# Patent

## Title
Isolation and application of BAD-1 for diagnosing infections with Blastomyces dermatitidis

## Abstract

Methods for obtaining highly pure native, recombinant or modified BAD-1 protein include the steps of culturing a population of microbes expressing BAD-1 protein in a culture medium, collecting the population of microbes from the culture medium, obtaining a BAD-1 protein-containing solution, and purifying the BAD-1 protein from the solution by combining the BAD-1 protein-containing solution with a nickel-chelating resin, washing the nickel-chelating resin to remove unbound matter, and eluting the BAD-1 protein from the nickel-chelating resin. Highly pure native BAD-1 protein may be used in diagnostic kits for detecting Blastomyces dermatitidis infections in animals.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims benefit from U.S. Provisional Application 61/579,390, filed Dec. 22, 2011 and U.S. Provisional Application 61/579,959, filed Dec. 23, 2011. These applications are incorporated herein by reference for all purposes.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH/DEVELOPMENT
This invention was made with government support under A1035681 awarded by the National Institutes of Health. The government has certain rights in the invention.


BACKGROUND OF THE INVENTION
Blastomyces dermatitidis is a fungus found primarily in soil and is endemic throughout the Southeast, Southcentral and Upper Midwestern U.S. and in parts of Canada, India and Africa. Most cases of blastomycosis in the U.S. occur in the Ohio and Mississippi river valleys, the southeastern states, and around the Great Lakes (Bradsher, Chapman, et al., 2003). Blastomycosis typically starts in the lungs after inhalation of conidia or hyphal fragments causing pneumonia-like symptoms and may lead to disseminated disease if not diagnosed and treated early (Brandhorst, et al., 2005). Dogs are particularly vulnerable to infection, especially hunting dogs.
Upon entry into the host, B. dermatitidis undergoes a temperature-dependent phase transition into a pathogenic yeast form. Upon transition, yeast phase cells secrete and display on their surface BAD-1 (Blastomyces adhesin 1), a 120-kDa multi-functional protein that promotes adherence to macrophages by binding CD11b/CD18 (CR3) and CD14 (Newman, Chaturvedi, et al., 1995) and deviates host pro-inflammatory responses by suppressing tumor necrosis factor-α (TNF-α) (Finkel-Jimenez, Wüthrich, et al., 2001; Brandhorst, Finkel-Jimenez, et al., 2004) and inducing transforming growth factor-β (Finkel-Jiminez, Wüthrich, et al., 2002). Soluble BAD-1 released by wild-type yeast enters macrophages via CR3 receptor-mediated endocytosis, and this event has likewise been demonstrated to suppress tumor necrosis factor-α responses and control of the infection (Finkel-Jiminez, Wüthrich, et al., 2002).
A variety of techniques have been used to aid in the clinical diagnosis of blastomycosis. These include microscopic detection of characteristic broad-based budding yeast forms in body fluids or tissue biopsies, isolation and identification of the organism in culture, detection of B. dermatitidis-specific antigens in urine, and detection of specific immunologic responses to infection (Rippon, 1988; Mongkolrattanothai, Peev, et al., 2006). However, clinical tests for blastomycosis suffer from potential false positives due to cross-reactivity with common fungal antigens. One approach to reduce the number of false positives in blastomycosis detection is to use BAD-1, a protein unique to B. dermatitidis, as a biomarker for diagnosing blastomycosis infection.
To date, methods for isolating BAD-1 have relied on recombinant BAD-1 proteins, either full length or truncated, that display a 6×His tag and are purified on nickel affinity agarose columns (Finkel-Jimenez, Wüthrich, et al., 2001; Hogan, et al., 1995).
SUMMARY OF THE INVENTION
In one aspect, the present invention relates to a method for the purification of native BAD-1 protein or protein fragments comprising the steps of: a). obtaining a native BAD-1 protein-containing or protein fragment-containing solution; and b). purifying the native BAD-1 protein or protein fragments from the solution by the steps of: i). combining the native BAD-1 protein or protein fragments-containing solution with a suitable divalent cation, ii). washing the suitable divalent cation to remove unbound matter, and iii). eluting the native BAD-1 protein or protein fragments from the nickel-chelating resin.
In another aspect, the present invention relates to a composition comprising highly pure native BAD-1 protein or protein fragments purified from Blastomyces dermatitidis, wherein the composition comprises less than about 0.21 mg mannoprotein per mg of native BAD-1 protein or protein fragments.
In another aspect, the present invention relates to a diagnostic method for detecting Blastomyces dermatitidis in a mammalian patient suspected of being infected, comprising the steps of: a). providing highly purified native BAD-1 protein or protein fragment, b). preparing a specimen from the mammalian patient and exposing the specimen to the highly purified native BAD-1 protein or protein fragments to form a reaction mixture, and c). analyzing the reaction mixture and determining a diagnosis by using a desired product as the reference, wherein the presence of the desired product in the reaction mixture indicates the mammalian patient is infected with Blastomyces dermatitidis. 
In another aspect, the present invention relates to a diagnostic kit for detecting an infection of Blastomyces dermatitidis in an animal comprising a highly pure native BAD-1 protein or protein fragment.



DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph of sodium dodecyl sulfate polyacryamide gel electrophoresis (SDS-PAGE) analysis showing relative protein enrichment by Ni-NTA resin. Coomassie stain of 10% PAGE gel (images collated from 2 separate gels). Lane 1—MW standard (kDa); lane 2—H2O extract of B. dermatitidis yeast; lane 3—protein not bound by Ni-NTA resin; lane 4—Ni-NTA elution fraction; lane 5—BAD-1 after extraction with 10 μg of a conA (concanavalin A)-agarose resin, dialysis against H2O and concentration via CENTRIPREP® (Millipore, Billerica, Mass.).
FIG. 2 is graph showing total protein at each step of enrichment process. Protein quantity was estimated by measuring absorbance at A280 for each step of the BAD-1 enrichment process. Absorbance was measured prior to adjustment for the empirically determined absorbance co-efficient of BAD-1.
FIG. 3 is a graph of SDS-PAGE analysis showing relative carbohydrate removal by concanavalin A (ConA)-agarose resin purification. Ten percent PAGE gel of BAD-1 enriched fractions (images collated from 2 separate gels) were stained for carbohydrate with ProQ® Emerald stain to visualize carbohydrates/glycoproteins. Lane 1—MW standard (kDa); lane 2—Ni-NTA elution fraction containing BAD-1; and lane 3—BAD-1 after extraction of contaminants via concanavalin-agarose resin (10 μg). BAD-1 migrates at 120 kDa, and primary mannoprotein contaminants migrate at  ̃220 kDa. Note that most of the 220 kDa contaminant has been removed.
FIG. 4 is a graph showing total carbohydrate at each step of the enrichment process. Phenol Sulfuric Acid (PSA) was used to determine total carbohydrate present in each step of the BAD-1 enrichment process. Columns 3-5 are represented in FIG. 5 at a more proportionate range of carbohydrate concentration.
FIG. 5 is a graph showing total carbohydrate at each step of enrichment process. Blow up of lanes 3-5 of FIG. 4.
FIG. 6 is the native BAD-1 protein sequence of the 26199 strain (SEQ ID NO:1).
FIG. 7 is the native BAD-1 protein sequence of the 14081 strain (SEQ ID NO:2).
FIG. 8 is the native BAD-1 protein sequence of the ER-3 strain (SEQ ID NO 3).
FIG. 9 shows a domain of the BAD-1 sequence where the known native 60636 strain matches perfectly with the 14081 strain.
FIG. 10 shows the protein sequence of a BAD-1 protein fragment, which is a native BAD-1 protein of the 26199 strain lacking the C-terminus EGF-like domain (Delta C-Term) (SEQ ID NO:4).
FIG. 11 shows the protein sequence of a BAD-1 protein fragment, which includes a four tandem repeat domain set after a six-histidine tag (TR4) (SEQ ID NO:5).



DETAILED DESCRIPTION OF THE INVENTION
A novel method for obtaining large quantities of BAD-1 protein without a 6×His tag or other capture means from batch-cultured microorganisms is described herein. The isolated BAD-1 protein may be native, non-recombinant BAD-1 or a modified recombinant BAD-1, for example, in which the C-terminal epidermal growth factor consensus sequence has been removed. A preferred subsequent purification step may remove contaminating carbohydrates to provide highly pure BAD-1 protein samples for use in diagnostic tests and the like.
As used herein, by use of the term “affinity tag,” e.g., a 6×His tag, or other “capture” means, we mean to exclude methods that use cation chelation as a capture mechanism.
As used herein, the phrase “a BAD-1 protein fragment” refers to a fragment or a modified version of wild type BAD-1 that retains at least 90% of the ability to interact with host antibodies of the wild type version of BAD-1. In one embodiment, one may wish to use only selected domains of the native BAD-1 protein, such as Delta C-Term which is a native BAD-1 protein of the 26199 strain lacking the C-terminus EGF-like domain (FIG. 10) and TR4 which includes four tandem repeats domain set after a six-histidine tag (FIG. 11). In another embodiment, one may wish to produce and use a BAD-1 protein or a BAD-1 protein fragment having an affinity or capture tag on the N-terminus or the C-terminus.
Although the purification of the present invention does not require an affinity tag, in one embodiment of the present invention, one may wish to purify the protein via both methods. It was reported that a BAD-1 protein having an affinity tag on the N-terminus or the C-terminus shows a similar activity to that of the native BAD-1 protein (Brandhorst, Wüthrich, et al., 2003). An affinity tag may be a six-histidine tag.
In another embodiment, a BAD-1 protein fragment refers to a modified, preferable conservative alterations, wild type BAD-1 sequence. Typically, a modified or a conservatively altered sequence will comprise 95% sequence identity of the wild type sequence or domain. Modifications may also take the form of deletions and truncations of the wild-type sequence.
As used herein, the term “activity” refers to both the chelating reactivity of the BAD-1 native protein or protein fragments with nickel or other suitable divalent cations and the ability of the BAD-1 native protein or protein fragments to interact with host antibodies. The ability to interact with host antibodies is characteristic of the primary structures of the BAD-1 native protein or protein fragments. Applicants note that the BAD-1 native protein or protein fragments may act as adhesions with related functions to suppress immune response, and the BAD-1 native protein or protein fragments may suppress T cells by ligation of CD47 in a heparin dependent fashion.
In brief, the present invention, as described below and in the figures, is directed to the isolation of BAD-1 protein. For example, highly pure native BAD-1 protein may be obtained using the methodology disclosed herein without the need for developing a recombinant protein, including, for example, a recombinant protein displaying a 6×His tag or other capture means. Consequently, highly pure native BAD-1 isolated using the disclosed method may be obtained more economically for use in diagnostic kits. Moreover, inclusion of highly pure native BAD-1 isolated by the disclosed methodology may be used to achieve greater sensitivity compared to BAD-1 isolated via other means.
In one embodiment, the present invention is the isolation of native BAD-1 protein or a BAD-1 protein fragment by nickel chelation without the addition of a histidine tag or other capture means added to the protein or protein fragment. In other embodiments, one may substitute nickel with other divalent cations such as manganese, copper, zinc, and others, which show similar affinity with the native protein or the protein fragment to nickel. We refer to these cations as “suitable divalent cations”.
To obtain a native protein, in one preferred embodiment, one would first obtain a cell culture, preferably Blastomyces dermatitidis strain 26199 (ATCC). In other embodiments, one may use other strains of Blastomyces dermatitidis suitable for the present invention. A suitable strain would be any of the various isolates that produces and secretes BAD-1. Applicants note that there are strains that do not produce BAD-1, such as African isolates. Clinical isolates may be preferred, as BAD-1 is a virulence factor, but any BAD-1 producing strain should suffice. For example, native 14081 strain and ER-3 strain are suitable for the present invention (FIG. 7 and FIG. 8).
The Examples below describe a typical culture preparation. The Examples below also describe the preparation of a typical “BAD-1 protein-containing solution.” One may obtain a BAD-1 protein-containing solution by exposing yeast to a low osmotic, divalent cation-free buffer (for example, distilled water [dH2O]). The BAD-1 containing solution is extracted in this solvent and centrifuged, followed by filter sterilization to remove yeast.
The BAD-1 containing solution is adjusted in pH and salinity, preferably by adding phosphate buffer, pH8 to a final concentration of 20 mM, and NaCl to a final concentration of 300 mM. One then exposes the BAD-1-containing solution to a nickel-chelating resin (for example, Ni-NTA). The Examples below disclose a ratio of 10 ml of Ni-NTA for every liter of original yeast volume. However, this ratio may be modified depending on desired yield of BAD-1 and the amount of starting materials. An optimal ratio of resin to BAD-1 protein or protein fragment may be determined empirically for any given BAD-1 protein or protein fragment isolate. Typically, the ratio may fall within the range between 1 ml of resin for every 10 mg BAD-1 protein or protein fragment to be isolated and 10 mls of resin for every 1 mg of BAD-1 protein or protein fragment to be isolated.
The Examples below disclose the combination of the Ni-NTA resin and the buffer extract containing BAD-1, preferably mixing with agitation for one hour at 4° C. The resin is preferably packed into a column and the columns are washed with buffer, preferably, 10 volumes of 20 mM phosphate buffer containing 300 mM NaCl at a temperature of 4° C. Without wishing to be bound by theory, it is believed that the higher pH and higher salinity of the preferred wash buffer may lead to more optimal purifications, compared to traditionally formulated PBS. For example, a PBS buffer with a pH of 8 and containing 300 mM NaCl is contemplated as an alternative buffer. The examples below disclose elution of the protein with 250 mM imidazole in PBS buffer at 4° C. As alternatives to an imidazole-containing buffer, it is contemplated that either a histidine-containing buffer or a buffer with a low pH may serve to elute BAD-1 from nickel-chelating resin.
Preferably, one would also wish to extract the mannoproteins from the solution. The Examples below describe a preferred extraction using concanavalin-agarose resin. However, any technique that might reduce and/or remove mannan is contemplated herein. For example, other techniques that may be used to purify BAD-1 from solution in order to minimize mannan and other contaminants include, for example, those that separate proteins based on anion exchange, saline gradients, size exclusion, hydrophobic interactions, and the like.
Preferably, imidazole is then removed from the collected eluate and the samples are dialyzed and concentrated. One may easily achieve protein concentrations at or above about 4 mg/ml, or about 1 mg/ml, or about 2 mg/ml, or about 3 mg/ml, or from about 1 mg/ml to about 4 mg/ml. In one embodiment, one may easily achieve protein concentrations at or above about 4 mg/ml, up to 12 mg/ml.
By “highly purified BAD-1 protein,” a preparation of BAD-1 protein that preferably comprises less than about 0.21 mg mannoprotein and at least about 1 mg BAD-1 protein per milliliter solution is meant. This concentration of mannoprotein is measured prior to extraction of the BAD-1 protein preparation with concanavalin-agarose resin. After extraction, the concentration of mannoprotein in the BAD-1 protein preparation is typically reduced to about 0.15 mg/ml. BAD-1 protein concentration in the preparation remains substantially unaffected by concanavalin-agarose resin extraction of mannoproteins. Preferably, BAD-1 protein concentration is at least about 1 mg/ml. These amounts may be achieved from a 1 liter culture of yeast.
We note that the same general methods can be used for purifying recombinant BAD-1 protein, modified BAD-1 protein or recombinant protein fragments without the addition of a histidine tag or any other capture means according to methods described above. The preparation of the present invention is especially useful for clinical diagnostic tests. The present invention is a simplified method of obtaining sufficient amounts of native BAD-1 proteins for testing.
Though both 6×His tagged and native BAD-1 proteins may be purified by nickel chelation, one advantage recognized by the present disclosure is that production of the recombinant form from yeast strains harboring the altered protein can be somewhat unstable, and these strains must be maintained in an antibiotic-containing medium (for example, chlorimuron ethyl). Even under such selection conditions, recombinant protein production levels can fall over time. This is less of a problem when working with native strains, as the present disclosure allows.
In another embodiment, the present invention is directed to a highly purified native BAD-1 protein or BAD-1 protein fragments. As disclosed in Brandhorst, Gauthier, et al., a native BAD-1 protein has three domains: 1) an N-terminus that harbors a secretion signal governing its trafficking; 2) a core domain of 25 amino acids arrayed in tandem in 30 to 40 copies, representing a so-called “tandem repeat region”; and 3) a C-terminus harboring an epidermal growth factor (EGF)-like consensus sequence.
In a preferred embodiment, the present invention relates to a highly purified native BAD-1 protein. FIGS. 6-8 depict the protein sequence of native BAD-1 proteins, obtained from ATCC wild-type strain 26199, 14081, and ER-3, respectively. Specifically, as shown in FIG. 6, the protein sequence of the 26199 native BAD-1 protein (SEQ ID NO:1) includes an N-terminal signal sequence of MPDIKSVSSILLLVSSSLVAAHPGARYPR, a 41 tandem repeat domain, and a C-terminal, EGF-like domain. As shown in FIG. 7, the protein sequence of the 14081 native BAD-1 protein (SEQ ID NO:2) includes an N-terminal signal sequence of MPDIKSVSSILLLVSSSLVAARPGARYPR, a 22 tandem repeat domain, and a C-terminal, EGF-like domain. As shown in FIG. 8, the protein sequence of the ER-3 native BAD-1 protein (SEQ ID NO:3) includes an N-terminal signal sequence of MPDIKSVSSILLLVSSSLVAAHPGGARYPR, a 42 tandem repeat domain, and a C-terminal, EGF-like domain. A comparison between these native BAD-1 protein sequences demonstrates that N-terminal and C-terminal regions are substantially conserved. Further, FIG. 9 shows that the domain of the known sequence for BAD-1 from the strain 60636 matches perfectly with that from the strain 14081.
A method to produce native BAD-1 proteins is well know in the art (e.g., Brandhorst, Wüthrich, et al., 2003; Brandhorst, Gauthier, et al., 2005), and a highly purified native BAD-1 protein may be obtained after the as-prepared native BAD-1 protein is purified using the protocol as discussed above.
In other embodiments, one may wish to obtain a highly purified native BAD-1 protein having a six-histidine affinity tag at the end of the C-terminal EGF-like domain. A highly purified native BAD-1 protein having a six-histidine tag at the end of C-terminus may be initially produced using a previous reported protocol (Brandhorst, Wüthrich, et al., 2003), and consequently following a purification process as discussed above.
In one embodiment, the present invention is directed to highly purified BAD-1 protein fragments. The highly purified BAD-1 protein fragments may include a native BAD-1 protein, e.g., those in FIGS. 6-8, lacking at least one part of each domain of the N-terminal signal sequence, the tandem repeat domain, and the C-terminal EGF-like domain. For instance, a BAD-1 protein fragment may be a native BAD-1 protein lacking a C-terminal EFG-like domain. A BAD-1 protein fragment may also be a native BAD-1 protein lacking part or complete domain of the tandem repeat domain. A BAD-1 protein fragment may also be a native BAD-1 protein lacking both part or complete domain of the tandem repeat domain and the C-terminal EGF-like domain. In other embodiments, one may wish to obtain a highly purified BAD-1 protein fragment, having a six-histidine affinity tag either at the beginning of the N-terminal signal sequence or at the end of the C-terminal EGF-like domain. BAD-1 protein fragments may be produced following methods reported in Brandhorst, Wüthrich, et al., 2003 or in Brandhorst, Gauthier, et al., 2005, and a highly purified BAD-1 protein fragment may be obtained after the as-prepared native BAD-1 protein is purified using the protocol as discussed above.
FIG. 10 shows one example of the BAD-1 protein fragment named Delta C-term. Delta C-term (SEQ ID NO:4) is a native BAD-1 protein of the 26199 strain lacking a C-terminal EGF-like domain. Essentially, Delta C-term replaces the last 95 amino acids of the native BAD-1 protein of the 26199 strain with a six-histidine tag.
FIG. 11 shows another example of the BAD-1 protein fragment denoted TR4 (SEQ ID NO:5). TR4 includes four tandem repeat set after a six-histidine tag.
In one embodiment, the present invention is directed to a diagnostic method using the highly purified BAD-1 protein or protein fragments. As the present invention provides a means to purify large amount of BAD-1 protein or protein fragments, and BAD-1 protein or protein fragments are proven to be an important antigen against the fungus of Blastomyces dermatitidis, the highly purified BAD-1 protein or protein fragments are potent biomarkers for diagnostic blastomycosis. The high purity of BAD-1 protein or protein fragments may offer unprecedented specificity and sensitivity to the diagnostic method.
As used herein, the term “patient” refers to a human or non-human mammalian patient at danger of suffering from a condition of Blastomycosis.
As used herein, the term “body fluid” refers to liquids originating from inside the bodies of living mammals. The body fluid includes fluids that are excreted or secreted from the body as well as body water that normally is not. The examples of body fluids may include urine, amniotic fluid, aqueous humour and vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, endolymph and perilymph, feces (diarrhea), female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), peritoneal fluid, pleural fluid, pus, saliva, sebum (skin oil), semen, sweat, synovial fluid, tears, vaginal secretion, vomit, urine, and others.
The present diagnostic method is generally applied to mammals. A mammal may be human or non-human mammal. For instance, the present invention is suitable for commercially important farm animals, such as cows, horses, pigs, rabbits, goats, and sheep. One may also wish to apply the present invention on companion animals, such as cats and dogs. In one embodiment, the present diagnostic method is applied to dogs.
Blastomycosis causes acute and chronic pneumonias and disseminated infection with cutaneous lesions as the major extrapulmonary manifestation. However, the vast majority of infected patients are asymptomatic or have mild respiratory symptoms that are not diagnosed as being caused by a fungal infection. Rarely, patients develop severe pulmonary infection that progresses to acute respiratory distress syndrome (ARDS), which has a high mortality rate. The sensitivity and specificity of a urinary or other body fluid antigen test to aid diagnostic Blastomycosis is dependent on the purity of the BAD-1 protein or protein fragments, as the antigen.
The current available urinary or other body fluid antigen test with BAD-1 protein or protein fragments having low purity is not specific and is positive in patients who have histoplasmosis as well as blastomycosis. Further, currently available antibody assays remain nonspecific and insensitive due to the same reason of lacking highly purified antigen. The confirmatory diagnostic test is still growth of the organism in culture, which is a time-consuming process.
Applicants envision that the highly purified BAD-1 protein or protein fragments provided in the present invention would offer diagnostic methods of high sensitivity, high specificity, and less detection time to urinary or other body fluid antigen test essays and antigen assays.
In one embodiment, the present invention is a detection method in which the protein preparations of the present invention are used in an antibody detection method. Urinary or other body fluid antigen test assays and antigen assays are well-known in the art. For instance, examples of antigen assays may include, but are not limited to, immunoassay (including enzyme immunoassay), radioimmunoassay, Spectrophotometry assay, Enzyme-linked immunosorbent assay (ELISA), Immune-chromatographic assay, or even PCR detection methods, etc.
In its simplest form, the assay is formatted with antigen as the target or bait to detect appropriately specific antibodies in sera of infected patient samples. This reaction is followed by anti-serum against the appropriate species, which is bound to a fluorescent tag or enzyme for detection. Alternatively, the protein antigen in question here is sought by using specific antiserum or monoclonal to detect its presence. The antigen-antibody complex would be detected as above with antiserum raised against the species in question. PCR based methods may be used to detect the protein in biological fluids, if aptamers specific for the protein sequence were available.
Applicants' initial results show that the diagnostic method using native BAD-1 protein as a biomarker has at least 99% sensitivity. The specificity is found to be at least 60%, but usually much higher. Further, after the identification of an EGF (epidermal growth factor) binding domain on the C-terminal end of the native BAD-1 protein, Applicants hypothesized that the EGF domain may be the cause of the sensitivity problem since a number of microbials and other cells express EGF receptor. As the EGF-deleted BAD-1 protein fragment still binds divalent cations, such as nickel, the EGF-deleted BAD-1 protein fragment may be easily purified in large quantity. Applicants' subsequent results show that a diagnostic method using the EGF-deleted BAD-1 protein fragment after removal of the C-terminal end of the native BAD-1 protein improves sensitivity to at least 90% with a slight loss in specificity.
In another embodiment, the present invention is directed to a diagnostic kit using diagnostic methods discussed above. In one embodiment, a diagnostic kit for detecting an infection of Blastomyces dermatitidis in a mammalian patient would comprise highly pure native BAD-1 protein or protein fragments, in some embodiments attached to a substrate such as alkaline phosphatase. The most preferred components of the kit reaction would include BAD-1 proteins or protein fragments as antigens purified as described above, a solid support or a substrate, patient antiserum (which would be supplied by the patient), and a means of signal generation and detection. The means of signal generation and detection may include, but not limited to, anti-human immunoglobulin linked to an enzyme or fluorescent tag, alkaline phosphatase, horse-radish peroxidase, or fluorescent signal. The means of signal generation and detection may further include, but not limited to, microscopy, protein immunostaining, Protein immunoprecipitation, Immunoelectrophoresis, Immunoblotting, BCA Protein Assay (to measure protein concentrations), Western blot, Spectrophotometry, or Enzyme assay. In such an assay, the protein could typically be linked to any solid support including a plate, bead, agarose matrix, etc.
One suitable kit may include the components of a direct fluorescent-antibody kit, such as Vet-IF (Cell Labs), IMAGEN (Celltech), Chlamydia-Direct IF (Bio Merieux), antigen detection enzyme-linked immunosorbent assay (ELISA) kits, such as Clearview (Unipath), Surecell (Kodak), Pathfinder (Kallestad), Chlamydia-EIA (Pharmacia), Chlamydiazyme (Abbott), and IDEIA (Celltech).
EXAMPLES
Materials
7H10 Medium:
Middlebrook 7H10 agar (Sigma, St. Louis, Mo.) with 10% oleic acid-albumin-dextrose-catalase complex (OADC).
HMM Medium:
F-12 nutrient mixture with I-glutamine, with phenol red, without sodium bicarbonate (Gibco BRL, Gaithersburg, Md.), supplemented with (per liter) 18.2 g of glucose, 1.0 g of glutamic acid, 84 mg of cystine, and 5.96 g of HEPES, adjusted to pH 7.5.
OADC Complex:
500 ml dH2O, 4.25 g NaCl, 25 g Bovine Serum Albumin Fraction V, 10 g D-dextrose, 20 mg catalase, and 0.25 g Oleic acid; Dissolve NaCl and BSA in dH2O; add D-dextrose, catalase, and oleic acid.
Protocol for Extraction of Native BAD1 from Blastomyces Yeast
1. Cell Culture
Blastomyces dermatitidis strain 26199 (ATCC) was inoculated from a fresh stock slant culture of 7H10 medium into 50 ml of HMM medium with penicillin/streptomycin in a 250 ml Erlenmeyer flask. This starter culture was incubated at 200 rpm at 37° C. until the culture became thick (about 2 days). The turbid culture was expanded by splitting into two 2-liter flasks, each containing 0.5 liters of HMM with penicillin/streptomycin and culturing for an additional 5 days at 37° C. 7H10 medium includes Middlebrook 7H10 agar (Sigma, St. Louis, Mo.) with 10% oleic acid-albumin-dextrose-catalase complex (OADC). OADC included 500 ml dH2O, 4.25 g NaCl, 25 g bovine serum albumin fraction V, 10 g D-dextrose, 20 mg catalase, and 0.25 g oleic acid. HMM includes F-12 nutrient mixture with L-glutamine, with phenol red, without sodium bicarbonate (Gibco BRL, Gaithersburg, Md.), supplemented with (per liter) 18.2 g of glucose, 1.0 g of glutamic acid, 84 mg of cystine, and 5.96 g of HEPES, adjusted to pH 7.5.
2. Isolation of BAD-1
Yeast were collected by centrifugation at 4000×g for 15 min. The supernatant was discarded and the pellet transferred into 50 ml falcon tubes and washed once, briefly, in PBS (50 mM Na+-phosphate buffer, pH 8, 150 mM NaCl). The yeast were spun down immediately and the yeast pellet saved. Two volumes of distilled water were added to the pellet and the pellet was resuspended. The yeast slurry was rocked at a rate to maintain an even slurry of yeast for 1 hour at 4° C. After the incubation, the yeast were pelleted at 4000×g for 5 min. The supernatant was collected and the water wash procedure was repeated twice more. All water extracts were pooled and filter sterilized to remove residual yeast. Pooled extracts often contain >75 micrograms BAD-1/ml and are only moderately contaminated by non-BAD-1 Blastomyces proteins that were removed in subsequent steps below.
To enrich for BAD-1 protein (with or without an engineered His tag), 10 ml of nickel resin (Ni-NTA) were prepared for every liter of original yeast culture volume. For every 1 liter of culture of 26199 yeast required a column containing 10 ml packed bed volume of Ni-NTA resin. The Ni-NTA resin was washed three times with PBS. Phosphate buffer, pH8 and NaCl are added to the buffered water extract containing BAD-1 to final concentrations of 20 mM and 300 mM respectively. The Ni-NTA resin and the buffered water extract containing BAD-1 were combined and agitated gently for 1 hour at 4° C. The slurry was then poured through a funnel into a column fitted with a frit, allowing the resin to pack the column evenly.
The first few milliliters of pass-though were collected and cycled back through the funnel to rinse residual resin into the column. The columns were then washed with 10 volumes of 20 mM phosphate buffer containing 300 mM NaCl. Because the BAD-1 protein being isolated lacked a 6×His tag, a lower stringency, imidazole-free PBS buffer was used to wash the loaded columns. A sample of the wash buffer was retained for analysis of BAD-1 content and the remainder discarded. The columns were eluted with 3 column volumes of 250 mM imidazole in 20 mM phosphate buffer containing 300 mM NaCl.
3. Extraction of Mannoproteins
To extract contaminating mannoproteins from BAD-1, PBS-washed concanavalin-agarose resin (Sigma, St. Louis, Mo.) and MgCl2 and CaCl2 salts to final concentrations of 1 mM each and MnCl2 to a final concentration of 0.1 mM were added to the BAD-1 eluate. One milligram of concanavalin-agaroseresin extracts approximately 12 micrograms of contaminating mannoprotein. Typically, between 20-50 mg of resin were required to clean up a BAD-1 sample derived from 1 liter of original culture volume. Optimal quantity of concanavalin-agarose resin should be determined empirically, contingent upon variations in scaling-up and application. The samples were incubated for 30 min, and the resin spun off prior to removing imidazole.
Imidazole was removed from the collected concanavalin-agarose resin eluate immediately by dialyzing against dH2O using dialysis tubing with a molecular weight cut off of 50,000 Da. The samples were dialyzed with three changes of dH2O with at least one hour between changes. The dialyzed samples were concentrated using a Centriprep® unit (Millipore Corp., Billerica, Mass.), reducing the volume to <2 ml. Concentrations of BAD-1 were measured on a spectrophotometer at A280 using an absorption coefficient of 0.15 (0.15 mg/ml BAD-1 protein has an A280 of 1.0 due to exceptionally high tryptophan content). The samples were sterilized with a syringe filter unit and preserved with azide to 0.1% for long-term storage at 4° C. or frozen for long-term storage at −20° C. At concentrations above 4 mg/ml, and in the presence of azide, sterilized BAD-1 remains quite stable for years at 4° C. For anticipated diagnostic uses of the BAD-1 protein, storage at −20° C. may be preferred. Alternatively, concanavalin extractions may be performed subsequent to imidazole removal to minimize the influence of high imidazole concentrations upon this extraction process.
4. Results and Discussion
Nickel Agarose Purification of BAD-1 and Carbohydrate Contaminant Removal
The BAD-1 adhesin of Blastomyces dermatitidis is of interest due to its indispensible role in B. dermatitidis virulence, its highly antigenic tandem-repeat sequences, and the fact that said tandem-repeats are distinctly unique to B. dermatitidis, with no reported homologues amongst the dimorphic fungi. During yeast-phase culture of B. dermatitidis, the 120 kDa BAD-1 adhesin accumulates upon the outer cell walls of yeast in a fashion that has been found to be calcium-dependent. This characteristic allows the extraction of BAD-1 into the aqueous by depletion of divalent cations. Our protocol accomplishes this by washing pelleted yeast once, briefly, in phosphate buffer prior to serial extraction into dH2O at 4° C. This material is impure, but BAD-1 is the principal protein component (FIG. 1, lane 2).
BAD-1 has a significant affinity for polysaccharide, in particular the polysaccharide chitin present in fungal cell walls. BAD-1 may also interact with mannoprotein components of the cell wall, and these components appear to co-purify with BAD-1. While the BAD-1 adhesin is unique to B. dermatitidis, it is known that some cell surface mannoproteins are conserved amongst the dimorphic fungi. These components could be responsible for observed cross-reactivity of sera from patients with blastomycosis, histoplasmosis, valley fever, etc. The challenge, therefore, is to enrich BAD-1 while minimizing the amount of cross-reactive mannoprotein present in the final product.
Under the conditions of the protocol disclosed herein, Ni-NTA resin binds BAD-1 with sufficient affinity that BAD-1 protein in not found in the unbound fraction or washes (FIG. 1, lane 3). This ability of BAD-1 to bind Ni-containing resin permits the removal of a significant amount of non-specific protein (FIG. 2, column 2). It is further contemplated that additional divalent cation-containing resins (for example, manganese, zinc, or copper) or other cation-supporting matrices may be used in the present disclosure along with or in place of Ni-resin. BAD-1 enriched by the current Ni-resin protocol, when examined by PAGE gel/Coomassie staining, shows no appreciable contaminating material (FIG. 1, lane 4), but cell wall glycoproteins rich in polymannose modifications are known to stain poorly by conventional methods. Optimally, detection of these contaminating glycoproteins is accomplished by carbohydrate specific stain, for example, ProQ Emerald staining kit, (Invitrogen) as is shown in FIG. 3 or Phenol Sulfuric Acid (PSA) assay in FIG. 4 following conventional techniques.
The Ni-resin enrichment step described removes approximately 99.8% of the carbohydrate present in the dH2O extracts (FIG. 4). Most of the material removed in this fashion is low molecular weight mannan. Elimination of contaminating mannan at this stage of the protocol is important, as mannan is a potent inhibitor of the concanavalin-agarose resin and severely interferes with the capacity of concanavalin-agarose resin to clean up the highly antigenic, high-molecular weight mannoprotein that remains as a contaminant (FIG. 3, lane 2, band migrating at  ̃220 kDa).
Incubation of the Ni-resin eluate fraction with 10 μg of concanavalin-agarose resin results in the loss of less than 10% of the A280 determined protein (FIG. 2, column 4). However, this treatment decreases total carbohydrate in the sample by about 14% (FIG. 5, column 2). Incubating the Ni-resin eluate fraction with 30 μg of concanavalin-agarose resin resulted in an 18% lower overall yield of protein by A280 (FIG. 2, column 5), but decreased total carbohydrate contaminant by 40% (FIG. 5, column 3). It is not known if contaminating carbohydrate can be completely eliminated. However, for the purposes of developing a diagnostic antigen, it is preferable to eliminate contaminating carbohydrates, preferably rendering an antigen 100% free of contaminating carbohydrates.
REFERENCES
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5. Brandhorst, et al. Calcium Binding by the Essential Virulence Factor BAD-1 of Blastomyces dermatitidis. J. Biol. Chem. 280(51), 42156-63, 2005.
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13. Brandhorst T, Wüthrich M, Finkel-Jimenez B, Klein B (2003) A C-terminal EGF-like domain governs BAD1 localization to the yeast surface and fungal adherence to phagocytes, but is dispensable in immune modulation and pathogenicity of Blastomyces dermatitidis. Mol Microbiol 48: 53-65

## Claims

1. A method for the purification of native Blastomyces Adhesion 1 (BAD-1) protein or protein fragments, comprising the steps of:
(a) obtaining a native BAD-1 protein or protein fragment-containing solution; and
(b) purifying the native BAD-1 protein or protein fragments from the solution in the absence of affinity tags, consisting essentially of the steps of:
(i) combining the native BAD-1 protein or protein fragments-containing solution with a suitable divalent cation,
(ii) washing the suitable divalent cation with an imidazole-free buffer to remove unbound matter, and
(iii) eluting the native BAD-1 protein or protein fragments with an imidazole-containing buffer from a nickel-chelating resin.

2. The method of claim 1, wherein the native BAD-1 protein or protein fragment-containing solution is obtained by the steps of:
(a1) culturing a population of microbes expressing native BAD-1 protein or protein fragment in a culture medium;
(a2) collecting the population of microbes containing the native BAD-1 protein or protein fragment from the culture medium, and
(a3) isolating the native BAD-1 protein or protein fragment from the microbes.

3. The method of claim 1, wherein the suitable divalent cation is a nickel-chelating resin.

4. The method of claim 2, wherein the microbes comprise Blastomyces dermatitidis.

5. The method of claim 4, wherein Blastomyces dermatitidis is selected from the group consisting of ATCC native strains of 26199, 14081, and ER-3.

6. The method of claim 1, wherein step (a) comprises extraction of the BAD-1 protein or protein fragments from yeast cell surfaces using a low osmotic strength and divalent cation-free buffer.

7. The method of claim 1, wherein the BAD-1 protein fragments are derived from one or more recombinant BAD-1 proteins.

8. A method for the purification of native Blastomyces Adhesion 1 (BAD-1) protein or protein fragments, comprising the steps of:
(a) obtaining a native BAD-1 protein or protein fragment-containing solution; and
(b) purifying the native BAD-1 protein or protein fragments from the solution in the absence of affinity tags, consisting essentially of the steps of:
(i) combining the native BAD-1 protein or protein fragments-containing solution with a suitable divalent cation,
(ii) washing the suitable divalent cation with an imidazole-free buffer to remove unbound matter, and
(iii) eluting the native BAD-1 protein or protein fragments with an imidazole-containing buffer from a nickel-chelating resin,
(iv) extracting contaminating mannoproteins from the eluted native BAD-1 protein or protein fragments.

9. A method for the purification of native Blastomyces Adhesion 1 (BAD-1) protein or protein fragments, comprising the steps of:
(a) obtaining a native BAD-1 protein or protein fragment-containing solution; and
(b) purifying the native BAD-1 protein or protein fragments from the solution in the absence of affinity tags, consisting essentially of the steps of:
(i) combining the native BAD-1 protein or protein fragments-containing solution with a suitable divalent cation,
(ii) washing the suitable divalent cation with an imidazole-free buffer to remove unbound matter, and
(iii) eluting the native BAD-1 protein or protein fragments with an imidazole-containing buffer from a nickel-chelating resin,
(iv) extracting contaminating mannoproteins from the eluted native BAD-1 protein or protein fragments by treating the eluted native BAD-1 protein or protein fragments with concanavalin-agarose resin.