii urantiacus), B. axarquiensis, B. azotofixans, B. azotoformans, B. badius, B. barbaricus, B. bataviensis, B. beijingensis, B. benzoevorans, B. bogoriensis, B. boroniphilus, B. potsdamiensisborstelenis), Bacillus butanolivorans, Bacillus carboniphilus, Bacillus cecembensis, Bacillus cellulosilyticus, Bacillus centrosporus, Bacillus chagannorensis, Bacillus chitinolyticus, Bacillus chondroitinus, Bacillus choshinensis, Bacillus foodii, Bacillus cibi), B.circulans, B.clarkii, B.clausii, B.coagulans, B.coahuilensis, B.cohnii, B.curdianolyticus, B.cycloheptanicus, B.decisifrondis, B.decolorationis, B.dipsosauri, B. Bacillus drentensis, Bacillus edaphicus, Bacillus ehimensis, Bacillus endophyticus, Bacillus farraginis, Bacillus fastidiosus, Bacillus firmus, Bacillus plexus, Bacillus foraminis, Bacillus fordii, Bacillus formosus, Bacillus fortis, Bacillus fumarioli, Bacillus funiculus, Bacillus fusiformis, Bacillus galactophilus, Bacillus galactosidilyticus, Bacillus gelatini, Bacillus gibsonii, Bacillus ginsengi, Bacillus ginsengihumi, Bacillus globisporus, Bacillus globisporus subsp. Globisporus, Bacillus globisporus marine subsp.globisporus subsp. Marinus), Bacillus glucanolyticus, Bacillus gordonae, Bacillus halmapalus, Bacillus haloalkaliphilus, Bacillus halodenitrificans, Bacillus halodurans, Bacillus halophilus, Bacillus hemicellulosilyticus, Bacillus herbersteinensis, Bacillus horikoshii, Bacillus halogenide ... horti), Bacillus hemi, Bacillus hwajinpoensis, Bacillus idriensis, Bacillus indicus, Bacillus infantis, Bacillus infernus, Bacillus insolitus, Bacillus isabeliae, Bacillus jeotgali, Bacillus kaustophilus, Bacillus kobensis, Bacillus koreensis, Bacillus kribbensis, Bacillus kribbensis, Bacillus krugeri, Bacillus horti krulwichiae), Bacillus laevolacticus, Bacillus larvae, Bacillus laterosporus, Bacillus lautus, Bacillus lehensis, Bacillus lentimorbus, Bacillus lentus, Bacillus litoralis, Bacillus luciferensis, Bacillus macaue nsis), B. macerans, B. macquariensis, B. machyae, B. malacitus, B. mannanilyticus, B. marinus, B. marisflavi, B. marismortui, B. massiliensis, B. methanolicae, B.methanolicus), B. migulanus, B. mojavensis, B. mucilaginosus, B. muralis, B. murimartini, B. mycoides, B. naganoensis, B. nealsonii, B. neidei, B. niabensis, B. niacini, B. niaconi (B.novalis), B.odysseyi, B.okhensis, B.okuhidensis, B.oleronius, B.oshimensis, B.pabuli, B.pallidus, B.illeg., B.panaciterrae, B.pantothenticus, B.parabrevis, B.pabuli asteurii), B. patagoniensis, B. peoriae, B. plakortidis, B. pocheonensis, B. polygoni, B. polymyxa, B. popilliae, B. pseudoalcaliphilus, B. pseudofirmus, B. pseudomycoides, B. psychrotrophicus Bacillus psychrodurans, Bacillus psychrophilus, Bacillus psychrosaccarolyticus, Bacillus psychrotolerans, Bacillus pulvifaciens, Bacillus pycnus, Bacillus qingdaonensis, Bacillus reuszeri, Bacillus runs, Bacillus safensis, Bacillus salarius, Bacillus halogenus ...salexigens), Bacillus saliphilus, Bacillus schlegelii, Bacillus selenatarsenatis, Bacillus selenitrireducens, Bacillus seohaeanensis, Bacillus shackletonii, Bacillus silvestris, Bacillus simplex, Bacillus sira lis), B.smithii, B.soli, B.sonorensis, B.sphaericus, B.sporothermodurans, B.stearothermophilus, B.stratosphericus, B.subterraneus, B.subtilis subsp.Spizizenii, B.subtilis subsp. subsp.Subtilis), Taiwan Bacillus (B.taeanensis), Tequilensis Bacillus (B.tequilensis), Thermoantarcticus Bacillus (B.thermantarcticus), Thermoaerophilus Bacillus (B.thermoaerophilus), Thermoamylovorans Bacillus (B.thermoantarcticus), Thermocatenulatus Bacillus (B.thermocloacae), Thermodenitrificans Bacillus (B.thermoglucoside) Bacillus thermoglucosidasius, Bacillus thermoleovorans, Bacillus thermoruber, Bacillus thermosphaericus, Bacillus thiaminolyticus, Bacillus thioparans, Bacillus thuringiensis, Bacillus tusciae, Bacillus validus, Bacillus vallismortis, Bacillus vedderi, Bacillus vedderi, Bacillus vedderi, Bacillus thiaminolyticus, Bacillus thioparans, Bacillus thuringiensis, Bacillus tusciae, Bacillus vallismortis, Bacillus vedderi ...velezensis), B. vietnamensis, B. vireti, B. vulcani, B. wakoensis, and B. weihenstephanensis.

[0198] In a particular embodiment, the Bacillus species cell is selected from the group consisting of Bacillus subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. radiata, and B. thuringiensis. As used herein, "Bacillus" includes Bacillus species that have been reclassified, including but not limited to B. stearothermophilus, which is now named Geobacillus stearothermophilus.

[0199] V. Molecular Biology

[0200] As generally set forth above and further described in the Examples below, certain embodiments of the present disclosure relate to recombinant Gram-positive bacterial cells expressing heterologous lectin proteins, recombinant polynucleotides (e.g., vectors, expression cassettes) encoding heterologous lectin proteins that are particularly suitable for introduction (e.g., transformation) into Gram-positive host cells (i.e., for expressing heterologous lectins), and the like. In other embodiments, the Gram-positive host cells of the present disclosure are constructed to be (made to be) deficient in the production of one or more endogenous proteins (e.g., enzymes). For example, in one or more embodiments or aspects of the present disclosure, Applicants contemplate that eliminating or reducing one or more (several) background enzymes (activities) will aid / facilitate the lectin downstream recovery and purification processes described herein (e.g., by reducing undesirable host cell background (native) protein contaminants).

[0201] Thus, in one or more embodiments or aspects, the recombinant host cell may comprise a genetic modification (e.g., deletion, disruption, interfering RNA, etc.) of one or more endogenous genes encoding one or more native background proteins (e.g., glycoside hydrolases native to the recombinant cell, proteases native to the recombinant cell, etc.). In certain one or more embodiments, the recombinant cells of the present disclosure are made defective in the production of one or more endogenous proteins, including, but not limited to, amylases, pullulanases, xylanases, proteases, etc. For example, in the case of a recombinant Bacillus subtilis cell expressing one or more lectin proteins, the cell may be made defective in the production of one or more endogenous proteins, including, but not limited to, α-amylases (e.g., amyE), β-glucanases (e.g., bglS), esterases (e.g., pnbA), lipases (e.g., lipA), mannosidases (e.g., gmuG), pectin lyases (e.g., pel), phytases (e.g., phy), proteases (e.g., aprE, nprE, etc.), pullulanases (e.g., amyX), etc.

[0202] Therefore, certain embodiments of the present disclosure are particularly directed to nucleic acids, polynucleotides (e.g., plasmids, vectors, expression cassettes), regulatory elements, etc. suitable for use in constructing recombinant Gram-positive host cells. Therefore, as shown in the examples and generally described herein, standard and conventional recombinant DNA and molecular cloning techniques well known in the art can be used by those skilled in the art to construct the recombinant cells of the present disclosure. Methods for genetically modifying cells include, but are not limited to, (a) introducing, replacing, or removing one or more nucleotides in a gene, or introducing, replacing, or removing one or more nucleotides in a regulatory element required for transcription or translation of a gene, (b) gene disruption, (c) gene conversion, (d) gene deletion, (e) gene downregulation, (f) site-specific mutagenesis, and / or (g) random mutagenesis.

[0203] In certain embodiments, the recombinant (modified) cells of the present disclosure can be constructed by reducing or eliminating the expression of a gene using methods well known in the art (e.g., insertion, disruption, substitution or deletion). The portion of the gene to be modified or inactivated can be, for example, a coding region or a regulatory element required for expression of the coding region.

[0204] Examples of such regulatory or control sequences may be promoter sequences or functional portions thereof (i.e., portions sufficient to affect the expression of the nucleic acid sequence). Other control sequences for modification include, but are not limited to, leader sequences, propeptide sequences, signal sequences, transcription terminators, transcription activators, and the like.

[0205] In some other embodiments, modified cells are constructed by gene deletion to eliminate or reduce the expression of genes.Gene deletion technology enables partial or complete removal of one or more genes, thereby eliminating their expression, or expressing non-functional (or reduced activity) protein products.In such methods, the deletion of one or more genes can be completed by homologous recombination using a plasmid constructed to continuously contain 5' and 3' regions flanking the gene.By way of example, continuous 5' and 3' regions can be introduced into the Bacillus cells associated with the second selectable marker at a permissive temperature (for example, on a temperature-sensitive plasmid (such as pE194)), to allow plasmids to be established in cells.Then the cells are transferred to a non-permissive temperature to select cells that integrate plasmids into one of the homologous flanking regions of the chromosome.The selection of plasmid integration is realized by selecting the second selectable marker.After integration, the recombination event at the second homologous flanking region is stimulated by moving the cells to the permissive temperature for several generations without selecting.Cells are plated to obtain single colonies, and it is checked whether the colonies lose two selectable markers. Thus, one skilled in the art can readily identify nucleotide regions (suitable for complete or partial deletion) in the coding sequence of a gene and / or in the non-coding sequence of a gene.

[0206] In other embodiments, modified cells are constructed by introducing, replacing or removing one or more nucleotides in a gene or a regulatory element required for its transcription or translation. For example, nucleotides can be inserted or removed to cause the introduction of a stop codon, the removal of a start codon or a frameshift of an open reading frame. Such modifications can be accomplished by site-directed mutagenesis or PCR-generated mutagenesis according to methods known in the art. Therefore, in certain embodiments, a gene of the present disclosure is inactivated by complete or partial deletion.

[0207] In another embodiment, modified cells are constructed by gene conversion techniques. For example, in gene conversion methods, the nucleotide sequence corresponding to one or more genes is induced in vitro to produce defective nucleic acid sequences, and then the defective nucleic acid sequences are transformed into parental Bacillus cells to produce defective genes. Through homologous recombination, defective nucleic acid sequences replace endogenous genes. It may be desirable that defective genes or gene fragments also encode markers that can be used to select transformants containing defective genes. For example, defective genes can be introduced into non-replicating or temperature-sensitive plasmids by association with selectable markers. The selection of plasmid integration is realized by selecting markers under conditions that do not allow plasmid replication. The selection of the second recombination event that causes gene replacement is realized by checking whether the bacterium colony loses the selectable marker and whether the gene obtained through mutation is realized. Alternatively, the defective nucleic acid sequence can contain the insertion, replacement or disappearance of one or more nucleotides of a gene, as described below.

[0208] In other embodiments, modified cells are constructed using nucleotide sequences complementary to the nucleotide sequence of the gene by the antisense technology established. More particularly, the expression of the gene of the host cell can be reduced (downregulated) or eliminated by introducing a nucleotide sequence complementary to the nucleotide sequence of the gene, which can be transcribed in the cell and can hybridize with the mRNA produced in the cell. Under conditions where the complementary antisense nucleotide sequence is allowed to hybridize with the mRNA, the amount of the translated protein is therefore reduced or eliminated. Such antisense methods include, but are not limited to, RNA interference (RNAi), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, etc., all of which are well known to skilled technicians.

[0209] In other embodiments, modified cells are constructed by random or specific mutagenesis using methods well known in the art (including but not limited to chemical mutagenesis and transposition).Gene modification can be performed by subjecting parental cells to mutagenesis and screening mutant cells whose gene expression has been reduced or eliminated.Specific or random mutagenesis can be performed, for example, by using suitable physical or chemical mutagens, using suitable oligonucleotides, or subjecting DNA sequences to mutagenesis produced by PCR. In addition, mutagenesis can be performed by using any combination of these mutagenesis methods.Examples of physical or chemical mutagens suitable for the purposes of the present invention include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrous acid, ethyl methanesulfonate (EMS), sodium bisulfite, formic acid, and nucleotide analogs. When such agents are used, mutagenesis is typically performed by incubating the parental cells to be mutagenized under appropriate conditions in the presence of the selected mutagenizing agent, and selecting for mutant cells that exhibit reduced or no expression of the gene.

[0210] PCT Publication No. WO 2003 / 083125 discloses methods for modifying Bacillus cells, such as using PCR fusion to generate Bacillus deletion strains and DNA constructs to bypass E. coli. PCT Publication No. WO 2002 / 14490 discloses methods for modifying Bacillus cells, including (1) construction and transformation of an integrating plasmid (pComK), (2) random mutagenesis of coding sequences, signal sequences, and propeptide sequences, (3) homologous recombination, (4) increasing transformation efficiency by adding non-homologous flanks to transforming DNA, (5) optimizing double crossover integration, (6) site-directed mutagenesis, and (7) markerless deletion.

[0211] Suitable methods for introducing polynucleotide sequences into bacterial cells (e.g., E. coli, Bacillus species) are well known to those skilled in the art. In fact, methods such as transformation, including protoplast transformation and mid-plate collection, transduction, and protoplast fusion, are known and suitable for use in the present disclosure. Transformation methods are particularly suitable for introducing the DNA constructs of the present disclosure into host cells.

[0212] In addition to common methods, in some embodiments, host cells are directly transformed (i.e., before being introduced into host cells, intermediate cells are not used to amplify DNA constructs or otherwise process DNA constructs). Introducing DNA constructs into host cells includes those physical and chemical methods known in the art for introducing DNA into host cells without inserting into plasmids or vectors. Such methods include but are not limited to calcium chloride precipitation, electroporation, naked DNA, liposomes, etc. In other embodiments, DNA constructs are co-transformed with plasmids without inserting into the plasmids. In other embodiments, by methods known in the art, selective markers are deleted or substantially excised from modified Gram-positive bacterial strains. In certain embodiments, the vector is decomposed from the host chromosome, the flanking regions are left on the chromosome, and the intrinsic chromosome regions are removed.

[0213] Promoters and promoter sequence regions for expressing genes, their open reading frames (ORFs) and / or their variant sequences in Gram-positive cells are generally known to those skilled in the art. The promoter sequences of the present disclosure are generally selected so that they function in Gram-positive host cells (e.g., Bacillus cells, such as Bacillus licheniformis cells, Bacillus subtilis cells, Bacillus amyloliquefaciens, etc.). For example, promoters that can be used to drive gene expression in Bacillus cells include, but are not limited to, the Bacillus subtilis alkaline protease (aprE) promoter, the α-amylase promoter of Bacillus subtilis (amyE), the α-amylase promoter of Bacillus licheniformis (amyL), the α-amylase promoter of Bacillus amyloliquefaciens, the neutral protease (nprE) promoter from Bacillus subtilis, a mutant aprE promoter, or any other promoter from Bacillus licheniformis or other related Bacillus. Methods for screening and generating promoter libraries with a range of activities (promoter strengths) in Bacillus cells are described in PCT Publication No. WO 2002 / 14490.

[0214] VI. Cultivation of Gram-positive cells for lectin production

[0215] In certain embodiments, the present disclosure provides recombinant cells capable of producing a desired lectin protein. More particularly, certain embodiments are (recombinant) gram-positive bacterial cells expressing relevant genetic modifications of heterologous lectins. Therefore, particular embodiments relate to culturing (fermenting) gram-positive cells for producing lectin proteins. Typically, gram-positive cells are fermented using fermentation methods well known in the art.

[0216] In certain embodiments, these cells are grown under batch or continuous fermentation conditions.Classic batch fermentation is a closed system, wherein the composition of the culture medium is set at the beginning of fermentation, and the composition does not change during fermentation.At the beginning of fermentation, the culture medium is inoculated with one or more required organisms.In this method, fermentation is allowed to occur without adding any component to the system.Typically, batch fermentation meets the qualification of "batch" about adding carbon source, and control factors (such as temperature, pH and oxygen concentration) are often attempted.The metabolites and biomass composition of batch systems are constantly changing until fermentation stops.In batch culture, cells progress to high growth logarithmic phase by static lag phase, and finally enter the stable phase where growth rate decreases or stops.If not treated, the cells in the stationary phase eventually die.Usually, cells in the later stage of logarithmic growth are responsible for the large-scale production of products.

[0217] The suitable modification of standard batch system is " fed-batch fermentation " system.In this modification of typical batch system, along with the progress of fermentation, substrate is added with increment.When catabolite repression may inhibit the metabolism of cell and in the case of expecting to have limited amount of substrate in culture medium, fed-batch system is useful.It is difficult and therefore it is estimated based on the variation of measurable factors in fed-batch system, and these measurable factors are partial pressures such as pH, dissolved oxygen and waste gas (such as CO , O ).Batch and fed-batch fermentation are commonly used and well known in the art.

[0218] Continuous fermentation is an open system, wherein the fermentation medium limited is continuously added to the bioreactor, and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation usually maintains the culture at a constant high density, wherein the cell is mainly in the logarithmic phase growth. Continuous fermentation allows one or more factors affecting cell growth and / or product concentration to be regulated. For example, in one embodiment, a limiting nutrient (such as a carbon source or a nitrogen source) is maintained at a fixed rate, and all other parameters are allowed to be regulated. In other systems, many factors affecting growth can be constantly changed, while the cell concentration measured by the turbidity of the culture medium remains unchanged. Continuous systems strive to maintain steady-state growth conditions. Therefore, the cell loss caused by extracting the culture medium should be balanced with the cell growth rate in the fermentation. The method for adjusting the nutrients and growth factors used for continuous fermentation technology and the technology for maximizing the product formation rate are well known in the field of industrial microbiology.

[0219] Cultivation / fermentation is typically accomplished in a growth medium comprising an aqueous mineral salts medium, organic growth factors, carbon and energy sources, molecular oxygen, and of course a starting inoculum of the microbial host to be used.

[0220] In addition to the carbon and energy sources, oxygen, assimilable nitrogen and the microbial inoculum, it is necessary to supply the appropriate amounts of mineral nutrients in the right proportions to ensure proper microbial growth, maximize the assimilation of the carbon and energy sources by the cells during microbial conversion, and obtain maximum cell yield and maximum cell density in the fermentation medium.

[0221] The composition of the aqueous mineral medium may vary within wide limits, depending in part on the microorganisms and substrates used, as is known in the art. In addition to nitrogen, these mineral media should also include suitable amounts of phosphorus, magnesium, calcium, potassium, sulfur and sodium in suitable soluble assimilable ionic and combined forms, and preferably certain trace elements such as copper, manganese, molybdenum, zinc, iron, boron and iodine, as well as others, also in suitable soluble assimilable forms, should also be present, all as known in the art.

[0222] The fermentation reaction is an aerobic process in which the required molecular oxygen is supplied by a molecular oxygen-containing gas such as air, oxygen-enriched air, or even substantially pure molecular oxygen, as long as the contents of the fermentation vessel are maintained at a suitable oxygen partial pressure effective to aid the growth of the microbial species in an active manner.

[0223] The fermentation temperature may vary somewhat, but for most Gram-positive cells it will generally be in the range of about 20°C to 40°C.

[0224] Microorganisms also need assimilable nitrogen sources. Assimilable nitrogen sources can be any nitrogenous compound or can release nitrogen in a form suitable for microorganisms to metabolize and utilize. Although various organic nitrogen source compounds such as protein hydrolysates can be used, cheap nitrogenous compounds such as ammonia, ammonium hydroxide, urea and various ammonium salts (such as ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride or various other ammonia compounds) can be used usually. Ammonia itself is convenient for large-scale operation, and can be used by bubbling through aqueous fermentation (fermentation medium) in a suitable amount. At the same time, such ammonia can also be used to help control pH.

[0225] The pH range in the aqueous microbial ferment (fermentation mixture) should be within the exemplary range of about 2.0 to 8.0. The preference for the microbial pH range depends to some extent on the culture medium employed and the specific microorganism, and thus varies slightly with changes in the culture medium, as can be readily determined by those skilled in the art.

[0226] In some aspects, fermentation is carried out in a manner that the carbon-containing substrate can be controlled as a limiting factor, thereby providing cells with a good conversion of the carbon-containing substrate and avoiding these cells to be polluted and inhibited by the unconverted substrate of the basic amount. The latter situation is not a problem for water-soluble substrates, because any remaining trace materials can be easily washed off. However, this may be a problem under the situation of non-water-soluble substrates, and the product treatment steps that need to be increased are such as suitable washing steps.

[0227] As mentioned above, the time to reach this level is not critical and may vary with the specific microorganism and fermentation process being performed. However, it is well known in the art how to determine the carbon source concentration in the fermentation medium and whether the desired carbon source level has been reached.

[0228] If desired, part or all of the carbon source and energy material and / or part of the assimilable nitrogen source (such as ammonia) may be added to the aqueous mineral culture medium before feeding the aqueous mineral culture medium to the fermentor.

[0229] Each stream introduced into the reactor is preferably controlled at a predetermined rate, or in response to demand which can be determined by monitoring, for example, the concentration of carbon and energy substrates, pH, dissolved oxygen, oxygen or carbon dioxide in the off-gas from the fermentor, cell density measurable by dry cell weight, light transmittance, etc. The feed rates of the various materials can be varied to obtain the fastest possible cell growth rate consistent with efficient utilization of the carbon source and energy source, to obtain the highest possible microbial cell yield relative to substrate variation.

[0230] In batch operation or preferably fed-batch operation, all equipment, reactors or fermentation apparatus, vessels or containers, piping, associated circulation or cooling equipment, etc. are initially sterilized, usually by the use of steam such as at about 121° C. for at least about 15 minutes. The sterilized reactor is then inoculated with a culture of the selected microorganism in the presence of all required nutrients, including oxygen and a carbon-containing substrate. The type of fermentor used is not critical.

[0231] VII. Lectin Recovery and Purification Process

[0232] As further detailed below and presented in the examples, the present disclosure further describes and exemplifies particularly suitable processes (methods) for harvesting, clarifying, recovering, purifying, etc., a fermentation broth in which one or more lectin proteins have been produced. Thus, certain embodiments relate, inter alia, to collecting broth at the end of fermentation, harvesting the collected broth, recovering one or more lectins from the harvested broth (e.g., such as clarifying the harvested broth, concentrating the clarified broth, purifying the clarified broth concentrate, etc.). In certain aspects, a purified lectin (protein) preparation is derived from a fermentation broth collected and harvested as described herein.

[0233] Certain other aspects of the present disclosure provide, inter alia, novel methods for recovering and optionally purifying lectins obtained from recombinant cells expressing lectins (e.g., recombinant Gram-negative cells, recombinant Gram-positive cells, recombinant plant (e.g., tobacco) cells, etc.). Certain other aspects of the present disclosure provide, inter alia, novel methods for recovering and optionally purifying lectins obtained from naturally occurring sources (e.g., see, Section III, Lectin Proteins).

[0234] Thus, in certain aspects, lectin protein preparations are recovered according to the compositions and methods of the present disclosure. In other aspects, lectin preparations are recovered and purified according to the methods of the present disclosure. As used herein, the terms "purified," "isolated," or "enriched" with respect to "lectin" (protein) mean that the lectin is converted from a less pure state by separating it from some or all of the contaminants with which it is associated. Contaminants include, but are not limited to, microbial cells, metabolites, solvents, chemicals, colors, inactive forms of the target lectin, aggregates, processing aids, inhibitors, fermentation media, cell debris, nucleic acids, proteins other than the target lectin, host cell proteins, cross-contaminants from production equipment, and the like.

[0235] Thus, in the context of "purified lectin" as used herein, purification can be accomplished by art-recognized separation techniques, including but not limited to ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, heat treatment, ammonium sulfate precipitation or other protein salt precipitation, crystallization, centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis, or gradient separation to remove whole cells, cell debris, impurities, extraneous proteins, or enzymes that are not desired in the final composition.

[0236] The purified or isolated lectin composition may then be further supplemented with ingredients that provide additional benefits, such as activators, anti-inhibitors, desired ions, compounds that control pH, or other enzymes or chemicals.

[0237] As used herein, "lectin" purity is a relative term and is not intended to be limiting when used in phrases such as "the recovered lectin has a higher purity, the same purity, or a lower purity than before the recovery process." For example, the relative "purity" of the lectin (protein) before and after the recovery process can be determined using methods known in the art, including but not limited to general quantitative methods (e.g., Bradford assay, UV-Vis, activity assay), electrophoresis analysis (SDS-PAGE), analytical HPLC, mass spectrometry, hydrophobic interaction chromatography, etc.

[0238] Non-limiting examples for assessing the relative purity of lectins include, but are not limited to, SDS-PAGE analysis and / or A analysis of non-lectins (impurities) relative to lectins. 280 ratio.

[0239] For example, relative lectin purity determined via SDS-PAGE can be determined by the visual abundance of lectin (protein) bands compared to non-lectin protein (unwanted contaminants; impurities) bands present in the preparation. Alternatively, the relative purity of the lectin can be determined by the (A) abundance of non-lectin to lectin. 280 ) ratio. For example, A 280 The ratio is a measure of the amount of absorbance at 280 nm contributed by non-lectin impurities for 1 unit of absorbance at 280 nm contributed by lectins in a protein preparation (e.g., non-lectin A 280 / lectin A 280 ), where a smaller number means a higher purity. More specifically, for determining the lectin (A 280 ) concentration can be measured by HPLC using purified lectin as a standard, where the concentration determined by HPLC is converted to lectin A using 1 mg / mL lectin at 280 nm absorbance = 0.936. 280 . Used to determine the non-agglutinin (A280 ) concentration method can be measured using a 1 cm path cuvette zeroed with MilliQ water and diluted to A with MilliQ water as needed. 280 <1, where Lectin A is subtracted from the preparation measurement 280 Non-lectin A 280 concentration.

[0240] Thus, according to certain aspects, a lectin (protein) preparation is recovered from a fermentation broth, wherein the recovered lectin preparation has a higher purity after undergoing one or more recovery processes described herein. For example, the fermentation broth (e.g., the whole broth at the end of fermentation) can be subjected to one or more protein recovery processes, including but not limited to a broth conditioning process, a broth clarification process, a protein enrichment and / or protein purification process (e.g., protein concentration, filtration, precipitation, crystallization, crystal separation, crystal sludge dissolution process, etc.), a buffer exchange process, a sterile filtration process, etc. In certain aspects, the fermentation broth is subjected to a broth treatment (broth conditioning) process to improve subsequent broth treatment characteristics.

[0241] In certain embodiments, such as when Gram-positive host cells have been constructed for intracellular lectin expression, the fermentation broth is subjected to a cell lysis process. For example, cell lysis processes include, but are not limited to, enzymatic treatment (e.g., lysozyme, proteinase K treatment), chemical means (e.g., ionic liquids), physical means (e.g., French press, ultrasound), simply maintaining the culture without feed, etc. Thus, in certain preferred embodiments, the broth lysis process releases the intracellular lectin into the (lysed) cell broth.

[0242] Thus, as described herein, the methods / processes disclosed herein are not intended to be limiting, as a skilled artisan can readily adapt or modify one or more of the compositions and / or methods disclosed herein for the recovery of specific lectin proteins and / or combinations thereof. In certain aspects, the fermentation broth obtained by fermenting Gram-positive cells expressing secreted lectins can be processed by harvesting, clarifying, and concentrating the broth, as generally described in Example 2. More particularly, as described in Example 2, the concentrate had a lectin (Q-GRFT) recovery of 65%, which was substantially purer than the broth supernatant.

[0243] Likewise, as demonstrated in Examples 3 and 4, the fermentation broth harvested, clarified and / or concentrated as described above can be further enriched or purified by pH treatment and / or heat treatment. More specifically, as described in Example 3, the ultrafiltration (UF) concentrate prepared in Example 2 was treated at different pH and temperature ranges, and these UF concentrate samples were subsequently centrifuged and the supernatants were collected for analysis by SDS-PAGE. As presented in this example, all pH treated supernatants (i.e., at 5° C., 22° C., and 55° C.) had higher purity than the initial whole broth sample.

[0244] Additionally, the ultrafiltration (UF) concentrate from Example 2 was subjected to low pH and heat treatment, further including the addition of activated carbon, as described in Example 4, wherein the low pH / heat treated sample was subsequently filtered and the purity of the collected filtrate was analyzed by SDS-PAGE. Significant purification of Q-GRFT was achieved with low pH treatment (conditioning) followed by filtration, wherein incubation of the sample at 60° C. further improved protein purity, and the addition of activated carbon further reduced the color of the collected filtrate (data not shown).

[0245] In certain other embodiments, fermentation broth obtained by fermenting Gram-positive cells expressing an intracellular lectin protein is processed by harvesting, lysing, clarifying and concentrating the broth as generally described in Example 5. As described in this example, the fermentation broth is harvested at the end of fermentation and treated with lysozyme, pH / heat treated and the cooled broth collected, wherein the collected broth is recovered using the same microfiltration and ultrafiltration procedures as outlined and described in Example 2. More specifically, as described in Example 5, a visually clear concentrate is produced, wherein the heat-treated broth supernatant provides a significant purity improvement over the untreated broth supernatant, wherein the Q-GRFT purity is maintained through processing steps such as microfiltration for cell separation and ultrafiltration (UF) for dewatering (data not shown).

[0246] In certain other embodiments, the fermentation broth of the present disclosure is processed as described in Example 6. More particularly, the fermentation broth of Gram-positive bacterial cells containing two (2) introduced copies of the Q-grft expression cassette is processed using the same lysozyme treatment, pH adjustment and heat treatment process as described above (see Example 5), wherein the broth is recovered as described in Example 6. More particularly, as described in Example 6, a visually clear concentrate is produced, wherein the pH-adjusted and heat-treated broth supernatant exhibits a significant increase in purity relative to the untreated broth supernatant (data not shown). Likewise, the Q-GRFT purity is maintained by the processing steps for cell separation, flocculation and Buchner filtration, and ultrafiltration (UF) for dehydration. The purity of the UF concentrate obtained is similar to that of the concentrate of Example 5 derived from a similarly treated broth but recovered using microfiltration for cell separation, and is higher in purity than the UF concentrate derived from the untreated broth (Example 2).

[0247] In yet other embodiments, fermentation broth obtained by fermenting Gram-positive cells expressing secreted lectins is processed by harvesting, clarifying, and concentrating the broth as generally described in Example 7. As described in that example, the fermentation broth is held for a sufficient amount of time (under specified conditions) at the end of fermentation, gently mixed, then heat treated for a sufficient amount of time (pH adjusted as needed), and then the broth is cooled and collected and recovered as described in Example 6. In particular, the pH-adjusted and heat-treated broth supernatant provides a significant increase in purity relative to the untreated broth supernatant, wherein the Q-GRFT purity is maintained through the processing steps (e.g., flocculation and Buchner filtration for cell separation and ultrafiltration (UF) for dewatering (data not shown)).

[0248] In other embodiments, one or more broth concentrates of the present disclosure may be subjected to one or more protein purification processes. More particularly, as described above and illustrated in Examples 8-10, the one or more protein purification processes include a protein crystallization process, wherein all concentrates processed according to Example 8 produced square to rectangular plate-like crystals (data not shown), all concentrates processed according to Example 9 formed crystals in the filtrate (see Table 1), and all concentrates processed according to Example 10 formed square to rectangular plate-like crystals in the filtrate (data not shown).

[0249] In other aspects, the present disclosure provides methods and compositions for producing or obtaining highly pure lectin (protein) preparations. More particularly, as described in Example 11, a crystal slurry is easily derived from a broth concentrate, which may include one or more crystal washing steps. As described in this example, after about 48 hours of crystallization, the crystal slurry was centrifuged and the supernatant was decanted, showing an apparent crystallization rate of about 92%. More particularly, the purification factor was increased by about 2.1 to 3.3 times relative to the initial UF concentrate (see Table 2). Figure 8 As presented in , a highly pure protein preparation was obtained by solubilizing the crystalline precipitate in sodium acetate followed by filtration, with Q-GRFT being the only band visible by SDS-PAGE in both the no-wash and 1× wash solubilization filtrates (see Figure 8 ). Similarly, Example 12 describes exemplary recovery of crystals and dissolution of crystal precipitates of various clarified broth concentrates of the present disclosure, wherein a filtrate of single-band or near single-band purity was obtained.

[0250] In other embodiments, the broth concentrate of the present disclosure is subjected to crystallization as generally described in Example 13, wherein suitable crystals are formed in solution under specified conditions with and without added salt.

[0251] In certain other embodiments, the present disclosure provides methods for obtaining / recovering a high purity lectin protein preparation, as generally described in Example 14. More particularly, as described in this example, a high purity lectin preparation is obtained by liquid-liquid (two-phase) extraction, wherein the lectin protein preferentially partitions into one of the fractions. For example, a lectin (Q-GRFT) preparation obtained / recovered by such a two-phase extraction method (Example 14; see Fig. 9 ) has a purity greater than 90%.

[0252] As further presented and described in Examples 15-17 below, the recombinant Gram-positive cells of the present disclosure can express / produce different classes of lectins, including but not limited to japanese lectin-like lectins, CVN-like lectins, OAA-like lectins, ricin-like lectins, galectin-1-like lectins, etc. More particularly, as set forth in Examples 15 and 16, one or more lectins described herein can be produced in the recombinant (modified) Gram-positive cells of the present disclosure for large-scale fermentation, production, recovery and optional purification of the expressed / produced one or more lectins.

[0253] For example, as described in Example 15, expression plasmids for thirty-five (35) different lectins were generated based on Bacillus subtilis strain CBS12 and their expression was evaluated in 2.5 L high efficiency flasks. In particular, SDS-Page analysis of supernatants from these strains revealed significant expression of many different lectins that exhibited a variety of protein folds, molecular weights, carbohydrate binding specificities, and species of origin ( Fig.11 ).

[0254] As described in Example 16, the recombinant lectins produced as described above can be fermented, recovered, and purified using exemplary purification methods, such as acid treatment, isoelectric precipitation, and ion affinity separation via chromatographic columns (Example 16, Table 4).

[0255] The generated (purified or crude) lectins are tested for functional activity using a hemagglutination assay as described in Example 17. For example, active lectins react with specific carbohydrate moieties on the surface of erythrocytes (resulting in the formation of a diffusion matrix), whereas inactive lectins are unable to bind to erythrocytes (resulting in the formation of distinct clumps), which allows for clear differentiation between active and inactive samples (e.g., Fig.12 As described in this example, hemagglutination ability of lectins was evaluated using erythrocytes from 15 sources ( Fig.13 ). It is expected that various lectins will react differently with erythrocytes from different species based on their carbohydrate specificity and carbohydrate binding affinity.

[0256] VIII. Exemplary Embodiments

[0257] Non-limiting examples of the compositions and methods disclosed herein are as follows:

[0258] 1. A method for producing a lectin in a Gram-positive bacterial cell, the method comprising (a) obtaining a Gram-positive bacterial cell and introducing an expression cassette encoding the lectin into the cell, wherein the cassette comprises an upstream promoter sequence operably linked to a downstream open reading frame (ORF) encoding the lectin, and (b) fermenting the cell under suitable conditions for producing the lectin.

[0259] 2. The method of embodiment 1, wherein the lectin is expressed intracellularly, and / or the lectin is expressed and secreted extracellularly.

[0260] 3. The method of embodiment 1, wherein the introduced cassette is integrated into the genome of the cell.

[0261] 4. The method of embodiment 1, wherein the cell comprises at least two introduced cassettes encoding the lectin, wherein the cassettes comprise an upstream promoter sequence operably linked to a downstream ORF encoding the lectin.

[0262] 5. The method of embodiment 4, wherein the at least two introduced cassettes are integrated into the genome of the cell.

[0263] 6. The method of embodiment 4, wherein the at least two introduced cassettes comprise the same ORF encoding the same lectin, or comprise different open reading frames (ORFs) encoding different lectins.

[0264] 7. A method for producing a lectin in a Gram-positive bacterial cell, the method comprising (a) obtaining a Gram-positive bacterial cell and introducing an expression cassette encoding the lectin into the cell, wherein the cassette comprises an upstream promoter sequence operably linked to a downstream nucleic acid encoding a signal (secretion) sequence, the signal (secretion) sequence being operably linked to a downstream open reading frame (ORF) encoding the lectin, and (b) fermenting the cell under suitable conditions for producing the lectin.

[0265] 8. The method of embodiment 7, wherein the lectin is expressed and secreted extracellularly.

[0266] 9. The method of embodiment 7, wherein the introduced cassette is integrated into the genome of the cell.

[0267] 10. The method of embodiment 7, wherein the cell comprises at least two introduced cassettes encoding the lectin, wherein the cassettes comprise an upstream promoter sequence operably linked to a downstream nucleic acid encoding a signal sequence, the signal sequence operably linked to a downstream ORF encoding the lectin.

[0268] 11. The method of embodiment 10, wherein the at least two introduced cassettes are integrated into the genome of the cell.

[0269] 12. The method of embodiment 10, wherein the at least two introduced cassettes comprise the same ORF encoding the same lectin, or comprise different open reading frames (ORFs) encoding different lectins.

[0270] 13. A method for producing a lectin in a Gram-positive bacterial cell, the method comprising (a) obtaining a Gram-positive bacterial cell and introducing into the cell a first and a second expression cassette encoding a lectin, wherein the first cassette comprises an upstream promoter sequence operably linked to a downstream open reading frame (ORF) encoding the lectin, and the second cassette comprises an upstream promoter sequence operably linked to a downstream nucleic acid encoding a signal (secretion) sequence, the signal (secretion) sequence operably linked to the downstream ORF encoding the lectin, and (b) fermenting the cell under suitable conditions for producing the lectin.

[0271] 14. The method of embodiment 13, wherein the lectin is expressed intracellularly, and / or the lectin is expressed and secreted extracellularly.

[0272] 15. The method of embodiment 13, wherein the introduced first box or the introduced second box is integrated into the genome of the cell.

[0273] 16. The method of embodiment 13, wherein the first and second introduced cassettes are integrated into the genome of the cell

[0274] 17. The method of embodiment 13, wherein the at least two introduced cassettes comprise the same ORF encoding the same lectin, or comprise different open reading frames (ORFs) encoding different lectins.

[0275] 18. The method of any one of embodiments 1, 7 or 13, wherein the ORF encodes a natural lectin or a variant lectin derived from a cyanobacterial cell, a eukaryotic cell or a bacterial cell.

[0276] 19. The method of embodiment 18, wherein the eukaryotic cell is selected from the group consisting of a plant cell, a fungal cell, and an animal cell.

[0277] 20. The method of any one of embodiments 1, 7 or 13, wherein the ORF encodes a lectin selected from the group consisting of: a natural griffithsin (GRFT) lectin or a variant GRFT lectin derived therefrom, a natural scytovirin (SVN) lectin or a variant SVN lectin derived therefrom, a natural cyanobacterial antiviral protein-N (CVN) lectin or a variant CVN lectin derived therefrom, a natural Kappaphycus alvarezii KAA-1 lectin or a variant KAA-1 lectin derived therefrom, a natural Kappaphycus alvarezii KAA-2 lectin or a variant KAA-2 lectin derived therefrom, a natural Microcystis viridis (MVL) lectin or a variant MVL lectin derived therefrom, a natural Boodlea hemispherica (Boodlea spp.) coacta) agglutinin (BCA) lectin or variant BCA agglutinin derived therefrom, natural Artocarpus heterophyllus (jackfruit lectin) lectin or variant jackfruit lectin derived therefrom, natural Musa acuminata (banana) lectin or variant banana lectin derived therefrom, natural Aaptospapilleta (sponge) lectin or variant sponge lectin derived therefrom, natural Abrus precatorius (abrus precatorius) (abrus precatorius) (abrus precatorius) (abrus precatorius) lectin or variant aegapodium podagraria (abrus precatorius) (abrus precatorius) lectin or variant aegapodium podagraria (abrus precatorius) (abrus precatorius) lectin or variant aegapodium podagraria (abrus precatorius) lectin or variant aegapodium podagraria (abrus precatorius) lectin, Agaricus bisporus (common mushroom) lectin or variant common mushroom lectin derived therefrom, natural Albizzia julibrissin) (Mimosa pudica seed) lectin or variants derived therefrom, Mimosa pudica seed lectin, natural Allomyrina dichotoma (Japanese beetle) lectin or variants derived therefrom, Japanese beetle lectin, natural Aloe arborescens (aloe plant) lectin or variants derived therefrom, Aloe plant lectin, natural Amphicarpaea bracteata (Amphicarpaea bracteata) (Amphicarpaea bracteata) lectin or variants derived therefrom, natural Anguilla (Eel) lectin or variants derived therefrom, eel lectin, natural Aplysia depilans (Mollusca) lectin or variants derived therefrom, mollusc lectin, natural Arachis hypogaea (Peanut) lectin or variants derived therefrom, peanut lectin, natural Bauhinia (Bauhiniapurpurea) (camel hoof tree) lectin or variant camel hoof tree lectin derived therefrom, natural Bryonia diocia (white bryonia) lectin or variant white bryonia lectin derived therefrom, natural Caragana Arborescens (Siberian pea tree) lectin or variant Siberian pea tree lectin derived therefrom, natural Carcinoscorpius rotundacauda (horseshoe crab) lectin or variant horseshoe crab lectin derived therefrom, natural Microcystis aeruginosa (cyanobacteria) microvirin (MVN) lectin or variant MVN lectin derived therefrom, natural Eucheuma serra (red algae) ESA-2 lectin or variant ESA-2 lectin derived therefrom, natural Musa acuminate) (banana) BanLec lectin or variant BanLec lectin derived therefrom, natural Aspidistra elatior (Aspidistra elatior) AEL lectin or variant AEL lectin derived therefrom, natural Chaetopterus variopedatus (marine worm) CVL lectin or variant CVL lectin derived therefrom, Vicia faba (broad bean) lectin or variant broad bean lectin derived therefrom, natural Lens culinaris (lentil) or variant lentil lectin derived therefrom, natural Pisum sativum (pea) lectin or variant pea lectin derived therefrom, jacaranda lectin-like lectin or variant jacaranda lectin-like lectin derived therefrom, including but not limited to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9. NO:39, the japanese lectin-like lectin, the CVN-like lectin or a variant CVN-like lectin derived therefrom, including but not limited to the CVN-like lectin listed in SEQ ID NO:10 and SEQ ID NO:11, the OAA-like lectin or a variant OAA-like lectin derived therefrom, including but not limited to the OAA-like lectin listed in one of SEQ ID NO:14-SEQ ID NO:23 or SEQ ID NO:29-SEQ ID NO:38, the galectin-1-like lectin or a variant galectin-1-like lectin derived therefrom, including but not limited to the galectin-1-like lectin listed in SEQ ID NO:25-SEQ ID NO:28, and the ricin-like lectin or a variant ricin-like lectin derived therefrom, including but not limited to the ricin-like lectin listed in SEQ ID NO:24.

[0278] 21. The method of any one of embodiments 1, 7 or 13, wherein the Gram-positive bacterial cell is selected from members of the phylum Firmicutes.

[0279] 22. The method of any one of embodiments 1, 7 or 13, wherein the Gram-positive bacterial cell is selected from a member of the Bacillaceae family.

[0280] 23. The method of any one of embodiments 1, 7 or 13, wherein the gram-positive bacterial cell is selected from the group consisting of: an alkali-tolerant Bacillus sp. cell, an Amphibacillus sp. cell, an anaerobic Bacillus sp. cell, a Bacillus sp. cell, a Caldalkalibacillus sp. cell, a Cerasilbacillus sp. cell, an Exiguobacterium sp. cell, a Filobacillus sp. cell, a Geobacillus sp. cell, a Gracilibacillus sp. cell, a Halobacillus sp. cell, a Halolactibacillus sp. cell, a sp. cells, Jeotgalibacillus sp. cells, Lentibacillus sp. cells, Marinibacillus sp. cells, Oceanobacillus sp. cells, Ornithinibacillus sp. cells, Paraliobacillus sp. cells, Paucisalibacillus sp. cells, Pontibacillus sp. cells, Sea Bacillus sp. cells, Saccharococcus sp. cells, Salibacillus sp. cells, Salinibacillus sp. cells, Tenuibacillus sp. cells sp. cells, Thalassobacillus sp. cells, Ureibacillus sp. cells, and Virgibacillus sp. cells.

[0281] 24. A method for recovering an intracellular lectin, the method comprising (a) constructing and fermenting a recombinant cell according to any one of embodiments 1, 7 or 13, (b) lysing the cells at the end of the fermentation to obtain a lysed cell broth, heat-treating the lysed broth at a pH between 1.5 and 8.5, then cooling the broth and harvesting the cooled broth, (c) subjecting the harvested cooled broth to a clarification process, and (d) subjecting the clarified broth to a concentration process, wherein the lectin is recovered in the clarified concentrated broth.

[0282] 25. A method for recovering a secreted lectin, the method comprising (a) constructing and fermenting a recombinant cell according to any one of embodiments 1, 7 or 13, and harvesting the broth, (b) subjecting the harvested cooled broth to a clarification process, and (c) subjecting the clarified broth to a concentration process, wherein the lectin is recovered in the clarified concentrated broth.

[0283] 26. A method for increasing the purity of a lectin concentrate, the method comprising (a) obtaining a lectin concentrate recovered according to the method of embodiment 24 or embodiment 25, adjusting the pH of the concentrate to between 1.5 and 8.5, (b) incubating the concentrate of step (a) at a temperature between about 5° C. and 55° C. for a sufficient amount of time, and (c) centrifuging the concentrate and collecting the supernatant, or filtering the concentrate and collecting the filtrate, wherein the collected supernatant or the collected filtrate comprises substantially purified lectin relative to the purity of the lectin concentrate obtained in step (a).

[0284] A method for increasing the purity of a lectin concentrate, the method comprising (a) obtaining a lectin concentrate recovered according to the method of embodiment 24 or embodiment 25, adjusting the pH of the concentrate to about pH 2, incubating the concentrate at pH 2 at a temperature between about 55° C. and 65° C. for a sufficient amount of time, and (b) centrifuging the incubated concentrate and collecting the supernatant, or filtering the concentrate and collecting the filtrate, wherein the collected supernatant or the collected filtrate comprises substantially purified lectin relative to the purity of the lectin concentrate obtained in step (a).

[0285] 28. A method for crystallizing a lectin, the method comprising (a) obtaining a lectin concentrate recovered according to any of the methods of embodiments 24-27, (b) adding a salt or a mixture of salts to the concentrate, the salt or mixture of salts comprising sodium, calcium, ammonium, sulfate or chloride ions, adjusting the pH of the salted concentrate to about pH 2 to 5, and mixing and incubating the concentrate at about 5°C to 50°C for an appropriate amount of time, wherein after mixing and incubating for an appropriate amount of time in step (b), the lectin crystallizes from the concentrate.

[0286] 29. A method for crystallizing a lectin, the method comprising (a) obtaining a lectin concentrate recovered according to any of the methods of embodiments 24-27, (b) adding 0.5% to 10% of a salt or a mixture of salts comprising sodium, calcium, ammonium, sulfate or chloride ions to the concentrate, adjusting the pH of the salted concentrate to about pH 2 to 5, and mixing and incubating the concentrate at about 5°C to 50°C for an appropriate amount of time, wherein after mixing and incubating for an appropriate amount of time in step (b), the lectin crystallizes from the concentrate.

[0287] 30. The method of embodiment 29, wherein the pH is about 2.5 to about 3.5, the salt is about 1% to about 5% sodium sulfate, and the incubation temperature is about 5°C to about 25°C.

[0288] 31. The method of embodiment 29, wherein the pH is about 2.8 to about 3.2, the salt is about 1.8% to about 2.5% sodium sulfate, and the incubation temperature is about 15°C to about 25°C

[0289] 32. A method for improving the purity of a lectin preparation, the method comprising (a) obtaining a lectin crystal slurry according to any one of embodiments 28-31, optionally washing the crystal slurry with water and mixing, and centrifuging the slurry for a sufficient amount of time, decanting and collecting the supernatant after centrifugation, adding an appropriate pH buffer to maintain a pH of about 4.8 to about 8.6 while mixing at room temperature for a sufficient amount of time, and (b) filtering the supernatant and collecting the filtered supernatant, wherein the collected supernatant comprises a high purity lectin preparation.

[0290] A method for recovering a secreted lectin from a fermentation broth, the method comprising (a) obtaining and harvesting a whole fermentation broth comprising a secreted lectin, (b) subjecting the harvested broth to a clarification process, and (c) subjecting the clarified broth to a concentration process, wherein the lectin is recovered in the clarified, concentrated broth.

[0291] 34. A method for recovering an intracellular lectin from a fermentation broth, the method comprising (a) obtaining and lysing a whole fermentation broth comprising an intracellular lectin, heat treating the lysed broth at a pH between about 1.5 and about 8.5, then cooling the broth and harvesting the cooled broth, (b) subjecting the harvested cooled broth to a clarification process, and (c) subjecting the clarified broth to a concentration process, wherein the lectin is recovered in the clarified concentrated broth.

[0292] A method for increasing the purity of a lectin concentrate, the method comprising (a) obtaining a lectin concentrate recovered according to the method of embodiment 33 or embodiment 34, adjusting the pH of the concentrate to between about 1.5 and about 8.5, (b) incubating the concentrate of step (a) at a temperature between about 5° C. and about 55° C. for a sufficient amount of time, and (c) centrifuging the concentrate and collecting the supernatant, or filtering the concentrate and collecting the filtrate, wherein the collected supernatant or the collected filtrate comprises substantially purified lectin relative to the purity of the lectin concentrate obtained in step (a).

[0293] 36. A method for increasing the purity of a lectin concentrate, the method comprising (a) obtaining a lectin concentrate recovered according to the method of embodiment 33 or embodiment 34, adjusting the pH of the concentrate to about pH 2, incubating the concentrate at about pH 2 at a temperature between about 55° C. and about 65° C. for a sufficient amount of time, and (b) filtering the incubated concentrate and collecting the supernatant, or filtering the concentrate and collecting the filtrate, wherein the collected supernatant or the collected filtrate comprises substantially purified lectin relative to the purity of the lectin concentrate obtained in step (a).

[0294] 37. A method for crystallizing a lectin, the method comprising (a) obtaining a lectin concentrate recovered according to the method of embodiment 35 or embodiment 36, (b) adding a salt or a mixture of salts to the concentrate, the salt or mixture of salts comprising sodium, calcium, ammonium, sulfate or chloride ions, adjusting the pH of the salted concentrate to about 2 to about 5, and mixing and incubating the concentrate at between about 5° C. and about 50° C. for an appropriate amount of time, wherein after mixing and incubating for an appropriate amount of time in step (b), the lectin crystallizes from the concentrate.

[0295] 38. A method for crystallizing a lectin, the method comprising (a) obtaining a lectin concentrate recovered according to the method of embodiment 35 or embodiment 36, (b) adding about 0.5% to about 10% of a salt or a mixture of salts comprising sodium, calcium, ammonium, sulfate or chloride ions to the concentrate, adjusting the pH of the salted concentrate to a pH of about 2 to about 5, and mixing and incubating the concentrate at about 5° C. to about 50° C. for an appropriate amount of time, wherein after mixing and incubating for an appropriate amount of time in step (b), the lectin crystallizes from the concentrate.

[0296] 39. The method of embodiment 38, wherein the pH is about 2.5 to about 3.5, the salt is about 1% to about 5% sodium sulfate, and the incubation temperature is about 5°C to about 25°C.

[0297] 40. The method of embodiment 38, wherein the pH is about 2.8 to about 3.2, the salt is about 1.8% to about 2.5% sodium sulfate, and the incubation temperature is about 15°C to about 25°C

[0298] 41. A method for improving the purity of a lectin preparation, the method comprising: (a) obtaining a lectin crystal slurry according to any one of embodiments 37-40, optionally washing the crystal slurry with water and mixing, and centrifuging the slurry for a sufficient amount of time, decanting and collecting the supernatant after centrifugation, adding an appropriate pH buffer to maintain a pH of about 4.8 to about 8.6 while mixing at room temperature for a sufficient amount of time, and (b) filtering the supernatant and collecting the filtered supernatant, wherein the collected supernatant comprises a high purity lectin preparation.

[0299] 42. A high-purity lectin preparation produced and obtained according to the method of any one of embodiments 1-41.

[0300] 43. A method for producing and recovering a high-purity griffithsin (GRFT) protein preparation, the method comprising (a) constructing a recombinant Gram-positive bacterial cell expressing a polynucleotide encoding the GRFT protein, (b) fermenting the recombinant cell to produce the GRFT protein, lysing the cells at the end of the fermentation to obtain a lysed cell broth, and treating the lysed cell broth by maintaining the broth at a pH of about 4.8 to about 5.2 and a temperature of about 50° C. to about 80° C. for about 1 to about 4 hours, (c) clarifying the broth of step (b) by a filtration or microfiltration process, and concentrating the clarified broth by an ultrafiltration process, (d) subjecting the concentrated broth of step (c) to a crystallization process, the crystallization process comprising adding about 2% sodium sulfate to the concentrate, adjusting the pH to about 3 and incubating at about 22° C. for a sufficient amount of time, (e) centrifuging the incubated concentrate of step (d), decanting the supernatant to harvest GRFT crystals, and decant the GRFT crystals at a pH of about 4.5 to about 5.5 in about 100 mM sodium acetate buffer to obtain an aqueous GRFT protein preparation, and centrifuging the GRFT preparation to remove any insoluble impurities therein and collecting the supernatant, or filtering the GRFT preparation to remove any insoluble impurities therein and collecting the filtrate, wherein the supernatant collected in step (e) or the filtrate collected in step (e) contains a high-purity GRFT protein preparation.

[0301] 44. A method for recovering a high-purity griffithsin (GRFT) protein preparation, the method comprising (a) obtaining a whole fermentation broth comprising recombinant cells expressing GRFT protein, lysing the cells in the cell broth, and treating the lysed broth by maintaining the broth at a pH of about 4.8 to 5.2 and a temperature of about 50° C. to 80° C. for about 1 to 4 hours, (b) clarifying the broth of step (b) by a filtration or microfiltration process, and concentrating the clarified broth by an ultrafiltration process, (c) subjecting the concentrated broth of step (c) to a crystallization process, the crystallization process comprising adding about 2% sodium sulfate to the concentrate, adjusting the pH to about 3 and incubating at about 22° C. for a sufficient amount of time, and (d) centrifuging the incubated concentrate of step (c), decanting the supernatant to harvest GRFT crystals, and concentrating the GRFT crystals at a pH of about 4.5 to about 5.5 in about 100 mM sodium acetate buffer to obtain an aqueous GRFT protein preparation, and centrifuging the GRFT preparation to remove any insoluble impurities therein and collecting the supernatant, or filtering the GRFT preparation to remove any insoluble impurities therein and collecting the filtrate, wherein the supernatant collected in step (d) or the filtrate collected in step (d) contains a high-purity GRFT protein preparation.

[0302] 45. A method as described in Example 43 or Example 44, wherein the high-purity GRFT preparation comprises a native GRFT protein or a variant GRFT protein preparation.

[0303] 46. ​​The method of embodiment 43 or embodiment 44, wherein the high purity GRFT preparation is at least 2.0 times purer than the recovered GRFT concentrate, such as by 280 The GRFT concentration was determined by measuring the GRFT concentration at nm.

[0304] 47. The method of embodiment 43 or embodiment 47, wherein the GRFT is the major band, or the only band of about 12.7 kDa, in the high purity GRFT preparation when visualized by SDS-PAGE.

[0305] 48. The method of embodiment 43 or embodiment 47, wherein the high purity GRFT has hemagglutination activity when assayed / screened against one or more animal red blood cells (erythrocytes).

[0306] 49. A protein preparation comprising high purity GRFT produced according to the method of any one of embodiments 43-48.

[0307] 50. A method for improving a lectin protein preparation, the method comprising (a) obtaining a lectin fermentation broth concentrate and adjusting the pH of the concentrate to about pH 2-4, adding a sufficient amount of salt to the concentrate, centrifuging the concentrate for a sufficient amount of time, and collecting the supernatant, (b) adding an appropriate amount of polymer to the supernatant and incubating for a sufficient amount of time to initiate liquid-liquid extraction, and centrifuging the liquid-liquid extract for a sufficient amount of time to precipitate the lectin protein, and (c) recovering the precipitated lectin protein precipitate, wherein the recovered precipitate contains the lectin of improved purity.

[0308] 51. The method of embodiment 50, wherein the salt is selected from the group consisting of phosphate, sulfate and citrate.

[0309] 52. The method of embodiment 50, wherein the salt is ammonium sulfate.

[0310] 53. The method of embodiment 50, wherein the polymer is selected from the group consisting of polyethylene glycols (PEGs) of different sizes, dextran of different sizes, chemically modified derivatives of one or more PEGs of different sizes, and / or chemically modified derivatives of one or more dextran of different sizes.

[0311] 54. The method of embodiment 50, wherein the pH is about 2.

[0312] 55. The method of embodiment 50, further comprising washing the recovered lectin precipitate with a suitable buffer and 30% PEG at about pH 2, followed by neutralizing and solubilizing the lectin with a suitable buffer to obtain a lectin protein preparation with improved purity.

[0313] 56. The method of embodiment 55, wherein the improved purity lectin preparation is at least 80% of the total protein composition.

[0314] 57. The method of any one of embodiments 1, 3 or 7, wherein the recombinant cell is defective in producing one or more endogenous (native) proteins selected from the group consisting of: amylase, xylanase, pullulanase, phytase, pectin lyase, glucanase, mannosidase, lipase, esterase

[0315] 58. A recombinant Gram-positive bacterial cell expressing a polynucleotide encoding a heterologous lectin.

[0316] 59. The recombinant cell of embodiment 58, comprising an introduced polynucleotide cassette encoding the lectin, wherein the cassette comprises an upstream promoter operably linked to a downstream nucleic acid encoding the lectin, or wherein the cassette comprises an upstream promoter operably linked to a downstream nucleic acid encoding a signal sequence operably linked to a downstream nucleic acid encoding the lectin.

[0317] 60. The recombinant cell of embodiment 58, comprising at least two introduced cassettes encoding the lectin, wherein the at least two introduced cassettes comprise an upstream promoter operably linked to a downstream nucleic acid encoding the lectin.

[0318] 61. The recombinant cell of embodiment 58, comprising at least two introduced cassettes encoding the lectin, wherein the introduced cassettes comprise an upstream promoter operably linked to a downstream nucleic acid encoding a signal sequence operably linked to a downstream nucleic acid encoding the lectin.

[0319] 62. The recombinant cell of embodiment 58, comprising at least two introduced cassettes encoding the lectin, wherein one of the introduced cassettes comprises an upstream promoter operably linked to a downstream nucleic acid encoding the lectin, and a second introduced cassette comprises an upstream promoter operably linked to a downstream nucleic acid encoding a signal sequence operably linked to the downstream nucleic acid encoding the lectin.

[0320] 63. The recombinant cell of embodiment 58, wherein the lectin is expressed in the cell

[0321] 64. The recombinant cell of embodiment 58, wherein the lectin is expressed and secreted extracellularly.

[0322] 65. The recombinant cell of embodiment 58, wherein the lectin is expressed intracellularly and expressed and secreted extracellularly.

[0323] 66. The recombinant cell of embodiment 58, wherein the introduced cassette is integrated into the genome of the cell.

[0324] 67. The recombinant cell of any one of embodiments 59-62, wherein at least one of the at least two introduced cassettes is integrated into the genome of the cell.

[0325] 68. The recombinant cell of any one of embodiments 59-62, wherein the at least two introduced cassettes are integrated into the genome of the cell.

[0326] 69. The recombinant cell of any one of embodiments 59-62, wherein the at least two introduced cassettes encode the same lectin or encode different lectins.

[0327] 70. The recombinant cell of embodiment 58, wherein the Gram-positive bacterial cell is selected from members of the phylum Firmicutes.

[0328] 71. The recombinant cell of embodiment 58, wherein the Gram-positive bacterial cell is selected from a member of the Bacillaceae family.

[0329] 72. The recombinant cell of embodiment 58, wherein the Gram-positive bacterial cell is selected from the group consisting of an alkali-tolerant Bacillus species cell, a bi-Bacillus species cell, an anaerobic Bacillus species cell, a Bacillus species cell, a thermoalkali Bacillus species cell, a cherry-like Bacillus species cell, an Exiguobacterium species cell, a line Bacillus species cell, a Geobacillus species cell, a tenuissus species cell, a halobacillus species cell, a halolactobacillus species cell, a saltwater seafood Bacillus species cell, Bacillus species cells, ... and Bacillus species cells.

[0330] 73. The recombinant cell of embodiment 58, wherein the heterologous lectin is derived from a bacterial cell, a eukaryotic cell, or a cyanobacterial cell.

[0331] 74. The recombinant cell of embodiment 73, wherein the eukaryotic cell is selected from the group consisting of a plant cell, a fungal cell, an insect cell, and an animal cell.

[0332] 75. The recombinant cell of embodiment 58, wherein the lectin is selected from the group consisting of: natural griffithsin (GRFT) lectin or variant GRFT lectin derived therefrom, natural scytovirin (SVN) lectin or variant SVN lectin derived therefrom, natural cyanobacterial antiviral protein-N (CVN) lectin or variant CVN lectin derived therefrom, natural Kappaphycus longifolia KAA-1 lectin or variant KAA-2 lectin derived therefrom -1 lectin, natural Kappaphycus longifolia KAA-2 lectin or variant KAA-2 lectin derived therefrom, natural Microcystis viridis (MVL) lectin or variant MVL lectin derived therefrom, natural B. hemispherica lectin (BCA) lectin or variant BCA lectin derived therefrom, natural Jackfruit (Jacaranda lectin) lectin or variant jacaranda lectin derived therefrom, natural Banana agglutinin or variant banana lectin derived therefrom, natural Aaptospapilleta (sponge) lectin or variants derived therefrom, sponge lectin, natural Abrus abrinus (abbreviation bean) lectin or variants derived therefrom, Abrus abrinus lectin, natural Ceratonia ovata (calyx bean) lectin or variants derived therefrom, Ceratonia ovata (calyx bean) lectin or variants derived therefrom, Agaricus bisporus (common mushroom) lectin or variants derived therefrom, common mushroom lectin, natural Albizia julibrissin (mimosa tree seed) lectin or variants derived therefrom, Mimosa tree seed lectin, natural Rhinoceros beetle (Japanese beetle) lectin or variants derived therefrom, Japanese beetle lectin, natural Aloe vera (aloe vera plant) lectin or variant aloe vera plant lectin derived therefrom, natural lectin of Dimorphotheca oleracea (isotype bean) or variant ditype bean lectin derived therefrom, natural lectin of Anguilla anguilla (eel) or variant eel lectin derived therefrom, natural lectin of Aplysia vulgaris (mollusc) or variant mollusc lectin derived therefrom, natural lectin of Arachis hypogaea (peanut) or variant peanut lectin derived therefrom, natural lectin of Bauhinia oleracea (camel hoof tree) or variant camel hoof tree lectin derived therefrom, natural lectin of Bryonia ovata (white bryonia) lectin or variants thereof, such as Bryonia leucoderma lectin, natural Caragana oleracea (Siberian pea tree) lectin or variants thereof, natural Horseshoe Crab (Horseshoe Crab) lectin or variants thereof, natural Microcystis aeruginosa (cyanobacteria) microvirin (MVN) lectin or variants thereof, natural Eucheuma dentata (red algae) ESA-2 lectin or variants thereof, natural Banana (Banana) BanLec lectin or variants thereof. variant BanLec lectin derived therefrom, natural Aegilops punctatus AEL lectin or variant AEL lectin derived therefrom, natural Trichoptera (marine worm) CVL lectin or variant CVL lectin derived therefrom, Vicia faba (broad bean) lectin or variant Vicia faba lectin derived therefrom, natural Lentil (lentil) or variant Lentil lectin derived therefrom, natural Pisum sativum (pea) lectin or variant Pisum sativum lectin derived therefrom, Artocarpus fasciatus-like lectin or variant Artocarpus fasciatus-like lectin derived therefrom, including but not limited to SEQ SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 39, CVN-like lectin or variant CVN-like lectin derived therefrom, including but not limited to SEQ ID NO: 10 and SEQ ID NO: 11, OAA-like lectin or variant OAA-like lectin derived therefrom, including but not limited to SEQ ID NO: 14-SEQ ID NO: 23 or SEQ ID NO: 29-SEQ ID NO: 30.OAA-like lectins listed in one of SEQ ID NO:38, galectin-1-like lectins or variant galectin-1-like lectins derived therefrom, including but not limited to galectin-1-like lectins listed in SEQ ID NO:25-SEQ ID NO:28, and ricin-like lectins or variant ricin-like lectins derived therefrom, including but not limited to the ricin-like lectin listed in SEQ ID NO:24.

[0333] 76. The recombinant cell of any one of embodiments 1, 3 or 7, wherein the recombinant cell is defective in producing one or more endogenous (native) proteins selected from the group consisting of: amylase, xylanase, pullulanase, phytase, pectin lyase, glucanase, mannosidase, lipase and esterase.

[0334] Examples

[0335] Certain aspects of the present disclosure may be further understood from the following examples, which should not be construed as limiting. Modifications to materials and methods will be apparent to those skilled in the art. Standard recombinant DNA and molecular cloning techniques used herein are well known in the art (Ausubel et al., 1987; Sambrook et al., 1989).

[0336] Example 1

[0337] Expression of griffithsin variant (M78Q) in Gram-positive bacterial cells

[0338] In the present example, applicants have designed, constructed, and evaluated exemplary Gram-positive host cells for their ability to express / produce heterologous (exogenous) lectins. More particularly, as described herein, it was unexpectedly observed that Gram-positive bacterial cells (e.g., Bacillus sp. cells) can express / produce significant amounts of a heterologous (eukaryotic) lectin called griffithsin (GRFT). As described and exemplified in the following sections, a host cell encoding a native griffithsin (GRFT) protein (SEQ ID NO: 1; Figure 1 A) Q-GRFT variant (SEQ ID NO: 2; Figure 1 B) polynucleotide (expression cassette).

[0339] Construction of expression cassette and Bacillus strain expressing Q-GRFT

[0340] Two (2) expression cassettes encoding Q-GRFT (SEQ ID NO: 2) were constructed for intracellular expression or secretory expression of mature Q-GRFT in Bacillus sp. cells (e.g., see Schematic Diagram). Figure 6 For example, to generate an expression cassette for intracellular expression ( Figure 6 A / 6B), a eukaryotic derived DNA sequence (Q-grft) encoding the Q-GRFT protein was codon-optimized (SEQ ID NO: 3) for expression in Bacillus subtilis cells and operably linked to a suitable upstream (5′) promoter (pro) sequence (e.g., 5′-[pro]-[Q-grft]-3′). To generate an expression cassette for extracellular (secreted) expression, the codon-optimized Q-grft DNA sequence (SEQ ID NO: 3) was operably linked to an upstream (5′) nucleic acid sequence encoding a (protein) signal sequence (ss) (e.g., aprE signal sequence) that functions in Bacillus subtilis cells, which was operably linked to a suitable upstream (5′) promoter (pro) region sequence (e.g., 5′-[pro]-[ss]-[Q-grft]-3′). In addition, an alanine racemase gene (alrA) was cloned in the expression cassette as a selection marker for transformation.

[0341] The exemplary Bacillus subtilis strains herein were transformed using a Q-grft cassette for directing secretion of Q-GRFT, making these strains defective in the production of one or more serine proteases and comprising a deleted alanine racemase (alrA) gene. More specifically, in this example, three (3) Q-GRFT producing strains were generated, including Bacillus subtilis strain CB462, which comprises a deletion of genes encoding aprE and nprE proteases; CB460, which comprises a deletion of genes encoding aprE, nprE, epr, isp, bpr, and wprA proteases; and CB447, which comprises a deletion of genes encoding aprE, nprE, epr, isp, bpr, wprA, vpr, mpr, and nprB proteases. Similarly, a Q-grft cassette for intracellular expression of Q-GRFT was used to transform a Bacillus subtilis strain containing deletions of the genes encoding aprE, nprE, epr, isp, bpr, wprA, vpr, mpr and nprB proteases, and containing a deleted alanine racemase (alrA) gene, resulting in the generation of Bacillus subtilis strain CB476.

[0342] Evaluation of secreted Q-GRFT produced in a 10 L bioreactor

[0343] Expression of secreted Q-GRFT protein (SEQ ID NO: 2) in Bacillus subtilis strains CB460, CB462, and CB447 was evaluated in a 10 L bioreactor and recovered as generally described herein.

[0344] Fermentation and recovery: The Q-GRFT producing strain is grown in a fermentation medium with cell growth nutrients containing carbon sources (such as sugars, alcohols, organic acids and amino acids), nitrogen sources (such as ammonium salts and nitrates), phosphates, magnesium salts, potassium salts, sodium salts, and trace metal salts such as iron, manganese, zinc, copper, and boron to produce a large amount of cell material. The fermentation medium is optimized for higher cell density by adding complex nutrients containing lipids, proteins, peptides, and amino acids that are typically present in by-products of soybean, corn, and yeast processes. The cell bank of the recombinant strain is maintained in a frozen state and first inoculated into a sterile fermentation medium to prepare seeds for starting a fermenter run. The seed cells are further grown in batches of nutrient salt fermentation medium supplemented with plant-derived protein hydrolysates and yeast extracts as described above.

[0345] The fermenter batch is also fed with controlled amounts of sugar and ammonia, wherein the pH is controlled at an optimal set point, and the temperature is controlled at an optimal set point. The fermentation temperature is maintained at 25°C-45°C, and in some aspects maintained at about 37°C, by cooling the water passing through the fermenter coils and jackets. When the pH drops below the set point of 6 to 8 (e.g., about 6.8-7.4), the pH of the fermenter is maintained by feeding NH3-water; if necessary, phosphoric acid or sulfuric acid is added to maintain the pH. The cell mass is increased by feeding more carbon and nitrogen sources, so that the yield of the expressed protein is higher. Nutrient salt medium is prepared in a stainless steel pressure vessel fermenter, and air is sprayed to fully supply dissolved oxygen (DO). DO is controlled at an optimal set point, and the air saturation value is 5%-50%. Secreted Q-GRFT is the most abundant protein in the fermenter broth after 24-48 hours (e.g., about 36 hours). Some contaminant proteins come from Bacillus cell debris and enzymes naturally secreted by Bacillus.

[0346] In the present example, the fermentation broth is recovered as a clarified concentrate, which can be carried out via a variety of methods, which generally start with a broth treatment that includes lysis (e.g., natural or chemically induced lysis, in this example natural), heat treatment, pH and temperature control, water or buffer dilution, and with or without flocculation. For example, cell separation can be carried out in a variety of methods, including but not limited to depth filtration or membrane-based operations. Concentration is carried out via ultrafiltration membrane operations. In some aspects, the resulting clarified concentrate is further purified, as described in the subsequent examples below. Additional details about the recovery process can be found in the subsequent examples and the specification of the present disclosure.

[0347] More specifically, the expression of Q-GRFT in ultrafiltrate (UF) concentrate, filtered broth, and broth supernatant was evaluated via SDS-PAGE, wherein ten microliters (10 μL) of 10-fold diluted samples along with See Blue Plus2 molecular weight standards and lysozyme protein standards were evaluated by SDS-PAGE, followed by staining and retention of the gel using standard molecular biology procedures (data not shown). As indicated on the SDS-PAGE gel (data not shown), the Q-GRFT protein appeared as a single band with a molecular weight between approximately 6 kDa and 14 kDa, where lane 1 is a His-tagged lysozyme standard, lane 2 is a concentrated ultrafiltrate (UF) from strain CB447 (grown in a fermentation medium containing 2% soy flour, 2% corn steep solids), lanes 3, 4 and 5 are filtered broth, supernatant broth and concentrated UF from strain CB447 (grown in a fermentation medium containing 1% corn steep solids), lanes 6, 7 and 8 are filtered broth, supernatant broth and concentrated UF from strain CB462 (grown in a fermentation medium containing 1% corn steep solids), and lanes 9, 10 and 11 are filtered broth, supernatant broth and concentrated ultrafiltrate from strain CB460 (grown in a fermentation medium containing 1% corn steep solids).

[0348] Thus, as demonstrated in this example, comparable Q-GRFT protein can be recovered from fermentation broths of Bacillus subtilis strains CB462 (with deletion of aprE, nprE), CB460 (with deletion of aprE, nprE, epr, isp, bpr, wprA), and CB447 (with deletion of aprE, nprE, epr, isp, bpr, wprA, vpr, mpr, and nprB), indicating that Q-GRFT protein can be efficiently produced in any Bacillus subtilis strain. More particularly, as described above, the use of certain strains lacking one or more side (background) activities can be beneficial in recovering and / or purifying Q-GRFT protein (or other lectin protein) from contaminant proteins and / or background enzyme activity, thereby potentially producing a cleaner target protein (lectin) product.

[0349] Evaluation of intracellular and secreted Q-GRFT in a 10-L bioreactor

[0350] Expression of intracellular Q-GRFT in strain CB476 and expression of secreted Q-GRFT in strain CB447 were evaluated in 10 L bioreactors and recovered as generally described herein.

[0351] Fermentation and recovery: The Q-GRFT producing strain is grown in a fermentation medium with cell growth nutrients containing carbon sources (such as sugars, alcohols, organic acids and amino acids), nitrogen sources (such as ammonium salts and nitrates), phosphates, magnesium salts, potassium salts, sodium salts, and trace metal salts such as iron, manganese, zinc, copper, and boron to produce a large amount of cell material. The fermentation medium is optimized by adding complex nutrients containing lipids, proteins, peptides, and amino acids that are typically present in by-products of soybean, corn, and yeast processes. The cell bank of the recombinant strain is maintained in a frozen state and first inoculated into a sterile fermentation medium to prepare seeds for starting a fermenter run. The seed cells are further grown in batches of nutrient salt fermentation medium supplemented with plant protein hydrolysates and yeast extracts as described above.

[0352] The fermentor batch is also fed with controlled amounts of sugar and ammonia, wherein the pH is controlled at an optimal set point, and the temperature is controlled at an optimal set point. The fermentation temperature is maintained at 25°C-45°C (e.g., about 37°C) by cooling the water passing through the fermentor coil and jacket. When the pH drops below the set point of 6 to 8 (e.g., about 6.8-7.4), the pH of the fermentor is maintained by feeding NH3-water; if necessary, phosphoric acid or sulfuric acid is added to maintain the pH. The cell mass is increased by feeding more carbon and nitrogen sources, so that the yield of the expressed protein is higher. Nutrient salt medium is prepared in a stainless steel pressure vessel fermentor, and air is sprayed to fully supply dissolved oxygen (DO). DO is controlled at an optimal set point, and the air saturation value is 5%-50%. Q-GRFT is the most abundant protein in Bacillus cells after 24-48 hours (e.g., about 36 hours). In the fermentation broth, some contaminant proteins other than Q-GRFT come from Bacillus cell debris and enzymes naturally secreted by Bacillus.

[0353] In the present example, the fermentation broth is recovered as a clarified concentrate. As generally described above, such processes can be carried out via a variety of methods, starting from a broth treatment including lysis (e.g., natural or chemically induced lysis, in this example natural) and heat treatment, pH and temperature control, water or buffer dilution, and with or without flocculation. Similarly, cell separation can be carried out in a variety of ways, including depth filtration or membrane-based operations. Concentration can be carried out via ultrafiltration membrane operations. In certain embodiments, the resulting clarified concentrate is further purified, as described in the subsequent examples below.

[0354] More specifically, the expression of Q-GRFT in broth supernatants from Bacillus subtilis strains CB447 and CB476 was evaluated via SDS-PAGE. Figure 7As presented in FIG. 1 , ten (10) μL of 10-fold diluted samples (lanes 1-6) were evaluated along with See BluePlus 2 molecular weight standards (labeled, "kDa") and lysozyme protein standards (MClab), followed by staining and retention of the gel using standard molecular biology procedures. Figure 7 As shown, lanes 1, 2 and 3 are the broth supernatants from strain CB447 at eighteen (18) hours, twenty-four (24) hours and thirty (30) hours of fermentation, respectively, and lanes 4, 5 and 6 are the broth supernatants from strain CB476 at eighteen (18) hours, twenty-four (24) hours and thirty (30) hours of fermentation, respectively, in which the Q-GRFT protein appears as a single band with a molecular weight of 12.7 kDa.

[0355] Thus, as described above, the exemplary Gram-positive (Bacillus subtilis) strains CB447 with secreted Q-GRFT expression / production and CB476 with intracellular Q-GRFT expression produced comparable amounts of Q-GRFT under large-scale test conditions. More particularly, this example demonstrates that Gram-positive bacterial cells / strains (e.g., Bacillus species) are particularly useful host strains for large-scale fermentation and production of lectin proteins.

[0356] Example 2

[0357] Clarified broth concentrate from untreated broth

[0358] In this example, fermentation broth from the above-described Bacillus subtilis strain CB447 (i.e., expressing / secreting Q-GRFT; SEQ ID NO: 2) was processed according to the materials and methods described herein. More particularly, the materials, compositions and / or methods set forth in Examples 2-14 are not intended to be limiting, as one skilled in the art can readily modify, adjust, improve, etc., such Gram-positive bacterial strains, materials and / or methods to suit the specific requirements of any given lectin protein of the present disclosure.

[0359] Material

[0360] Materials included the following: (i) fermentation broth from strain CB447 (Example 1), (ii) 180 μm mesh, microfilter equipped with 0.1 μm 80 mil spacer 4" diameter, 17" length spiral wound membrane, (iii) ultrafiltration filter with C-spacer cartridge 5K molecular weight cutoff (MWCO), sulfuric acid, and (iv) SDS-PAGE system and reagents. In this example, the SDS-PAGE system was 4%-12% Bis-Tris reducing and MES running buffer.

[0361] Methods and Results

[0362] The CB447 fermentation broth was typically filtered through a 180 μm screen, where the filtered (screened) broth was subsequently diluted with 1.1 parts of DI water. The diluted broth was transferred to a microfilter feed tank, and the microfiltration process was performed by operating the diafiltration at a constant feed tank level at 10°C with 4x feed volumes of DI water pH 7.4 (adjusted with sulfuric acid), where the permeate was collected. The collected microfiltration permeate was concentrated to 8x the initial broth volume using a 5KMWCO membrane. The Q-GRFT recovery was 65%, where a visually clear concentrate was produced (data not shown), and the concentrate obtained by SGS-PAGE was higher in purity than the broth supernatant.

[0363] Example 3

[0364] Purification of clarified broth concentrate by pH adjustment

[0365] In this example, the clarified broth concentrate recovered in Example 2 was further purified by pH adjustment according to the materials and methods described herein.

[0366] Material

[0367] Materials included the following: (i) clarified broth UF concentrate from Example 2, (ii) sulfuric acid, (iii) sodium hydroxide, (iv) pH meter, (v) shaker with temperature control, (vi) centrifuge, and (vii) SDS-PAGE system and reagents.

[0368] Methods and Results

[0369] 100 mL of UF concentrate prepared in Example 2 was adjusted to pH 2 using sulfuric acid. The pH was readjusted (raised) back to high values ​​using sodium hydroxide, and approximately 25 mL samples were taken during the adjustment period when pH 5.5, pH 6.8, and pH 8 were reached. The pH adjusted samples were then each divided into three (3) portions for incubation at 5° C., 22° C., and 55° C. for twenty-four (24) hours. The incubated samples were centrifuged at 14,000 rpm for ten (10) minutes, and the supernatant was collected for analysis by SDS-PAGE.

[0370] All pH treated supernatants had higher purity than the initial whole broth samples (data not shown).

[0371] Example 4

[0372] Purification of clarified broth concentrate by low pH treatment

[0373] In this example, the clarified broth concentrate recovered in Example 2 was further purified by low pH treatment (ie, pH 2) according to the materials and methods described herein.

[0374] Material

[0375] Materials included the following: (i) clarified broth concentrate from Example 2, (ii) sulfuric acid, (iii) activated charcoal, (iii) pH meter, (iv) shaker with temperature control, (v) benchtop Nalgene 0.2 mm filters, and (vi) SDS-PAGE system and reagents.

[0376] Methods and Results

[0377] 150 mL of the clarified broth concentrate (Example 2) was adjusted to pH 2 using sulfuric acid, and the pH adjusted sample was divided into two portions. 1% activated carbon was added to one of the portions, the pH was checked and adjusted as needed to maintain pH 2. Zero time (T0) samples were taken from both portions. Each portion was divided into three tubes for incubation for eighteen (18) hours at 5°C, 22°C, and 60°C. The incubated samples were filtered, the filtrate was collected, and the purity of the filtrate was analyzed by SDS-PAGE.

[0378] In particular, significant purification of Q-GRFT was achieved by adjusting the pH to 2 followed by filtration, where sample incubation at 60°C further improved protein purity (data not shown), and addition of activated carbon further reduced the color of the filtrate (data not shown).

[0379] Example 5

[0380] Clarified broth concentrate from pH 5-5.5 and heat treated broth recovered using microfilter for cell separation

[0381] In this example, fermentation broth from the Bacillus subtilis strain CB488 described above (ie, Q-GRFT expressed intracellularly and extracellularly; SEQ ID NO: 2) was processed according to the materials and methods described herein.

[0382] Material

[0383] Materials included the following: (i) fermentation broth from strain CB488 expressing Q-Grft, (ii) sulfuric acid, (iii) lysozyme, 20% stock (BioSeutica), (iv) 180 mm mesh, (v) microfilter equipped with 0.1 mm 80 mil spacer 4" diameter, 17" length spiral wound membrane, (vi) ultrafilter with C-spacer cartridge 5K MWCO, and (vii) SDS-PAGE system and reagents. In this example, the SDS-PAGE system was 4%-12% Bis-Tris reducing SDS-PAGE MES running buffer.

[0384] Methods and Results

[0385] In the present method, lysozyme treatment was performed at the end of fermentation by adding 1.5 mL of 20% lysozyme stock solution / L broth and allowed to mix at 37°C for four (4) hours. The pH was adjusted to 5.0-5.5 using sulfuric acid. Heat treatment was performed by heating the tank to 60°C for four (4) hours. The pH adjusted and heat treated broth was harvested by cooling the broth to 15°C and transferring to a container. The harvested broth was recovered using the same microfiltration and ultrafiltration procedures outlined and described in Example 2.

[0386] A visually clear concentrate was produced. For example, the heat treated broth supernatant provided a significant purity improvement relative to the untreated broth supernatant (data not shown). Likewise, the Q-GRFT purity was maintained through the processing steps (e.g., microfiltration for cell separation and ultrafiltration for dehydration), wherein the UF concentrate obtained had a higher purity than the untreated broth derived UF concentrate in Example 2 recovered using a similar process.

[0387] Example 6

[0388] Clarified broth concentrate from pH 5-5.5 and heat treated broth recovered using a Buchner filter for cell separation

[0389] In this example, fermentation broth from Bacillus subtilis strain CZ438 was processed according to the materials and methods described herein.

[0390] As generally described herein, CZ438 cells contain two (2) introduced (transformed) copies of the Q-grft expression cassette integrated into the aprE locus (1st copy) and skfABCEFGH locus (2nd copy) of the Bacillus subtilis cells. For example, the Q-grft cassette contains an upstream (5') DNA sequence (aprE) encoding the AprE signal sequence (i.e., for secretion of mature Q-GRFT) operably linked to a downstream (3') DNA sequence (Q-grft) encoding the Q-GRFT protein. In the present example, the Bacillus subtilis strain contains a deleted alanine racemase (alrA) gene for selection.

[0391] Material

[0392] Materials included the following: (i) fermentation broth from strain CZ438 expressing Q-GRFT, (ii) sulfuric acid, (iii) lysozyme, 20% stock solution (BioSeutica), (iv) 781G flocculent polymer, (v) FW12 diatomaceous earth, (vi) Buchner filter equipped with HR900 pad, (vii) ultrafilter with C-spacer cartridge 5K MWCO, and (viii) SDS-PAGE system and reagents. In this example, the SDS-PAGE system was 4%-12% Bis-Tris reducing SDS-PAGE MES running buffer.

[0393] Methods and Results

[0394] The fermentation broth from strain CZ438 was processed using the same lysozyme treatment, pH adjustment and heat treatment process as described in Example 5 above. In this example, the harvested broth was recovered as follows. An equal portion of DI water was added to the harvested broth, and the pH was adjusted as needed to maintain a pH of 5.5. 0.2% 781G flocculent polymer (as a 20% solution) was added and mixed for five (5) minutes, then 7.5% FW12 was added and mixed thoroughly. Vacuum filtered through a Buchner filter equipped with a HR900 filter pad, and the filtrate was collected. The collected filtrate was concentrated using an ultrafilter.

[0395] Produces a visually clear concentrate ( Fig. 9 ), wherein the pH-adjusted and heat-treated broth supernatant ( Fig. 9 , lanes 3 and 4) showed a significant improvement in purity relative to the untreated broth supernatant ( Fig. 9 , lane 1). The purity of Q-GRFT was maintained through processing steps for cell separation, flocculation and Buchner filtration, and ultrafiltration for dehydration. The UF concentrate obtained ( Fig. 9 , lane 7) is similar in purity to that of the concentrate of Example 5 derived from similarly treated CB488 strain broth but recovered using microfiltration for cell separation; and is higher in purity than the UF concentrate derived from untreated broth (Example 2).

[0396] Example 7

[0397] Clarified broth concentrate from pH 4.8-5.2 and heat treated broth recovered using a Buchner filter for cell separation

[0398] In this example, fermentation broth from the Bacillus subtilis strain CB447 described in Example 2 was processed according to the materials and methods described herein.

[0399] Material

[0400] Materials included the following: (i) fermentation broth from strain CB488 expressing Q-GRFT, (ii) sulfuric acid, (iii) 781G flocculent polymer, (iv) FW12 diatomaceous earth, (v) Buchner filter equipped with HR900 pad, (vi) ultrafilter with C-spacer cartridge 5K MWCO, and (vii) SDS-PAGE system and reagents. In this example, the SDS-PAGE system was 4%-12% Bis-Tris reducing SDS-PAGE MES running buffer.

[0401] Methods and Results

[0402] In the present method, the end of fermentation hold is performed as follows. The glucose and ammonia feeds are stopped, the air flow is reduced, and the broth is held at 37°C for four (4) hours with gentle mixing. The pH is adjusted to 4.8-5.2 using sulfuric acid. Heat treatment: The tank is heated to 60°C and held for four (4) hours. Broth harvest: The broth is cooled to 15°C and transferred to a container. Recovery procedure: The same as outlined in Example 6. A visually clear concentrate is produced, wherein the pH adjusted and heat treated broth supernatant provides a significant purity improvement relative to the untreated broth supernatant (data not shown). Similarly, Q-GRFT purity is maintained through the processing steps for cell separation, flocculation and Buchner filtration, and ultrafiltration for dewatering. In particular, the UF concentrate obtained has the highest purity improvement when compared to UF concentrates obtained from broths with different broth treatments (e.g., see Example 2, no heat treatment; and Example 5, with pH 5-5.5 and heat treatment).

[0403] Example 8

[0404] Crystallization of clarified broth concentrate using pH 3, 2% sodium sulfate

[0405] In this example, clarified UF concentrates from Examples 2, 5, 6, and 7 were processed according to the materials and methods described herein.

[0406] Material

[0407] Materials included the following: (i) clarified UF concentrate from Example 2, Example 5, Example 6, and Example 7, (ii) sodium sulfate, (iii) sulfuric acid, and (iv) microscope.

[0408] Methods and Results

[0409] In this example, the clarified concentrate was processed as follows. 2% sodium sulfate was added to the clarified broth concentrate, the pH was adjusted to 3, incubated at 22°C while mixing, and the morphology was monitored over time. More specifically, all concentrates processed according to such methods produced square to rectangular plate-like crystals (data not shown).

[0410] Example 9

[0411] Crystallization of pH 2 treated clarified broth concentrate filtrate with sodium sulfate

[0412] In this example, the filtrate from the pH 2 treatment of Example 4 was processed according to the materials and methods described herein.

[0413] Material

[0414] Materials included the following: (i) filtrate from pH 2 treatment of Example 4, (ii) 10% sodium sulfate, (iii) sodium hydroxide, (iv) pH meter, (v) oscillator with temperature control, and (vi) microscope and supplies.

[0415] Methods and Results

[0416] In this example, the pH 2 treated filtrate was processed as follows. For crystallization, the filtrate was adjusted from pH 2 to pH 3 using sodium hydroxide. 10% sodium sulfate stock solution was added to reach 2% sodium sulfate in the prepared solution, the pH was checked (and adjusted if the pH was not within the range of 2.8-3.2) and incubated at 22°C while mixing. The morphology was monitored by microscopic observation. Crystals were formed in all filtrates processed according to these methods as generally summarized in Table 1 below.

[0417] Table 1 Q-GRFT protein crystals

[0418]

[0419] Example 10

[0420] Crystallization of pH 2 treated clarified broth concentrate filtrate with sodium sulfate

[0421] In this example, the filtrate from the pH 2 incubation of Example 4 was processed according to the materials and methods described herein.

[0422] Materials, methods, and results

[0423] Materials included the following: filtrates from the pH 2 (5° C.) and pH 2 (60° C.) incubations of Example 4. Filtrates were prepared using procedures similar to those outlined in Example 4, except adjusted to pH 3 and incubated at 5° C. and 60° C. Adjusted filtrate samples were held at 5° C. and the morphology monitored over time, with square to rectangular plate-like crystals formed in all filtrates after forty-eight (48) hours at 5° C. (data not shown).

[0424] Example 11

[0425] Crystal recovery and dissolution of crystal precipitates

[0426] This example describes the recovery of crystals and dissolution of crystal precipitates according to the materials and methods described herein.

[0427] Material

[0428] Materials included the following: (i) crystal slurry derived from the clarified broth concentrate of Example 2 (see Example 7), (ii) centrifuge, (iii) 100 mM Tris buffer (pH 8.6), (iv) 100 mM Bis Tris (pH 6.5), (v) 100 mM sodium acetate (pH 5), (v) 0.2 μm Nalgene benchtop filter, and (vii) SDS-PAGE system and reagents.

[0429] method

[0430] A. No-wash crystal recovery

[0431] (1) After forty-seven (47) hours of crystallization, 20 g of crystal slurry was aliquoted into 3 centrifuge tubes, (2) centrifuged at 3000 rpm for 15 minutes, (3) the supernatant was decanted, (4) the following buffer was added to each tube to achieve an initial weight of 20 g: 100 mM Tris buffer (pH 8.6), 100 mM Bis Tris (pH 6.5), and 100 mM sodium acetate (pH 5), (5) mixed at room temperature for one (1) hour, and (6) filtered through a 0.2 μm Nalgene benchtop filter.

[0432] B. 1x Washing Crystal Recovery

[0433] Same process as above, except in step (1), 20 g of DI water was added and mixed.

[0434] result

[0435] Crystallinity = 92%

[0436] Table 2 shows the absorption wavelength (A) of the dissolved crystal precipitate filtrate at 280 nm. 280 ) relative to the lectin protein, where the numbers in parentheses are the purification factors relative to the initial clarified broth concentrate. As presented in Table 2, all values ​​are lower than the initial clarified broth concentrate ratio of 4.96, where the purity of the obtained lectin preparations is at least 2.6 to 6.8 times higher.

[0437] Table 2 Impurities and Lectin A in the filtrates from the dissolution of the unwashed and 1X washed crystal precipitates 280 ratio

[0438]

[0439] <PN 243677>

[0440]

[0441] More specifically, if Figure 8 As presented in , a high purity protein preparation is obtained by dissolving the crystalline precipitate in 100 mM sodium acetate (pH 5.5) followed by filtration. For example, the buffers of Table 2 described above (i.e., 100 mM Tris buffer, pH 8.6; 100 mM Bis Tris, pH 6.5; or 100 mM sodium acetate, pH 5) are Figure 8 Lanes 3, 6, 10, 13 (100

[0442] mM Tris buffer, pH 8.6), lanes 4, 7, 11, 14 (100 mM Bis-Tris, pH 6.5) and lanes 5, 8, 12, 15 (100 mM sodium acetate, pH 5.5).

[0443] like Figure 8 As shown, Q-GRFT was the only band present in the no wash and 1x wash solubilization filtrates on SDS-PAGE. In addition, all filtrates were very clear, with the darkest color observed in the case of pH 8.6 solubilization, and the color of the pH 5 filtrate being very light (data not shown).

[0444] Example 12

[0445] Crystal recovery and precipitate dissolution from crystal slurries obtained from various strains harvested and recovered herein

[0446] This example describes the recovery of crystals and dissolution of crystal precipitates according to the materials and methods described herein.

[0447] Material

[0448] The materials include the following: (i) a crystal slurry prepared from the clarified broth concentrate of Example 4 (see Example 7), (ii) a crystal slurry prepared from the clarified broth concentrate of Example 5 (see Example 7), (iii) a crystal slurry prepared from the clarified broth concentrate of Example 6 (see Example 7),

[0449] (iv) a crystal slurry prepared from the filtrate of Example 2 using the procedure (Example 7), (v) a centrifuge, (vi)

[0450] 100 mM Tris buffer (pH 8.6), (vii) 100 mM Bis Tris (pH 6.5), (viii) 100

[0451] mM sodium acetate (pH 5), (ix) 0.2 μm Nalgene benchtop filter, and (x) SDS-PAGE system and reagents.

[0452] method

[0453] A. No-wash crystal recovery

[0454] (1) After forty-seven (47) hours of crystallization, 20 g of crystal slurry was aliquoted into 3 centrifuge tubes, (2) centrifuged at 3000 rpm for 15 minutes, (3) the supernatant was decanted, (4) the following buffer was added to each tube to achieve an initial weight of 20 g: 100 mM Tris buffer (pH 8.6), 100 mM Bis Tris (pH 6.5), and 100 mM sodium acetate (pH 5), (5) mixed at room temperature for one (1) hour, and (6) filtered through a 0.2 μm Nalgene benchtop filter.

[0455] B. 1x Washing Crystal Recovery

[0456] Same process as above, except in step (1), 20 g of DI water was added and mixed.

[0457] result

[0458] Filtrates of single-band or near single-band purity were obtained from crystal slurries derived from various strains, harvested and recovered according to the methods outlined in the Examples above (data not shown).

[0459] Example 13

[0460] Crystallization of clarified broth concentrate by varying pH, temperature and salt addition

[0461] This example describes the crystallization of the clarified broth concentrate recovered in Example 8 according to the materials and methods described herein.

[0462] Material

[0463] Materials included the following: (i) clarified broth concentrate prepared using the procedure described in Example 6, (ii) sodium sulfate, (iii) ammonium sulfate, (iv) sodium chloride, (v) calcium chloride, (vi) sulfuric acid, and (vii) microscope.

[0464] Methods and Results

[0465] For each salt to be tested, 2% of the salt was added to the clarified broth concentrate, divided into three (3) portions. The pH of one portion was adjusted to pH 4.2, another portion to pH 3, and another portion to pH 2 using sulfuric acid; and each pH adjusted portion (i.e., pH 4.2, 3, and 2) was separated for incubation at 10°C, 22°C, and 50°C while mixing, with morphology monitored over time via microscopy. The same test was set up for conditions without salt addition. Between about four (4) and ninety (90) hours, plate-like crystals ranging from square, rectangular to diamond-shaped were formed.

[0466] More specifically, crystals were formed in solutions without salt addition at about pH 2 at 10°C, 22°C and 50°C, and at pH 3 at 50°C. Crystals were also formed in solutions with sodium sulfate or ammonium sulfate at pH 2, pH 3 and pH 4.2 at 50°C, and at pH 2 and pH 3 at 20°C and 22°C. Likewise, crystals were formed in solutions with sodium chloride or calcium chloride at pH 2 at 10°C, 22°C and 50°C, and at pH 4.2 at 22°C and 50°C.

[0467] Example 14

[0468] Method for producing high-purity griffithsin preparations from fermentation broth

[0469] This example describes a method for recovering a lectin such as Q-GRFT in a substantially pure form from a fermentation broth in which a recombinant host cell (e.g., a bacterial cell, a plant cell, an insect cell, etc.) has been fermented. More particularly, such exemplary purification methods provide a combination of a water-soluble polymer and a salt that spontaneously separates into two (2) fractions, one of which contains the lectin. Such methods may be referred to as liquid-liquid extraction (or two-phase extraction), wherein the target substance (lectin) is preferentially partitioned into one of these fractions. Thus, the materials and methods described herein are not intended to be limiting, as such materials and methods may be readily modified, adjusted, improved, etc. by one skilled in the art to accommodate the specific requirements of any given lectin protein of the present disclosure. For example, commonly used water-soluble polymers for such liquid-liquid (two-phase) extractions include, but are not limited to, polyethylene glycols (PEGs) of various sizes, dextran of various sizes, and chemically modified derivatives thereof. Commonly used salts include, but are not limited to, phosphates, sulfates, and citrates. More particularly, the combination and concentration of salts and polymers are adjusted for optimal recovery and purity of the target lectin.

[0470] In particular, as illustrated herein, purification of lectins (Q-GRFT) from heterologous hosts is greatly aided by low pH treatment. In the context of the present invention, a pH of about pH 2-4 is most useful for separating host proteins from target lectins (Q-GRFT). For example, in some aspects, liquid-liquid (two-phase) separation can be performed by centrifugation, or in some cases, separation can occur spontaneously over the course of several hours. In addition, when performed at low pH, liquid-liquid (two-phase) extraction methods are particularly effective, resulting in excellent recovery and purity compared to the same extraction at neutral pH. Therefore, a particular benefit of the liquid-liquid extraction method of the present invention is that there is no need to utilize a time-consuming and expensive chromatography step.

[0471] More specifically, in this example, the concentrated clarified fermentation broth was prepared with 12% (w / w) ammonium sulfate and adjusted to pH 2 by adding hydrochloric acid (HCl) or sulfuric acid (H2SO4). The precipitated material (impurities) was separated by centrifugation, and the supernatant was prepared with 30% (w / v) polyethylene glycol (PEG) 3350 to induce liquid-liquid extraction. After incubation at 4°C overnight, the material was centrifuged and the precipitated precipitate material was recovered. The recovered precipitate was washed once (1x) with 20mM glycine (pH2), 30% PEG, followed by neutralization and solubilization by adding 40mM Tris base. Fig. 9 As presented in , the purity of the obtained / recovered lectin (Q-GRFT) preparation was greater than 90%.

[0472] Example 15

[0473] Jacaranda lectin-like, CVN-like, OAA-like and galectin-1 lectins in Gram+

[0474] Expression in bacterial cells

[0475] In this example, applicants have designed, constructed, and evaluated exemplary Gram-positive host cells for their ability to express / produce heterologous (exogenous) lectins. More particularly, as described herein, it was unexpectedly observed that Gram-positive bacterial cells can express / produce significant amounts of heterologous lectins, referred to as jacaranda lectin-like, CVN-like, OAA-like, ricin-like, and galectin-1-like lectins. As described and exemplified in the following sections, polynucleotides (expression cassettes) encoding jacaranda lectin-like, CVN-like, OAA-like, ricin-like, and galectin-1-like lectins were constructed and evaluated in 2.5 L high-efficiency flasks.

[0476] Construction of expression plasmids and Bacillus strains expressing jacaranda lectin-like, CVN-like, OAA-like, ricin-like and galectin-1-like lectins

[0477] Thirty-five (35) expression plasmids encoding jacaranda lectin-like, CVN-like, OAA-like, ricin-like, and galectin-1-like lectins were constructed (Table 3) for expression of mature lectins in a Bacillus subtilis host. More specifically, in this example, thirty-five (35) lectin production strains were generated, which were based on Bacillus subtilis strain CBS12. For example, the gene (ORF) encoding the lectin sequence (i.e., without the secretion (signal) sequence) was codon-optimized based on Bacillus subtilis codon preference and cloned into the p3JM vector using a double digestion and ligation method with SpeI and HindIII restriction enzymes ( Fig.10 ). The plasmid contains the Bacillus subtilis aprE promoter operably linked to a codon-optimized nucleotide sequence (ORF) encoding the lectin protein and chloramphenicol acetyltransferase (CAT) as a selection marker for transformation. The constructed vector is subjected to rolling circle amplification and transformed into the host Bacillus subtilis CBS12 and plated on a Luria agar plate supplemented with 5ppm chloramphenicol. Therefore, in some or more embodiments, when fermented under suitable conditions for expressing / producing lectins, a gram-positive host cell containing an imported expression vector / cassette (e.g., p3JM) encoding the lectin protein without using a secretory sequence can express and retain the lectin intracellularly and / or secrete the lectin extracellularly (e.g., secreted into broth via a general secretory pathway).

[0478] Evaluation of secreted lectins produced in high-efficiency flasks

[0479] The expression of lectin protein in Bacillus subtilis strain CBS12 was evaluated in 2.5 L high efficiency flasks and recovered as generally described herein. More specifically, the characterization of lectins expressed in recombinant Bacillus host cells is summarized in Table 3 below ( Fig.11 In particular, certain OAA-like lectins described herein (e.g., SEQ ID NO: 14, SEQ ID NO: 18-20) (Table 3) were expressed in similar amounts in cell broth and cell lysate (i.e., when the cell pellet was lysed), indicating low secretion efficiency of certain lectins without the use of a signal (secretion) sequence.

[0480] Table 3 Characterization of lectins expressed in Gram-positive host cells of the present disclosure

[0481]

[0482] Table 3 (continued) Characterization of lectins expressed in Gram-positive host cells of the present disclosure

[0483]

[0484]

[0485] Y: purified; N: not purified.

[0486] Fermentation and recovery

[0487] As presented in Table 3 above, recombinant host cells expressing lectin proteins are grown in a fermentation medium (Grant's II medium) with cell growth nutrients containing carbon sources (such as sugars, alcohols, organic acids and amino acids), nitrogen sources (such as ammonium salts and nitrates), phosphates, magnesium salts, potassium salts, sodium salts, and trace metal salts such as iron, manganese, zinc, copper, cobalt, molybdate, calcium, and boron to produce a large amount of cell material. The cell bank of the recombinant strain (cell) is maintained in a frozen state and first inoculated into sterile LB medium to prepare seeds for efficient flask fermentation. The seed cells are further grown in batch fermentation medium. The strains are cultured in 2.5L flasks in an Infos shaker rotating at 220rpm. The fermentation temperature is maintained at 37°C for 16 hours, then reduced to 32°C and cultured for another 32 hours. Some contaminant proteins come from Bacillus (host) cell debris and naturally secreted host cell enzymes.

[0488] In the present example, the fermentation broth is recovered as a clarified concentrate, which can be recovered by a variety of methods, which generally start with a broth treatment that includes lysis (e.g., natural or chemically induced lysis, in this example natural), heat treatment, pH and temperature control, water or buffer dilution, and with or without flocculation. For example, cell separation can be performed in a variety of methods, including but not limited to centrifugation, depth filtration, or membrane-based operations. Concentration is performed via an ultrafiltration membrane operation. In some aspects, the resulting clarified concentrate is further purified, as described in the subsequent examples below. Additional details about the recovery process can be found in the subsequent examples and the specification of the present disclosure.

[0489] For example, the jacaranda lectin-like ( Figure 2 ), CVN-like ( Figure 3 ), OAA-like ( Figure 4 ), ricin-like ( Figure 5 ) and galectin-1-like ( Figure 5) lectin in broth supernatant (data not shown), where fifteen microliters (15 μL) of sample were evaluated by SDS-PAGE along with Invitrogen broad spectrum molecular weight standards or Sangon RealBand protein standards, followed by staining and retention of the gel using standard molecular biology procedures. In particular, the jacaranda lectin-like proteins (SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 39) were expressed as a single band with a molecular weight between about 10 kDa and 31 kDa on SDS-PAGE. In certain aspects, the molecular weight of the jacaranda lectin-like lectin of SEQ ID NO: 39 was observed to be ( Figure 2 E) is about 31 kDa (data not shown), which is twice its theoretical molecular weight, which may be caused by the strong binding of two monomers.

[0490] Similarly, CVN-like protein ( Figure 3 ; SEQ ID NO: 10 and SEQ ID NO: 11;) appeared as a single band with a molecular weight between about 10 kDa and 15 kDa on SDS-PAGE (data not shown), and OAA-like protein ( Figure 4 ; SEQ ID NO:14-23 and SEQ ID NO:29,30,31,32,33,34,35,36,37,38) showed a single band with a molecular weight between about 10 kDa and 40 kDa on SDS-PAGE (data not shown). Ricin-like lectin ( Figure 5 ; SEQ ID NO: 24) appears as a single band with a molecular weight between about 10 kDa and 40 kDa on SDS-PAGE, and the galectin-like protein ( Figure 5 ; SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28) appeared as a single band with a molecular weight between about 10 kDa and 15 kDa on SDS-PAGE.

[0491] Unlike other classes of lectins, similar amounts of OAA-like lectins comprising SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 were found in both the broth and the cell lysate when the cell pellet was lysed, indicating their low secretion efficiency and certain challenges in secreting (or binding) certain lectin proteins.

[0492] Example 16

[0493] Lectin recovery and purification

[0494] This example describes a general method for recovering one or more expressed / produced lectins in substantially pure form from a fermentation broth in which recombinant host cells (e.g., bacterial cells, plant cells, insect cells, etc.) have been fermented. More particularly, such exemplary purification methods include acid treatment by addition of sulfuric acid, isoelectric precipitation, ion affinity separation via a chromatographic column, which separates into two (2) or more fractions, wherein one of these fractions contains the lectin. The methods for purification of each lectin are listed in Table 4 below.

[0495] For example, as shown in Table 4, the fermentation crudes of certain OAA-like and galectin-1 lectins (SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 27, and SEQ ID NO: 28) were concentrated and ammonium sulfate was added to a final concentration of 1 M. The solution was loaded onto a HIC column pre-equilibrated with 20 mM Tris-HCl (pH 7.5) supplemented with 1 M ammonium sulfate. The target lectin protein was eluted by a gradient with 0-1 M ammonium sulfate in 20 mM Tris-HCl (pH 7.5) buffer. The fractions containing the lectin protein were concentrated, buffer exchanged, and combined into one (1) fraction. The final concentrated samples of lectins (SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 27 and SEQ ID NO: 28) were formulated with 20 mM Tris pH 7.5, 150 mM NaCl buffer and 40% w / w glycerol. The final concentrated sample of lectin (SEQ ID NO: 14) was formulated with 20 mM NaPi pH 7.5, 150 mM NaCl buffer and 40% w / w glycerol. A special case is the galectin-1 lectin (SEQ ID NO: 27 and SEQ ID NO: 28) which is sensitive to low pH (e.g., pH 2.0). Both lectins formed a precipitate when the pH was adjusted to pH 2.0 by dropwise addition of 1 molar sulfuric acid (H2SO4). In other embodiments (Table 4), purification of lectins from heterologous hosts (SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 25) was greatly aided by low pH treatment. In the context of the present invention, a pH of about pH 2 is most useful for separating (e.g., background) host proteins from one or more target lectin proteins. For example, host proteins are precipitated at pH 2, where the host proteins and target lectins can be separated using a centrifuge. The lectins retained in the supernatant are collected, and the pH is adjusted to neutral by adding 2 molar sodium hydroxide, and ammonium sulfate is added to a final concentration of 1 M. The sample containing the lectins is then loaded onto a HIC column as described above. The final concentrated sample is formulated with 20 mM Tris pH 7.5, 150 mM NaCl buffer and 40% w / w glycerol.

[0496] In certain other embodiments (Table 4), purification of ricin-like lectin (SEQ ID NO: 24) from heterologous hosts is greatly aided by low pH treatment. In the context of the present invention, a pH of about pH 2 is most useful for separating host proteins from target lectins. Host proteins are precipitated at pH 2, where host proteins and target lectins can be separated using a centrifuge. The lectins in the supernatant are collected and the pH is adjusted to neutral by adding 2 molar sodium hydroxide. The final concentrated sample is formulated with 20 mM sodium citrate pH 3.5, 150 mM NaCl buffer and 40% w / w glycerol.

[0497] In one or more other embodiments (Table 4), the purified product of the jacaranda lectin-like lectin (SEQ ID NO: 8) from a heterologous host was ultrafiltered and ammonium sulfate was added to a final concentration of 1 M. The solution was loaded onto a HIC column pre-equilibrated with 20 mM NaAc pH 5.0 supplemented with 1 M ammonium sulfate. The target lectin protein was eluted with a 0 to 1 M ammonium sulfate gradient. The fractions containing the target protein were combined and buffer exchanged into 20 mM Tris-HCl pH 7.5, and then incubated overnight at 4°C to promote isoelectric precipitation. The resulting target protein precipitate was resuspended in 20 mM sodium citrate pH 3.5, 150 mM NaCl buffer and 40% w / w glycerol.

[0498] In certain other embodiments (Table 4), the purification of CVN-like lectin (SEQ ID NO: 11) from a heterologous host was ultrafiltered and ammonium sulfate was added to a final concentration of 1 M. The solution was loaded onto a HIC column pre-equilibrated with 20 mM Tris pH 7.5 supplemented with 1 M ammonium sulfate. The target protein was eluted with a 0 to 1 M ammonium sulfate gradient. The fractions containing the target protein were combined and buffer exchanged into 20 mM NaAc pH 5.0 and then applied to an AIEX column pre-equilibrated with 20 mM NaAc pH 5.0. The target protein was eluted with a 0 to 0.5 M NaCl gradient. The resulting target protein fractions were then combined and concentrated via a 5K AmiconUltra device and then stored at -20°C in 20 mM Tris pH 7.5, 150 mM NaCl buffer and 40% w / w glycerol.

[0499] In yet other embodiments (Table 4), the purification of OAA-like lectin (SEQ ID NO: 15) from a heterologous host was ultrafiltered and buffer exchanged into 20 mM NaPi (pH 6.0) and then applied to an AIEX column pre-equilibrated with 20 mM NaPi (pH 6.0). The target protein was eluted with a 0 to 0.5 M NaCl gradient. The resulting target protein fractions were then combined and concentrated via a 5K Amicon Ultra device and then stored at -20°C in 20 mM Tris pH 7.5, 150 mM NaCl buffer and 40% w / w glycerol.

[0500] In certain other embodiments (Table 4), the purified product of OAA-like lectin (SEQ ID NO: 21) from a heterologous host is ultrafiltered and ammonium sulfate is added to a final concentration of 1 molar. The solution is loaded onto a HIC column pre-equilibrated with 20mMTris pH 7.5 supplemented with 1M ammonium sulfate. The target protein is eluted with a 0 to 1M ammonium sulfate gradient. The fractions containing the target protein are combined and buffer exchanged into 20mM NaAc pH 5.5 and then applied to an AIEX column pre-equilibrated with 20mM NaAc pH 5.5. The target protein is eluted with a 0 to 0.5M NaCl gradient. The resulting target protein fractions are combined and buffer exchanged into 20mM NaAc pH 5.0 and then applied to a CIEX column pre-equilibrated with 20mM NaAc pH 5.0. The target protein is eluted with a 0 to 0.5M NaCl gradient. The resulting target protein fraction was concentrated via a 5K Amicon Ultra device and then stored in 20 mM NaPi pH 7.0, 150 mM NaCl buffer and 40% w / w glycerol at -20°C. In addition, OAA-like lectin (SEQ No 21) was completely precipitated after pH adjustment to 2, and the protein remained insoluble after pH adjustment to neutrality.

[0501] In certain other embodiments (Table 4), the purified product of galectin-1 lectin (SEQ ID NO: 26) from a heterologous host was ultrafiltered and exchanged into 20mM NaPi pH 7.0. The solution was loaded onto a CIEX column pre-equilibrated with 20mM NaPi pH 7.0. The target protein was eluted with a 0 to 0.5M NaCl gradient. The fractions containing the target protein were combined and buffer exchanged into 20mM NaPi pH 7.0 and then applied to an AIEX column pre-equilibrated with 20mM NaPi pH 7.0. The target protein was eluted with a 0 to 0.5M NaCl gradient. The fractions containing the target protein were combined and buffer exchanged into 20mM Tris pH 7.5, and ammonium sulfate was added to a final concentration of 1 molar and then applied to a HIC column pre-equilibrated with 20mM Tris pH 7.5 supplemented with 1M ammonium sulfate. The target protein was eluted with a 0 to 1M ammonium sulfate gradient. The fractions containing the target protein were combined and buffer exchanged into 20mM Tris pH 7.5 and then applied to an AIEX column pre-equilibrated with 20mM Tris pH 7.5. The target protein was eluted with a 0 to 0.5M NaCl gradient. The resulting target protein fractions were then combined and concentrated via a 5K Amicon Ultra device and then stored at -20°C in 20mM Tris pH 7.5, 150mM NaCl buffer and 40% w / w glycerol. Similar to galectin-1-like lectins (SEQ No 27 and 28), galectin-1 lectin (SEQ No 26) was also sensitive to pH 2, and when the pH of the broth was adjusted to 2, a high ratio of lectins precipitated.

[0502] Table 4 Methods for lectin purification

[0503]

[0504]

[0505] Table 4 (Continued) Methods used for lectin purification

[0506]

[0507]

[0508] The columns used for lectin purification listed in Table 4 are: HIC: column HiPrepTM Phenyl FF (high substitution) 16 / 10; AIEX: column HiPrepTM Q FF 16 / 10; CIEX: column HiPrepTM SP FF 16 / 10

[0509] Example 17

[0510] Determination of hemagglutination activity of lectins

[0511] This example describes a method for screening the binding activity of one or more of the above-described purified lectins using a hemagglutination assay. The principle is that active lectins react with specific carbohydrate moieties on the surface of erythrocytes, resulting in the formation of a diffusion matrix, while inactive lectins are unable to bind to erythrocytes, resulting in the formation of distinct clumps. This allows for a clear visual distinction between active and inactive lectin samples. More specifically, erythrocytes from fifteen (15) different animal sources (i.e., dog, rabbit, guinea pig, mouse, rat, human, chicken, turkey, duck, goose, pig, cattle, horse, sheep, and goat) were used to evaluate the hemagglutination ability (activity) of the lectins and processed according to the materials and methods described herein.

[0512] Material

[0513] Materials included the following: (i) animal erythrocytes in Alsever's solution (Sbjbio company, Nanjing, China), including dog, rabbit, guinea pig, mouse, rat, human, chicken, turkey, duck, goose, pig, cow, horse, sheep, and goat erythrocytes, (ii) 200 mM phosphate buffer (PB, pH 7.4), and (iii) 96-well round-bottom microplates (Nunc, Thermo Scientific, USA).

[0514] method

[0515] (i) Hemagglutination assay was used to detect lectins in purified and crude samples. Purified lectin samples were diluted to an initial concentration of 200-400 μg / ml using PB buffer, and 50 μL of each sample was then dispensed into the sample wells of a 96-well round-bottom microplate. Crude samples were diluted by estimating protein expression levels based on SDS-PAGE gels. (ii) 50 μL of host strain supernatant was dispensed into the control wells of the above microplate and used as a negative control. A 50 μL PB buffer column was also included in the above microplate to determine possible buffer effects. (iii) Animal erythrocytes were diluted to an initial concentration of 2% (v / v) using PB buffer, and 50 μL of each erythrocyte was then added to each of the above samples. (iv) The mixture was pipetted for 30 seconds to merge, and the plate was then allowed to stand at room temperature for 60 minutes. (v) Hemagglutination activity was determined by visual inspection. Active lectins are characterized by the formation of a diffusion network, while inactive lectins were observed to form deposited buttons at the bottom of the wells.

[0516] result

[0517] like Fig.12As shown in , the hemagglutination ability of the lectin samples can be easily determined by visually distinguishing a homogenous suspension with no signs of clumping wells for the positive samples and a button-like sediment at the bottom of the wells for the negative samples. PB buffer and host strain supernatants showed no hemagglutination ability ( Fig.12 A and Fig.12 B) When different doses of lectin were used on 1% mouse erythrocytes, a dose-response pattern of hemagglutination activity of the active samples was detected, indicating that the hemagglutination ability of lectin was dose-dependent ( Fig.12 A). This shows that the hemagglutination assay is useful for distinguishing strongly positive samples (e.g. Fig.12 A, 200 μg / ml of SEQ ID NO: 1), weak positive samples (e.g., Fig.12 A 0.78 μg / ml of SEQ ID NO: 5), intermediate positive samples (e.g., Fig.12 A), and negative samples (e.g., Fig.12 At least two parallel plates were prepared for each test to ensure data accuracy, and the results showed good reproducibility ( Fig.12 B). Hemagglutination by lectin is stable for approximately 1-2 hours at room temperature, and observation and interpretation may vary with the test, animal red blood cell batch, type and concentration of lectin, incubation time and temperature used in the assay. Therefore, in this example, the hemagglutination assay is not intended to be used as a quantitative method, but rather to detect positive samples. The most obvious positive results are recorded in Fig.13 and the corresponding valid animal red blood cells are summarized in Table 5 below.

[0518] Table 5 Hemagglutination activity of the generated lectins

[0519]

[0520] Table 5 (Continued) Hemagglutination activity of the generated lectins

[0521] 28 Purified 50 Guinea pigs, mice, rats, pigs, cattle, horses, goats 29 Crude product - Rabbit, guinea pig, mouse, rat, pig, cow, horse, goat 30 Crude product - Mouse, Rat 31 Crude product - Mouse, Rat 32 Crude product - Rabbit, mouse, rat, pig, cow, horse, goat 33 Crude product - Rabbit, guinea pig, mouse, rat, pig, cow, horse, goat 34 Crude product - - 35 Crude product - Mouse, Rat 36 Crude product - Mouse, Rat 37 Crude product - Mouse, Rat 38 Crude product - Rabbit, guinea pig, mouse, rat 39 Crude product - -

[0522] ND - Not Detected Yet.

[0523] References PCT Publication No. WO 2002 / 14490 PCT Publication No. WO 2003 / 083125 PCT Publication No. WO 2005 / 118627 PCT Publication No. WO 2007 / 064844 PCT Publication No. WO 2008 / 022303 PCT Publication No. WO 2010 / 014248 PCT Publication No. WO 2014 / 197650 PCT Publication No. WO 2016 / 130628 PCT Publication No. WO 2019 / 108656

[0524] US Patent Publication No. US20020127675

[0525] U.S. Patent Publication No. US20040204365

[0526] U.S. Patent Publication No. US20110189105

[0527] US Patent Publication No. US20110263485

[0528] Ausubel et al., "Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).

[0529] Ayouba et al., "Legume lectins interact with muramic acid and N-acetylmuramic acid", FEBS, 289(1):12-104, 1991.

[0530] Cole et al., "Differentiation of Bacillus anthracis and Other BacillusSpecies by Lectins", J. Clinical Microbiol., 19(1):48-54, 1984.

[0531] El-Araby et al., "Characterization and antimicrobial activity oflectins purified from three Egyptian leguminous seeds", AMB Express, 10:90(pages1-14), 2020.

[0532] Gengenbach et al.,“Comparison of microbial and transient expression(tobacco plants and plant-cell packs)for the production and purification ofthe anticancer mistletoe lectin viscumin”,Biotechnology and Bioengineering,116,pages 2236-2249,2019.

[0533] Hirayama et al.,“High-Mannose Specific Lectin and Its Recombinantsfrom a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIVActivity Through High-Affinity Binding to the Viral Envelope Glycoproteingp120”,Mar.Biotechnol.,Vol 18,pages 144-160,2016.

[0534] Lagarda-Diaz et al.,“Legume Lectins:Proteins with DiverseApplications”,Int.J.Mol.Sci.,18,1242,2017.

[0535] O’Keefe et al.,“Scaleable manufacture of HIV-1entry inhibitorgriffithsin and validation of its safety and efficacy as a topicalmicrobicide component”,PNAS,Vol.106,No.15,pages 6099-6104,2009.

[0536] Petrova et al.,“Engineering Lactobacillus rhamnosus GG and GR-1toexpress HIV-inhibiting griffithsin”,International Journal of AntimicrobialAgents,volume 52,issue 5,pages 599-607,2018.

[0537] Petrova et al.,“The lectin-like protein 1 in LactobacillusrhamnosusGR-1 mediates tissue-specific adherence to vaginal epithelium andinhibitsurogenital pathogens”,Scientific Reports 6,Article No.37437,2016.

[0538] Peumans and Van Damme,“Plant Lectins:Versatile Proteins withImportantPerspectives in Biotechnology”,Biotechnology and GeneticEngineering Reviews,15:1,199-228,1998.

[0539] Pevzner et al.,“Differences in amino acid sequences of mistletoelectinI and III B-subunits determining carbohydrate binding specificity”,Biochimica et Biophysica Acta 1675:155-164,2004.

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Claims

1. A method for producing a lectin in a Gram-positive bacterial cell, the method comprising: (a) obtaining a Gram-positive cell and introducing an expression cassette encoding a lectin into the cell, wherein the cassette comprises an upstream promoter operably linked to a downstream open reading frame (ORF) encoding the lectin, and (b) fermenting the cells to produce the lectin.

2. The method of claim 1, wherein the cell comprises at least two introduced cassettes encoding the lectin, wherein the cassette comprises an upstream promoter sequence operably linked to a downstream ORF encoding the lectin.

3. A method for producing a lectin in a Gram-positive bacterial cell, the method comprising: (a) obtaining a Gram-positive cell and introducing an expression cassette encoding a lectin into the cell, wherein the cassette comprises an upstream promoter operably linked to a downstream nucleic acid encoding a signal sequence, the signal sequence being operably linked to a downstream open reading frame (ORF) encoding the lectin, and (b) fermenting the cells to produce the lectin.

4. The method of claim 3, wherein the cell comprises at least two introduced cassettes encoding the lectin, wherein the cassette comprises an upstream promoter sequence operably linked to a downstream nucleic acid encoding a signal sequence, the signal sequence operably linked to a downstream ORF encoding the lectin.

5. A method for producing a lectin in a Gram-positive bacterial cell, the method comprising: (a) obtaining a Gram-positive cell and introducing a first and a second expression cassette encoding a lectin into the cell, wherein the first cassette comprises an upstream promoter operably linked to a downstream open reading frame (ORF) encoding the lectin, and the second cassette comprises an upstream promoter sequence operably linked to a downstream nucleic acid encoding a signal sequence, the signal sequence being operably linked to the downstream ORF encoding the lectin, and (b) fermenting the cells to produce the lectin.

6. The method of any one of claims 1, 3 or 5, wherein the ORF encodes a natural lectin or variant lectin derived from a cyanobacterial lectin, a plant lectin, a fungal lectin, a bacterial lectin, an insect lectin, or an animal lectin.

7. A method for recovering intracellular lectins produced by Gram-positive bacterial cells, the method comprising: (a) constructing and fermenting Gram-positive cells according to any one of claims 1, 3 or 5, (b) lysing cells at the end of said fermentation to obtain a lysed cell broth, heat-treating said lysed cell broth at a pH between 1.5 and 8.5, then cooling the broth and harvesting said cooled broth, (c) subjecting said harvested cooled broth to a clarification process, and (d) subjecting the clarified broth to a concentration process, wherein said lectin is recovered in said clarified concentrated broth.

8. A method for recovering a secretory lectin produced by a Gram-positive cell, the method comprising: (a) constructing and fermenting Gram-positive cells according to any one of claims 1, 3 or 5, and harvesting the broth, (b) subjecting said harvested broth to a clarification process, and (c) subjecting the clarified broth to a concentration process, wherein said lectin is recovered in said clarified concentrated broth.

9. A method for increasing the purity of a lectin concentrate, the method comprising: (a) obtaining a lectin concentrate recovered according to claim 7 or claim 8, adjusting the pH of the concentrate to between 1.5 and 8.5, (b) incubating the concentrate of step (a) at a temperature between about 5°C and 55°C for a sufficient amount of time, and (c) centrifuging the concentrate and collecting the supernatant, or filtering the concentrate and collecting the filtrate, wherein said collected supernatant or said collected filtrate comprises substantially purified lectin relative to the purity of said lectin concentrate obtained in step (a).

10. A method for increasing the purity of a lectin concentrate, the method comprising: (a) obtaining a lectin concentrate recovered according to claim 7 or claim 8, adjusting the pH of the concentrate to about pH 2, incubating the concentrate at pH 2 at a temperature between about 55°C and 65°C for a sufficient amount of time, and (b) centrifuging the incubated concentrate and collecting the supernatant, or filtering the concentrate and collecting the filtrate, wherein said collected supernatant or said collected filtrate comprises substantially purified lectin relative to the purity of said lectin concentrate obtained in step (a).

11. A method for crystallizing a lectin, the method comprising: (a) obtaining a lectin concentrate recovered according to any one of claims 7 to 10, (b) adding a salt or a mixture of salts to the concentrate, the salt or mixture of salts comprising sodium, calcium, ammonium, sulfate or chloride ions, adjusting the pH of the salted concentrate to about pH 2 to 5, and mixing and incubating the concentrate at between about 5°C and 50°C for an appropriate amount of time, wherein after mixing and incubating for an appropriate amount of time in step (b), the lectin crystallizes from the concentrate.

12. A method for crystallizing a lectin, the method comprising: (a) obtaining a lectin concentrate recovered according to any one of claims 7 to 10, (b) adding 0.5% to 10% of a salt or a mixture of salts comprising sodium, calcium, ammonium, sulfate or chloride ions to the concentrate, adjusting the pH of the salted concentrate to about pH 2 to 5, and mixing and incubating the concentrate at between about 5°C and 50°C for an appropriate amount of time, wherein after mixing and incubating for an appropriate amount of time in step (b), the lectin crystallizes from the concentrate.

13. A method for improving the purity of a lectin preparation, the method comprising: (a) obtaining a lectin crystal slurry according to the method of claim 11 or claim 12, optionally washing the crystal slurry with water and mixing, and centrifuging the slurry for a sufficient amount of time, decanting and collecting the supernatant after centrifugation, adding an appropriate pH buffer to maintain a pH of about 4.8 to about 8.6, while mixing at room temperature for a sufficient amount of time, and (b) filtering the supernatant and collecting the filtered supernatant, wherein the collected supernatant contains a highly pure lectin preparation.

14. A method for producing and recovering a high-purity griffithsin (GRFT) protein preparation, the method comprising: (a) constructing and fermenting recombinant Gram-positive bacterial cells expressing heterologous GRFT protein, (b) lysing cells at the end of said fermentation to obtain a lysed cell broth, and treating said lysed cell broth by maintaining the broth at a pH of about 4.8 to about 5.2 and a temperature of about 50° C. to about 80° C. for about 1 to about 4 hours, (c) clarifying the broth of step (b) by a filtration or microfiltration process, and concentrating the clarified broth by an ultrafiltration process, (d) subjecting the concentrated broth of step (c) to a crystallization process, the crystallization process comprising adding about 2% sodium sulfate to the concentrate, adjusting the pH to about 3 and incubating at about 22° C. for a sufficient amount of time, (e) centrifuging the incubation concentrate of step (d), decanting the supernatant to harvest GRFT crystals, dissolving the GRFT crystals in about 100 mM sodium acetate buffer at about pH 4.5 to about 5.5 to obtain an aqueous GRFT protein preparation, and centrifuging the GRFT preparation to remove any insoluble impurities therein and collecting the supernatant, or filtering the GRFT preparation to remove any insoluble impurities therein and collecting the filtrate, wherein the supernatant collected in step (e) or the filtrate collected in step (e) contains a high-purity GRFT protein preparation.

15. The method of claim 14, wherein the highly pure GRFT preparation comprises a native GRFT protein or a variant GRFT protein.

16. The method of claim 14, wherein the high purity GRFT preparation is at least 2.0 times purer than the recovered GRFT concentrate, such as by 280 The method may be carried out as determined by the GRFT concentration measured at 100 nm and / or wherein the GRFT is the major band, or the only band of about 12.7 kDa, in the high purity GRFT preparation when visualized by SDS-PAGE.

17. A recombinant Gram-positive bacterial cell expressing a polynucleotide encoding a heterologous lectin.

18. The recombinant cell of claim 17, comprising an introduced polynucleotide cassette encoding the lectin, wherein the cassette comprises an upstream promoter operably linked to a downstream nucleic acid encoding the lectin, or wherein the cassette comprises an upstream promoter operably linked to a downstream nucleic acid encoding a signal sequence operably linked to a downstream nucleic acid encoding the lectin.

19. The recombinant cell of claim 17, comprising at least two introduced cassettes encoding the lectin, wherein the at least two introduced cassettes comprise an upstream promoter operably linked to a downstream nucleic acid encoding the lectin.

20. The recombinant cell of claim 17, comprising at least two introduced cassettes encoding the lectin, wherein the introduced cassettes comprise an upstream promoter operably linked to a downstream nucleic acid encoding a signal sequence operably linked to a downstream nucleic acid encoding the lectin.

21. The recombinant cell of claim 17, comprising at least two introduced cassettes encoding the lectin, wherein one introduced cassette comprises an upstream promoter operably linked to a downstream nucleic acid encoding the lectin, and a second introduced cassette comprises an upstream promoter operably linked to a downstream nucleic acid encoding a signal sequence operably linked to the downstream nucleic acid encoding the lectin.

22. The recombinant cell of claim 17, wherein the heterologous lectin is derived from a cyanobacterial lectin, a plant lectin, a fungal lectin, a bacterial lectin, or an animal lectin.

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