# Patent

## Title
Thimerosal removal device

## Abstract

A device for the removal of heavy metals and heavy metal complexes, such as thimerosal, from medications is provided wherein the device comprises a solid support having at least one substantially purified metallothionein protein associated therewith.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a continuation-in-part of U.S. patent application Ser. No. 11/624,059, filed Jan. 17, 2007 which claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 60/759,671 filed Jan. 17, 2006, and is a continuation-in-part of U.S. patent application Ser. No. 11/255,427 filed Oct. 19, 2005, now U.S. Pat. No. 7,273,962, which in turn claims priority to U.S. Provisional Patent Application No. 60/620,528 filed Oct. 19, 2004 and is a continuation-in-part of U.S. patent application Ser. No. 10/797,748, filed Mar. 9, 2004, now U.S. Pat. No. 7,135,605 which is a divisional of U.S. patent application Ser. No. 09/948,495 filed Sep. 6, 2001, now U.S. Pat. No. 6,750,056, the entire contents of all of which are incorporated by reference herein in their entirety.


FIELD OF THE INVENTION
The present invention relates to compositions and methods for removing heavy metals and heavy metal complexes from biological materials. More specifically the present invention relates to removing thimerosal from bioactive materials intended to be administered to patients at the time of administration using metallothionein proteins associated with dosing devices.
BACKGROUND OF THE INVENTION
Thimerosal, which is approximately 50% mercury by weight, has been one of the most widely used preservatives in vaccines. It is metabolized or degraded to ethylmercury and thiosalicylate. Ethylmercury is an organomercurial that should be distinguished from methylmercury, a related substance that has been the focus of considerable study.
At concentrations found in vaccines, thimerosal meets the requirements for a preservative as set forth by the United States Pharmacopeia; that is, it kills the specified challenge organisms and is able to prevent the growth of the challenge fungi (U.S. Pharmacopeia 2004). Thimerosal in concentrations of 0.001% (1 part in 100,000) to 0.01% (1 part in 10,000) has been shown to be effective in clearing a broad spectrum of pathogens. A vaccine containing 0.01% thimerosal as a preservative contains 50 micrograms of thimerosal per 0.5 mL dose or approximately 25 micrograms of mercury per 0.5 mL dose.
Thimerosal is a mercury-containing organic compound (an organomercurial). Since the 1930s, it has been widely used as a preservative in a number of biological and drug products, including many vaccines, to help prevent potentially life threatening contamination with harmful microbes. Over the past several years, because of an increasing awareness of the theoretical potential for neurotoxicity of even low levels of organomercurials, concerns about the use of thimerosal in vaccines and other products have been raised. Indeed, because of these concerns, the United States Food and Drug Administration (FDA) has worked with, and continues to work with, vaccine manufacturers to reduce or eliminate thimerosal from vaccines.
Thimerosal has been removed from or reduced to trace amounts in all vaccines routinely recommended for children 6 years of age and younger, with the exception of inactivated influenza vaccine. Some vaccines such as Td (tetanus and diphtheria vaccine), which is indicated for older children (≧7 years of age) and adults, are also now available in formulations that are free of thimerosal or contain only trace amounts. Vaccines with trace amounts of thimerosal contain 1 microgram or less of mercury per dose.
The various mercury guidelines are based on epidemiological and laboratory studies of methyl mercury, whereas thimerosal is a derivative of ethyl mercury. Because they are different chemical entities—ethyl- versus methylmercury—different toxicological profiles are expected. There is, therefore, an uncertainty that arises in applying the methylmercury-based guidelines to thimerosal. Lacking definitive data on the comparative toxicities of ethyl- versus methylmercury, the FDA considered ethyl- and methyl-mercury as equivalent in its risk evaluation.
Allergic responses to thimerosal are described in the clinical literature, with these responses manifesting themselves primarily in the form of delayed-type local hypersensitivity reactions, including redness and swelling at the injection site (Cox N H, Forsyth A. Thimerosal allergy and vaccination reactions. Contact Dermatitis 18:229-233, 1988). Such reactions are usually mild and last only a few days.
In 2001, the Institute of Medicine (10M) convened a committee (the Immunization Safety Review Committee) to review selected issues related to immunization safety. One such review focused on a potential relationship between thimerosal use in vaccines and neurodevelopmental disorders (Institute of Medicine, Thimerosal-containing vaccines and neurodevelopmental disorders, Washington D.C.: National Academy Press, 2001). In its report, the 10M's Immunization Safety Review Committee concluded that the evidence was inadequate to either accept or reject a causal relationship between thimerosal exposure from childhood vaccines and the neurodevelopmental disorders of autism, attention deficit hyperactivity disorder (ADHD), and speech or language delay. Additional studies were needed to establish or reject a causal relationship. The Committee did conclude that the hypothesis that exposure to thimerosal-containing vaccines could be associated with neurodevelopmental disorders was biologically plausible.
Therefore there exists a need for methods and systems to easily remove thimerosal from injectable materials at the point of service.
Metallothioneins (MTs) are small metal binding proteins ubiquitously distributed throughout the animal kingdom. They have high metal binding affinities and are believed to be important in controlling the intracellular levels of free metal ions. The structural features of MTs include a high cysteine composition and lack of aromatic amino acids. The cysteine residues are responsible for the protein's high affinity metal binding to heavy metals including arsenic, zinc, copper, cadmium, mercury, cobalt, lead, nickel, chromium, uranium, platinum, gold, silver and their complexes. In general, MTs from divergent species have a high degree of amino acid sequence similarity. If fact, the amino acid residues responsible for metal binding are essentially invariant between species.
Accordingly, an object of the present invention is to provide methods and devices to remove thimerosal from medications and bioactive materials at the time of administration using metallothionein-based systems.
SUMMARY OF THE INVENTION
Devices and methods for removing metals, such as mercury-containing thimerosal, from medications and bioactive materials using metallothionein proteins are provided.
In one embodiment, a device is provided for the removal of thimerosal from a medication to be administered to a subject comprising a solid support having associated therewith at least one substantially purified metallothionein protein, wherein the at least one substantially purified metallothionein protein binds thimerosal from the medication resulting in a substantially thimerosal-free medication.
In another embodiment, the medication is selected from the group consisting of vaccines, immunogenic compositions, liquid pharmaceutical compositions, colloidal pharmaceutical compositions, suspension pharmaceutical compositions, aerosols and dry powders.
In another embodiment, the solid support is selected from the group consisting of filters, membranes, nanoparticles, beads, solid support particulates, and polymer coatings. In yet another embodiment, the solid support comprises a biocompatible polymer. In another embodiment, the biocompatible polymer is selected from the group consisting of fluorinated polymers, polyolefins, polystyrene, substituted polystyrenes, polysulfones, polyesters, polyacrylates, polycarbonates; vinyl polymers, copolymers of butadiene and styrene, fluorinated ethylene-propylene copolymers, ethylenechlorotrifluoroethylene copolymers, nylon and mixtures thereof.
In another embodiment at least one substantially pure metallothionein protein is associated with a plurality of beads or nanoparticles. In another embodiment, the solid support is a filter.
In another embodiment, the at least one substantially purified metallothionein (MT) protein, or a portion thereof, is from an organism selected from the group consisting of mammals, fish, mollusks, echinoderms, crustaceans, reptiles, nematodes, grains, plants, yeast, and fungi. In yet another embodiment, the crustacean is brine shrimp (Artemia). In another embodiment, the MT protein has an amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 21 and SEQ ID NO. 23.
In one embodiment, a method is provided for removing thimerosal from a medication to be administered to a subject comprising contacting a thimerosal-containing medication with at least one substantially purified metallothionein protein associated with a support; and separating the support having thimerosal bound thereto from thimerosal free medication.
In one embodiment, a system is provided for removing thimerosal from a medication to be administered to a subject comprising a device having at least one metallothionein protein associated therewith; wherein passage of a medication through the device results in binding of the thimerosal to the metallothionein protein and a substantially thimerosal-free medication
In another embodiment, a device is provided for the removal of thimerosal from a medication to be administered to a subject comprising a solid support associated with at least one substantially purified metallothionein protein, wherein the at least one substantially purified metallothionein protein binds thimerosal from the medication resulting in a substantially thimerosal-free medication.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an elution profile of exemplary metal binding proteins of the present invention illustrating co-elution of metal binding proteins with the heavy metal zinc.
FIG. 2 illustrates metallothionein (MT) protein selectively binding heavy metals in solution.
FIG. 3 illustrates MT proteins coupled to a solid support.
FIG. 4 illustrates the removal of heavy metals from water.
FIG. 5 illustrates the selectivity and affinity metallothionein proteins for binding heavy metals.
FIG. 6 depicts the sequence homology in the cysteine metal binding motifs between metallothionein proteins isolated from divergent species.
FIG. 7A-C illustrates three embodiments of a thimerosal removal device in the form of syringe dosing devices.
FIG. 8 illustrates an embodiment of a thimerosal removal device of the present invention in the form of an inhalation device.
FIG. 9 illustrates an embodiment of a thimerosal removal device of the present invention in the form of an air gun injection device.
FIG. 10 depicts the formation of metallothionein/thimerosal complexes.
FIG. 11 depicts the elution profile of metallothionein and thimerosal from a chromatography column.
FIG. 12 depicts the elution profile of a mixture of metallothionein and thimerosal from a chromatography column.
FIG. 13 depicts the elution profile of bovine serum albumin and thimerosal from a chromatography column.
FIG. 14 depicts chemical groups derivatized on a solid support for immobilization of metallothionein.
FIG. 15 depicts conversion of surface primary amines to iodoacetate functional groups a solid support for immobilization of metallothionein.
FIG. 16 depicts the covalent cross-linking of metallothionein to a solid support with surface iodoacetate functional groups.
FIG. 17 depicts a metal binding molecule comprising multiple copies of metallothionein linked through a nonapeptide.
FIG. 18 illustrates an embodiment of a thimerosal delivery device for removing thimerosal from bulk materials.



DETAILED DESCRIPTION OF THE INVENTION
Systems, devices and methods for removing metals, such as mercury-containing thimerosal, from medications using metallothionein proteins are provided. In one embodiment, the thimerosal is removed prior to the dosing procedure from a bulk biological material.
The dosing devices, systems and methods disclosed herein remove substantially all of the thimerosal from the medication. In one embodiment, greater than about 90% of the thimerosal is removed. In another embodiment, greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the thimerosal is removed. In another embodiment, the present methods produce medications having less than about 1 μg of thimerosal per dose of medication. In another embodiment, each dose of medication contains less than about 0.7 μg. In another embodiment, each dose of medication contains less than about 0.5 μg of thimerosal. In yet another embodiment, each dose of medication contains less than about 0.1 μg of thimerosal.
As used herein, the term “medication” refers to any pharmaceutical preparation to be administered to a subject and which contains a heavy metal or heavy metal-containing compound including, but not limited to, thimerosal. The term medication includes, but is not limited to, injectable medications such as, but not limited to, vaccines, immunogenic compositions, gene therapy agents and any type of injectable agent, liquid pharmaceutical compositions, colloidal pharmaceutical compositions, suspension pharmaceutical compositions, aerosols and dry powders. For the purposes of this disclosure, medication and bioactive material are used interchangeably.
Metal binding proteins such as metallothioneins (MTs) that have been isolated from various species such as humans, mice, bacteria species, crabs, fish, yeast and chickens, are known to have very similar structural characteristics such as similar size (about 6.0-6.8 kDa), high amino acid sequence conservation, and a high percentage of cysteine residues in the proteins' total amino acid compositions (FIG. 6). It is the cysteine composition of these MTs that accounts for the protein's binding affinity for heavy metals including, but not limited to, arsenic, zinc, copper, cadmium, mercury, cobalt, lead, nickel, chromium, uranium, platinum, silver and gold. Unless otherwise stated, the term protein refers to proteins, polypeptides and peptides and includes metal binding domains. The MT proteins also bind heavy metal complexes in which the heavy metals are associated with a protein or other molecule.
For example, the MT proteins, and devices comprising them, disclosed herein are useful in connection with the treatment of any material having a concentration of at least one metal, such as a heavy metal or a heavy metal complexes, for example thimerosal. In particular, the MT proteins and devices are useful for removing heavy metal containing complexes, such as the mercury-containing thimerosal, from injectable materials. Furthermore, the MT proteins specifically bind certain heavy metals and do not bind other metals such as biologically required metals such as, but not limited to, calcium and magnesium (FIG. 2 and FIG. 4).
As used herein, medication is defined as any material suitable for administration to a mammal through any route. Non-limiting examples of medications include vaccines, plasma-derived products such as immune globulin and anti-toxins or anti-venoms and drugs including chemicals and biologicals including, but not limited to, proteins, peptides, hormones, polysaccharides, etc. Medications can also refer to immunogenic compositions, liquid pharmaceutical compositions, colloidal pharmaceutical compositions, suspension pharmaceutical compositions, aerosols and dry powders. Medication and bioactive material may be used interchangeably and are considered equivalent terms for the purposes of this disclosure. Routes of administration addressed by the devices and methods of the present invention include, but are not limited to, intravenous, subcutaneous, intradermal, and intramuscular injection; intravenous infusion; oral; inhalation; intraocular and other routes of administration known by medical professionals.
In general, a substrate from which one or more heavy metals or heavy metal complexes such as, but not limited to, thimerosal, are to be removed is contacted with an MT protein bound to a solid support, where the MT protein has an affinity for the heavy metal. The solid support forms a matrix to which the MT protein is bound (FIG. 3). In one embodiment, the MT protein is bound irreversibly to the solid support. In another embodiment, the MT protein is bound reversibly to the solid support.
The solid support can be in the form of a membrane, beads or solid support particulates, nanoparticles, a coating on a medical device or a dosing device, or any other form commonly used in biological or biochemical separations. If a membrane is used as the solid support, the MT-solid support composition can be incorporated into a contacting device comprising a housing, e.g., cartridge, containing the MT protein, by causing a solution containing a heavy metal to flow through an inlet port into the cartridge and thus come in contact with the MT protein before traveling out through an outlet port. In one embodiment, the inlet port and the outlet port can be the same port. Various housings may be used instead of a cartridge such as, but not limited to, a cassette, syringe, unit, canister, column or filter holder.
In one illustrative embodiment, the solid support is in the form of a membrane. Preferably, the membrane is a biocompatible polymer, and more preferably is a member selected from the group consisting of fluorinated polymers, polyolefins, polystyrene, substituted polystyrenes, polysulfones, polyesters, polyacrylates, polycarbonates; vinyl polymers, copolymers of butadiene and styrene, fluorinated ethylene-propylene copolymers, ethylenechlorotrifluoroethylene copolymers, nylon and mixtures thereof. In one embodiment, the membrane configuration is a pleated membrane, although other membrane configurations, such as flat sheet, mesh, pleated sheets, stacked disk, stacked sheets, and hollow fibers may be used as well as other configurations known to persons of ordinary skill in the art. A detailed discussion of solid supports, methods of binding MT proteins to solid supports and their use are disclosed in co-pending U.S. Pat. No. 7,273,962, which is incorporated by reference for all it contains regarding MT proteins and solid supports.
In one embodiment of the present invention, a dosing device has MT associated therewith to remove thimerosal from a thimerosal-containing solution passing through the dosing device. One exemplary dosing device depicted in FIG. 7 is a syringe. The MT can be associated with the dosing device in several ways. FIG. 7 depicts a syringe 100 generally having a plunger 102, a barrel 104 and a luer tip 106 for attachment to a needle 108. Needle 108 typically has a complimentary luer hub 124 for attaching to luer tip 106. Syringes can be manufactured to a variety of specifications and can have more or less components than depicted in FIG. 7. Optionally a gasket 120 is present to provide a seal between plunger 102 and barrel 104. In FIG. 7A, MT is bound to a solid support in the form of a bead 122, and a plurality of beads 122 having at least one MT protein bound thereto are disposed within barrel 104 of syringe 100 and/or luer hub 124 of needle 108 prior to a thimerosal-containing solution entering syringe 100. Injectable materials drawn into syringe 100 contact MT-coated beads 122 and thimerosal in the injectable material becomes bound to the MT protein. The beads can be of any size or shape compatible with their use. Beads useful within the luer hub 124 of needle 108 may be of a different size than those used within a reservoir or syringe barrel 104. Furthermore, beads within the barrel 104 of syringe 100 or luer hub 124 of needle 108 are retained within the barrel 104 or luer hub 124 after passage of the injectable material and do not pass into the subject or patient. The beads are retained by means including, but not limited to, presence of a membrane or mesh in barrel 104 or luer hub 124 with pore sizes smaller than the beads and/or the size of the beads exceeding the size of any exit ports of the barrel 104 or luer hub 124 such that the beads do not leave the barrel 104 or luer hub 124 with the injection material.
Beads suitable for coating with MT proteins include, but are not limited to, biocompatible polymers such as fluorinated polymers, polyolefins, polystyrene, substituted polystyrenes, polysulfones, polyesters, polyacrylates, polycarbonates; vinyl polymers, copolymers of butadiene and styrene, fluorinated ethylene-propylene copolymers, ethylenechlorotrifluoroethylene copolymers, nylon and mixtures thereof.
In yet another embodiment of a dosing device, MT is coated on a solid support is in the form of a filter. Filters with molecular weight cut offs sufficiently large to allow the injectable material to pass through are suitable for use in the present invention. FIG. 7B depicts a syringe 100 having disposed between luer tip 106 of syringe 100 and luer hub 124 of needle 108 a filter housing 130. At least one substantially pure MT protein is covalently bound to the biocompatible filter 136 contained within filter housing 130. An exemplary filter housing comprises a biocompatible plastic with an inlet port 132 and an outlet port 134 and a filter 136 disposed within the filter housing 130 having MT proteins bound thereto. The inlet port 134 and outlet port 136 have luer lock configurations to allow filter housing 130 to attach to syringe 100 and to needle 108. Preferable the filter housing 130 and filter 136 are stable to sterilization.
In still another embodiment, the solid support is in the form of a coating on the interior of the dosing device. In FIG. 7C, additional embodiments of a syringe dosing device are depicted. In non-limiting example, the interior of barrel 104 of syringe 100 is coated with a polymeric coating 140 to which at least one MT protein is bound. In another embodiment, the interior portion of luer hub 124 of needle 108 is coated with a polymeric coating 140 to which at least one MT protein is bound or incorporated therein. In yet another embodiment, the surface of rubber gasket 120 is coated with a polymeric coating 142 to which at least one MT protein is bound. In another embodiment, the interior of luer hub 124 of needle 108 is coated with a polymeric coating 144 to which at least one MT protein is bound. Injecteable materials containing thimerosal pass through barrel 104 and luer tip 106 of syringe 100 and luer hub 124 of needle 108 and thimerosal in the injectable material binds to the MT protein such that a substantial amount of the thimerosal is removed from the injectable material prior to entering the patient.
FIG. 8 graphically depicts an inhalation dosing device 800 comprises a body 802 in which is contained a medication to be administered to a patient by inhalation and an outlet 804. Suitable inhalation devices can deliver medications to the pulmonary system by inhaling through the nose or the mouth. The medication can be administered as an aerosol, a dry powder, or any form suitable for administration to the pulmonary system of a patient. Typically, the medication from the body 802 is propelled through a passageway 806 to an outlet 804 where it enters the nose or mouth of the patient. In one embodiment of the present invention presented in FIG. 8A, the interior of the passageway 806 and/or outlet 804 is coated with a polymer coating 808 to which at least one MT protein is bound. Inhalable medications containing thimerosal pass through passageway 806 and outlet 804 and thimerosal in the inhalable medication binds to the MT protein on polymer coating 808 such that a substantial amount of the thimerosal is removed from the inhalable medication prior to entering the patient. In another embodiment presented in FIG. 8B, a filter 810 having at least one MT protein bound thereto is disposed within passageway 806 such that inhalable medications containing thimerosal pass though filter 810 and thimerosal is removed from the inhalable medications prior to entering the patient. Inhalation dosing devices are well known in the art and exemplary devices are disclosed in U.S. Pat. Nos. 7,047,967, 7,143,764, 7,077,130, 7,032,594, 7,007,689, 6,955,169, 6,745,761, 6,557,550, 6,345,617, 6,131,566, 5,860,416, 5,571,246, 5,263,475, and 4,083,368, all of which are incorporated by reference for all they disclose regarding inhalation dosing devices.
FIG. 9 depicts another exemplary dosing device, a needleless injection device. An exemplary needleless injection device 900 comprises a reservoir chamber 902 connected through a dispensing passageway 904 to an orifice 906 through which a medication is injected into a patient. Typically, the medication contents of chamber 902 are propelled through dispensing passageway 904 and orifice 906 with sufficient force such that the medication penetrates the skin of the patient. In one embodiment of the present invention depicted in FIG. 9A, the interior of the dispensing passageway 904 and/or outlet 906 are coated with a polymer coating 908 to which at least one MT protein is bound. Injectable medications containing thimerosal pass through dispensing passageway 904 and outlet 906 and thimerosal in the injectable medication binds to the MT protein on polymer coating 908 such that a substantial amount of the thimerosal present in the injectable medication is removed from the injectable medication prior to entering the patient. In another embodiment depicted in FIG. 9B, reservoir 902 contains beads 922 to which at least one MT protein is bound. Injectable medications containing thimerosal contact beads 922 in reservoir 902 such that a substantial amount of the thimerosal present in the injectable medication is bound to beads 922 prior to the injectable medication leaving the reservoir 902 and entering the patient. In another embodiment presented in FIG. 9C, a filter 910 having at least one MT protein bound thereto is disposed within dispensing passageway 904 such that injectable materials containing thimerosal pass though filter 910 and thimerosal is removed from the injectable medications prior to entering the patient. Needleless injection devices are well known in the art and exemplary devices are disclosed in U.S. Pat. Nos. 7,156,822, 7,150,409, 7,056,300, 7,029,457, 6,939,323, 6,471,669, 5,993,412, 5,520,639, 4,342,310, and 3,948,266, all of which are incorporated by reference for all they disclose regarding needleless injection devices.
In another embodiment, the medication or bioactive material is mixed with a plurality of beads or nanoparticles having at least one MT protein bound thereto and the heavy metal-MT-bead complex is removed from the medication. Exemplary, non-limiting means for removal of heavy metal-bound beads from the mediation includes, but is not limited to, filters, membranes, meshes, affinity columns, and other means known to persons of ordinary skill in the art.
In yet another embodiment, a filter unit, as depicted in FIG. 7B, is provided to remove thimerosal from a thimerosal-containing liquid. The filter housing 130 contains at least one MT protein bound to a solid support 136 enclosed with a housing having an inlet port 132 and an outlet port 134. The filter unit can be attached to commercially available syringe or other device to remove thimerosal from a thimerosal-containing liquid. The thimerosal is retained within the filter unit and a substantially thimerosal-free medication results from passage of the medication through the filter unit.
Polymeric coatings suitable for coating the interior of a dosing device should not be able to dissociate from the surface of the dosing device, be capable of irreversibly binding metallothionein and be able to be sterilized.
The MT proteins are associated with the support, such as a polymer membrane or bead, by covalent bonding of the MT protein to the support or incorporation of the MT proteins into the polymer matrix.
Many derivatized solid supports designed specifically for protein binding are commercially available and are well known to the skilled practitioner. Certain of these materials have surface aldehyde groups for linking proteins by way of a primary amine. In other cases, the support has been derivatized with either a primary amine or a carboxyl group (FIG. 14). For example, and not intended as a limitation, metallothionein can be i) linked directly to the material or ii) an appropriate linker can be used to orientate the MT away from the surface of the membrane to remove any potential protein/membrane steric interactions that would block the active site and prevent a ligand from binding to the MT. In the case of MT, the use of a linker may result in an increased efficiency of thimerosal binding.
The C-terminal amino acid of MT is a histidine. Since this residue is i) not part of the structural domains responsible for the metal binding activity and ii) is located away from the surface of the protein, it presents itself as an excellent site for coupling MT to a solid support. In one embodiment of the present invention, MT is coupled to a solid support by first reacting the support with N-succinimidyl iodoacetate (Formula 1).




The N-hydroxysuccinamide (NHS) portion of the molecule reacts with the primary amines on the surface of the material to convert the surface functional groups to iodoacetate (FIG. 15). At a pH between 5 and 6, the iodoacetate functional groups react with the MT histidine, covalently linking the protein to the membrane (FIG. 16).
Additionally, increasing the number of MT molecules on the membrane can increase the total thimerosal binding capacity of the membrane. Since the membrane has a defined surface area with a finite number of functional groups (iodoacetate) available for linking protein to the membrane, another method will be required to increase the number of MT molecules on the membrane. This can be accomplished by cloning multiple copies of the Artemia MT gene within a standard cloning vector to form a polymeric MT gene sequence. In one embodiment of the present invention, the final expressed MT “protein” would be composed of multiple copies of MT separated by a linker such as, but not limited to, a nonapeptide linker (SSG4SDI, SEQ ID NO. 24) (FIG. 17).
The linker sequence is designed to i) allow the MT sequences to fold into their native conformation and retain their thimerosal binding activity and ii) impart a degree of rigidity that prevents the individual MT protein domains from aggregating. Using the exemplary MT sequence depicted in FIG. 16, the thimerosal binding capacity would increase four-fold.
The polymeric MT gene sequence would be constructed using overlapping PCR primers standard recombinant DNA technology. The construct can then be PCR amplified with primers containing unique restriction sites i) not present in the construct and ii) compatible with the multiple cloning site of a suitable expression vector. The resulting PCR product is then cloned into the expression vector such as, but not limited to, the pET expression vector. The recombinant plasmid is then used to transform suitable expression cells such as, but not limited to, BL2(D3) cells. If the expression vector contains an inducible promoter, protein expression is induced by the addition of a molecule such as, but not limited to, IPTG. The resultant protein is then purified as described below for monomeric MT.
The expressed MT proteins are purified using standard techniques. Techniques for purification of cloned proteins are well known in the art and need not be detailed further here. One particularly suitable method of purification is affinity chromatography employing an immobilized antibody to a metal binding protein. Other protein purification methods include chromatography on ion-exchange resins, gel electrophoresis, isoelectric focusing, and gel filtration, among others. Alternatively, the MT proteins of the present invention can be purified following their expression from modified organisms by methods such as precipitation with reagents (e.g. ammonium sulfate, acetone or protamine sulfate as well as other methods known in the art).
The MT proteins of the present invention can be isolated easily and efficiently from natural sources or synthetically produced. In one embodiment of the present invention, the MT proteins are isolated from brine shrimp (Artemia). Artemia MT comprise a family of metal binding proteins that are referred to as “isomers”. Analysis of these proteins' unique amino acid compositions showed each isoform to be essentially equivalent. At least five individual Artemia MT isoforms have been identified. Unlike MTs from other organisms which share a high degree of sequence homology or similarity, the Artemia metal binding proteins have unexpectedly different structural characteristics but possess a high degree of sequence homology to one another.
Amino acid sequence analysis indicated that the metal binding motif of the first six cysteine residues of the Artemia metal binding protein was conserved when compared to rabbit and human MTs, indicating the importance of these amino acid residues in the protein's metal binding function (Hamer D H, Metallothionein. Ann. Rev. Biochem. 55:813-51, 1986). This conservation of the cysteine-rich metal binding motif is seen across a wide variety of divergent species (FIG. 6).
FIG. 1 details an exemplary elution profile utilizing an exemplary MT protein of the present invention. This profile was obtained utilizing the following exemplary protocol. E. coli (Strain ER 2566) were transformed with a plasmid expression vector containing the MT gene sequence of SEQ ID NO. 1 in pTMZ. Bacteria were grown in LB broth containing 1% glucose at 37° C. to an A600 of 0.60. The bacterial cells were collected and resuspended in LB broth containing 0.1% glucose and incubated for 45 minutes at the same temperature. Isopropyl β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM. The bacterial cells were incubated for about 16 hours. Non-transformed bacteria were used as controls. The cells were collected by centrifugation and sonicated in 10 mM Tris, pH 8.0, 5 mM dithiothreitol (DTT) and 0.5 mM phenylmethylsulfonylfluoride (PMSF). The homogenate was centrifuged at 150,000×g for 1 hour at 4° C. The supernatant was collected and incubated with 2 μCi of 109Cd at room temperature. The radiolabeled supernatant was then applied to a G-50 molecular exclusion column and eluted with 50 mM Tris, pH 8.0. Five milliliter fractions were collected and assayed for radioactivity (CPM) and zinc (Zn), the zinc being an endogenous metal that associates with the exogenous metal binding protein expressed by the transformed bacteria. Each fraction eluting from the column was assayed for Zn by ICPMS (Inductively Coupled Plasma Mass Spectroscopy). Other nucleotide sequence that encode a functional metal binding protein, including, but not limited to SEQ ID NO. 3, may also be utilized, as provided and disclosed by the teachings of the present invention.
A substantially purified MT protein in accordance with the teachings of the present invention has an amino acid sequence analogous to:








SEQ ID NO. 2










MET ASP CYS CYS LYS ASN GLY CYS THR CYS ALA PRO ASN CYS LYS
15



 


CYS ALA LYS ASP CYS LYS CYS CYS LYS GLY CYS GLU CYS LYS SER
30


 


ASN PRO GLU CYS LYS CYS GLU LYS ASN CYS SER CYS ASN SER CYS
45


 


GLY CYS HIS
48






Also within the scope of the present invention are substantially purified MT proteins that are variants of the sequence of the above SEQ ID NO. 2 that preserve the protein's metal binding affinity. In particular, conservative amino acid substitutions within the scope of the present can include any of the following: (1) any substitution of isoleucine for leucine or valine, leucine for isoleucine, and valine for leucine or isoleucine; (2) any substitution of aspartic acid for glutamic acid and of glutamic acid for aspartic acid; (3) any substitution of glutamine for asparagine and of asparagine for glutamine; and (4) any substitution of serine for threonine and of threonine for serine.
A “conservative amino acid substitution” as used herein, refers to alteration of an amino acid sequence by substituting an amino acid having similar structural or chemical properties. Those skilled in the art can determine which amino acid residues may be substituted, inserted or altered without the metal binding properties of the proteins of the present invention.
Other substitutions can also be considered conservative, depending upon the environment of the particular amino acid. For example, glycine and alanine can be interchangeable, as can be alanine and valine. Methionine, which is relatively hydrophobic, can be interchanged frequently with leucine and isoleucine, and sometimes with valine. Lysine and arginine are interchangeable in locations in which the significant feature of the amino acid residue is its charge and the different pKs of these two amino acid residues and where their different sizes are not significant. Still other changes can be considered “conservative” in particular environments, as known in the art.
For example, if an amino acid on the surface of a protein is not involved in a hydrogen bond or salt bridge interaction with another molecule, such as another protein subunit or a ligand bound by the protein, negatively charged amino acids such as glutamic acid and aspartic acid can be substituted with positively charged amino acids such as lysine or arginine and vice versa. Histidine, which is more weakly basic than arginine or lysine, and is partially charged at neutral pH, can sometimes be substituted for these more basic amino acids as well. Additionally, the amides glutamine and asparagine can sometimes be substituted for their carboxylic acid homologues, glutamic acid and aspartic acid.
For example, these MT proteins are capable of heavy metal binding under a range of temperature conditions such as, for example, a temperature range of about 4° C. to about 100° C. and therefore particularly ideal for many applications. Those skilled in the art will appreciate that depending on a particular application or operation in which the MT proteins are to be utilized, a particular temperature range may be preferred for practical or economic reasons.
Turning now to an exemplary discussion of the genetic engineering of the novel metal binding proteins disclosed herein, a nucleotide sequence for one of the isoforms of an MT protein from a brine shrimp (Artemia) was identified, as discussed above. Generally, the isolation process comprises: (1) preparation of one or more sample(s) containing nucleic acids from brine shrimp (Artemia); (2) isolation of total RNA from Artemia; (3) preparation of cDNA from the total RNA; (4) amplification of metal binding protein gene sequences; and (5) cloning, sequencing and verification of an isolated nucleic acid sequence as an MT protein gene from Artemia. 
The above procedure yielded the entire coding sequence for Artemia MT. This sequence is:








SEQ ID NO. 1










5′-ATG GAC TGC TGC AAG AAC GGT TGC ACC TGT GCC CCA AAT TGC AAA
 45



 


TGT GCC AAA GAC TGC AAA TGC TGC AAA GGT TGT GAG TGC AAA AGC
 90


 


AAC CCA GAA TGC AAA TGT GAG AAG AAC TGT TCA TGC AAC TCA TGT
135


 


GGT TGT CAC TGA-3′
147






Species as divergent as humans and wheat express metallothionein proteins with similar binding affinities for heavy metals. These MT proteins contain from 12 to 22 cysteine residues, which are conserved across divergent species. These cysteine residues form metal binding motifs responsible for the metal binding function of the proteins (Hamer D H, Metallothionein. Ann. Rev. Biochem. 55:813-51, 1986). Therefore, one embodiment of the present invention provides MT proteins immobilized on solid supports, such as membranes, wherein the MT are isolated from organisms including, but not limited to, mammals, plants, fish, mollusks, echinoderms, crustaceans, reptiles, nematodes, grains, bacteria, algae, yeast and fungi. Non-limiting examples of these organisms include, but are not limited to, brine shrimp (Artemia), rabbit (Oryctolagus cuniculus), green monkey (Cercopithecus aethiops), human (Homo sapiens), channel catfish (Ictalurus punctatus), African clawed frog (Xenopus laevis), blue mussel (Mytilus edulis), painted sea urchin (Lytechinus pictus), fruit fly (Drosophila melanogaster), roundworm (Caenorhabditis elegans), rice (Oryza sativa), wheat (Triticum aestivum) and yeast (Candida glabrata).
One embodiment provides one or more nucleic acid sequences encoding a substantially purified MT protein having amino acid sequence analogous to at least one metallothionein protein from an organism including, but not limited to, Artemia, mammals and marine species, or other species having a metallothionein protein with conserved amino acid sequence homology in the cysteine residues, e.g. the metal binding motifs, as compared to Artemia MT (FIG. 6).
Another embodiment provides one or more amino acid sequences encoding a substantially purified MT protein analogous to at least one metallothionein protein from an organism including, but not limited to, Artemia, mammals and marine species, or other species having a metallothionein protein with conserved amino acid sequence homology in the cysteine residues, e.g. the metal binding motifs, as compared to Artemia MT (FIG. 6). Exemplary amino acid sequences include the sequences of SEQ ID NO. 2 and SEQ ID NOs. 11-23 (FIG. 6).
Alternatively, an isolated nucleic acid can comprise the minimal DNA sequences sufficient to allow translation of a functional MT protein. A functional MT protein need not be the entire native MT protein but can be just those portions or regions that encode a protein, or a synthetic chemical complex, capable of binding to heavy metals, in a non-limiting example the sequence of SEQ. ID. NO. 3. Therefore, the invention also includes isolated nucleic acids including DNA having at least 80% sequence identity to a DNA molecule having the sequence of nucleotide residues 1 to 66 of SEQ ID NO. 1.
Also within the scope of the present disclosre is a nucleic acid sequence encoding any one of the MT proteins. Such MT proteins can have molecular weight of about 5,800 daltons and are able to bind with high affinity to heavy metal ions such as arsenic, zinc, copper, cadmium, mercury, cobalt, lead, nickel, platinum, gold, silver and complexes thereof. The MT proteins include therein an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ. ID. NOs. 11-23 and sequences incorporating one or more conservative amino acid substitutions thereof wherein the conservative amino acid substitutions are any of the following: (1) any of isoleucine, leucine and valine for any other of these amino acids; (2) aspartic acid for glutamic acid and vice versa; (3) glutamine for asparagine and vice versa; and (4) serine for threonine and vice versa. Alternative nucleic acid sequences can be determined using the standard genetic code; the alternative codons are readily determinable for each amino acid in this sequence. Additionally, it is within the scope of the present disclosure to make additional mutations, deletions or additions to the amino acid or nucleic acid sequences of the MT proteins disclosed herein which do not effect the metal binding capabilities of the resultant MT protein.
The MT proteins used in these metal binding processes can be provided as a product purified from its natural source or can be produced by bioengineering techniques. For example, the MT proteins can be produced by transgenic or modified organisms. Modified organisms include, but are not limited to, transgenic animals, bacteria, plants, yeast, mammalian cells, insect cells and algae.
Methods for reducing the concentration of heavy metals in a substrate include contacting an MT protein with a substrate having heavy metals. In a non-limiting example, an MT protein having an amino acid sequence analogous to at least one metal binding protein sequence from brine shrimp (Artemia) can be contacted with a substance having a concentration of at least one heavy metal to bind the heavy metal to the MT protein. Subsequently, the bound heavy metal can be separated from the substrate, reducing the concentration of heavy metals in the original substrate.
Methods for reducing the concentration of heavy metals in a substance include producing the metal binding proteins in a modified organism. Modified organisms include, for example, transgenic organisms or transgenic hosts. For example, hosts or organisms such as shrimp, plants, bacteria, yeast or algae can be modified using molecular and genetic engineering techniques well known in the art. Using these techniques, which are described for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Press, 2001); Ausubel et al. Current Protocols in Molecular Biology (Wiley Interscience Publishers, 1995); US Dept Commerce/NOAA/NMFS/NWFSC Molecular Biology Protocols (URL:http://research.nwfsc.noaa.gov/protocols.html); or Protocols Online (URL:www.protocol-online.net/molbio/index.htm), organisms whose genomes are modified so as to result in expression of an MT protein are provided.
A modified organism producing an MT protein includes a modified organism producing at least one MT protein having an amino acid sequence substantially similar to a metal binding protein from a brine shrimp (Artemia). A modified organism also includes an organism producing an MT protein having an amino acid sequence substantially similar to SEQ ID NO. 2 or conservative amino acid substitutions thereof.
Alternatively, production or expression of the MT proteins from modified organisms is not limited to genomic expression of the metal binding proteins, but also includes epigenetic expression from the modified organisms. Methods and techniques for obtaining epigenetic expression from a modified organism include, for example, adenoviral, adeno-associated viral, plasmid and transient expression techniques which are known in the art.
Methods for producing the MT proteins are also disclosed herein. For example, a method for producing an MT protein having an amino acid sequence analogous to at least one MT protein from a brine shrimp (Artemia) includes providing an expression system, producing an MT protein using the expression system and purifying or isolating the MT proteins.
Expression systems can be systems such as traditional manufacturing plants. For example, organisms such as brine shrimp can be grown and the MT proteins purified or extracted from the tissues of the brine shrimp. Alternatively, biomanufacturing systems using genetically engineered organisms (produced as described herein) capable of producing the MT proteins can be used. For example, bacteria containing an MT protein expression vector can be cultured on large or small scale (depending on the particular need). The MT proteins can then be purified from the bacterial broth and used to remove heavy metals from a variety of substrates.
Therefore, an MT protein can be produced by expression of a nucleic acid sequence encoding an MT protein in a modified organism or host cell. Such a nucleic acid sequence includes, for example, a MT gene such as SEQ ID NO. 1 or a sequence encoding a fragment or functional metal binding domain of a MT gene.
A further understanding of the present invention will be accorded to those skilled in the art from a consideration of the following non-limiting Examples. It is emphasized that these Examples are illustrative of the principles and teachings of the present invention and are not intended to limit the scope of the invention.
The metal binding proteins disclosed herein can also be utilized as a naked composition or in association with a support or dispersal means to aid in either the dispersal, handling, packaging or function of the metal binding protein in metal recovery, metal remediation or metal binding processes. Such metal binding proteins are particularly useful in metal recovery, metal remediation and metal binding processes because they can be more easily and safely used as compared to other methodologies, such as chemical extraction, which exposes the user to toxic or other potentially dangerous types of chemicals.
A variety of solid supports to aid in the handling or dispersal of the novel metal binding proteins can be used and include a hydrophilic membrane, partially hydrophilic membrane, composite membrane, porous organic solid support, nonporous organic solid support, porous inorganic solid support, nonporous inorganic solid supports and combinations thereof. If the solid support is a membrane, exemplary membranes are described in U.S. Pat. Nos. 5,618,433 and 5,547,760, both of which are herein incorporated by reference in their entirety. If the solid support is an inorganic or organic particulate solid support, preferred solid supports include sand, silicas, silicates, silica gel, glass, glass beads, glass fibers, alumina, zirconia, titania, nickel oxide polyacrylate, polystyrene, polyphenol and others as described in U.S. Pat. Nos. 4,943,375, 4,952,321, 4,959,153, 4,960,882, 5,039,419, 5,071,819, 5,078,978, 5,084,430, 5,173,470, 5,179,213, 5,182,251, 5,190,661, 5,244,856, 5,273,660 and 5,393,892 which are herein incorporated by reference. Specific examples include flexible membranes, beads or particulates, filters, or any other solid supports known in the art that are useful for separations.
In one illustrative embodiment, the solid support is in the form of a membrane. Preferably, the membrane is a polymer, and more preferably is a member selected from the group consisting of fluorinated polymers, polyolefins, polystyrene, substituted polystyrenes, polysulfones, polyesters, polyacrylates, polycarbonates; vinyl polymers, copolymers of butadiene and styrene, fluorinated ethylene-propylene copolymers, ethylenechlorotrifluoroethylene copolymers, nylon and mixtures thereof.
The metal binding proteins are associated with a support, such as a polymer membrane, by covalent bonding of the metal binding protein to the polymer to by non-covalent binding such as, but not limited to, electrostatic attractions, dispersion forces and solvent-mediated forces.
In one embodiment, at least one metal binding protein is associated with a solid support such that a regenerative metal binding support is provided, wherein the regenerative metal binding support can bind heavy metals from a substrate, the heavy metals can be released from the regenerative metal binding support and the regenerative metal binding support can be reused to bind heavy metals.
In general, a substrate from which one or more heavy metal species are to be removed is contacted with a metal binding protein bound to a solid support, where the metal binding protein has an affinity for the heavy metal. The solid support forms a support for the metal binding protein and can be in the form of a membrane, beads or solid support particulates, or any other form commonly used in biochemical or chemical separations. If a membrane is used as the solid support, the metal binding protein-solid support composition can be incorporated into a contacting device comprising a housing, e.g., a cartridge, containing the metal binding proteins. A bulk material containing undesirable heavy metals (such as bulk vaccine product containing thimerosal) can be passed through the membrane whereby the heavy metals are retained on the membrane and the cleaned bulk material passes through the membrane. In one embodiment, the membrane configuration is a pleated membrane, although other membrane configurations, such as flat sheet, stacked disk or hollow fibers may be used. However, various contact apparatus may be used instead of a cartridge such as but not limited to a cassette, syringe, unit, canister, multi-well plate or filter holder. If a solid support is used, separation columns can be used as are known in the art.
FIG. 18 illustrates an exemplary embodiment of a bulk material thimerosal removal system. For example, a volume of a thimerosal-containing material 181, contained in container 180, passes through conduit 183 to spigot 185 into aseptic dosing vials 186. Disposed within conduit 183 or spigot 185 is as aseptic filter. A metallothionein-containing support can be disposed within the system at the junction of the container and the conduit 181, at the junction of the conduit and the spigot 184, or at any location within conduit 183 or spigot 185.
It should be noted, that an additional characteristic feature of the metal binding proteins are that they are also capable of reversible heavy metal binding. For example, bound metals can be eluted off or away from the metal binding proteins using acidic conditions or by instantaneous exchange reactions or inorganic chelators. For example, during incubation of a metal binding protein with radioactive Cd, the 109Cd metal exchanges for endogenous metal bound to the metal binding protein. At about pH 1.0, the metal is released from the protein. Bringing the pH of the solution up to about pH 8.0 regenerates the metal binding activity of the protein. Therefore, due to the reversible binding characteristics of the novel metal binding proteins, the present invention also provides compositions, formulations, powders, liquids, devices or apparatuses comprising the substantially purified metal binding proteins which can be utilized more than once.
Metal binding proteins of the present invention, when used in methods for reducing the concentration of a metal in a substrate can be provided in such a way as is appropriate for the particular use, situation, mode of administration or environment in which the metal binding proteins are to be used. For example, when used in metal remediation, or in pollution control, the metal binding proteins can be coupled to a support, such as a powder and used, for example, as a flocculent to provide a convenient and efficient means of dispersing the metal binding proteins.
Alternatively, the metal binding proteins can be provided coupled to a membrane, a semi-permeable membrane, a filter, or any other means appropriate for allowing sufficient exposure of the metal binding proteins to the heavy metal containing substrate so as to bind or sequester the heavy metals from the substrate. A membrane or filter comprising the metal binding proteins provides a particularly efficient means of treating ground water or waste water, as contaminated water can be purified by passage through the membrane or filter without further clean up as is required in chemical extraction processes. Coupling the metal binding proteins to a support or supporting matrix also affords easier handling of the metal binding proteins especially when used in large scale or industrial applications.
EXAMPLE 1
Isolation of MT from Artemia 
In accordance to the teachings of the present invention, the following exemplary protocols illustrate methods useful in the production, purification and analysis of MT proteins.
Sample Preparation
As a preliminary step in the isolation of the metal binding proteins, Artemia brine shrimp were grown in artificial seawater (AS) (422.7 mM NaCl, 7.24 mM KCL, 22.58 mM MgCl2.6H2O, 25.52 mM MgSO4.7H2O, 1.33 mM CaCl2.2H2O and 0.476 mM NaHCO3). Artemia cysts (2.5 g) were incubated for 48 hours in 250 mL of AS supplemented with antibiotics at 30° C. and rotation at 125 rpm. After 24 hrs, phototropic Artemia were collected, cultured for an additional 24 hrs and then collected by cloth filtration. The shrimp were weighed and if not used immediately, stored at −80° C.
The Artemia were then homogenized in homogenization buffer (HB) (10 mM Tris-HCl (pH 8.0), 0.1 mM DTT, 0.5 mM PMSF and 10 μg/ml Soybean Trypsin Inhibitor) and resuspended in HB at 4 mL/gm wet weight of shrimp. The homogenate was passed through a Yamato LH-21 homogenizer three times at a setting of 800 rpm, filtered through Miracloth (Calbiochem) and the filtrate centrifuged in a Sorvall SA-600 rotor at 14,300 rpm, 4° C. for 30 min. The lipid layer on top of the supernatant was removed by vacuum aspiration and the lower supernatant layer collected and centrifuged in a Beckman 50.2TI rotor at 40K rpm, 4° C. for 90 min. Again, the upper lipid layer was removed and the lower supernatant recentrifuged at 150,000×g (150K sup). The 150K sup was then used immediately or stored at −80° C. If used immediately, this product was then subjected to gel filtration as follows. The gel filtration studies verified the metal binding proteins' ability to bind to heavy metals.
Gel Filtration Studies
The 150K sup was filtered through a HPLC certified 0.45 micron LC13 acrodisc filter (Gelman Sciences). A 20 mL aliquot of filtered 150K supernatant was incubated at 4° C. for 20 min with 2 μL of 109Cd (0.066 μCi) to radiolabel the metal binding proteins. The sample was then applied to a Sephadex G-50 molecular weight exclusion column (2.6 cm×94 cm) previously equilibrated with 50 mM Tris-HCl (pH 8.0) saturated with N2. One molar DTT (2 μL) was added to fractions 60-100 prior to sample loading in order to maintain reducing conditions in the fractions containing the low molecular weight metal binding proteins. The column was eluted with 50 mM Tris (pH 8.0) at a flow rate of 20 mL/hr while monitoring the eluate at 280 nm. During the elution period, the buffer reservoir was continually purged with N2. Samples used for amino acid analysis were not radiolabeled.
The 109Cd content (CPM) of the column fractions was determined with an Auto-Logic gamma counter (ABBOTT Laboratories). Zinc content was measured by Flame or Furnace Atomic Absorption Spectroscopy and expressed as PPB zinc/fraction. Prior studies indicated that two classes of metal binding proteins were present, one class being a high molecular weight fraction. However, the majority of 109Cd eluted with a low molecular weight class of zinc-containing metal binding protein. As shown in FIG. 1, radioactive metal binding protein had a elution peak corresponding to that for zinc (roughly, fraction #50) indicating the presence of endogenously bound zinc. The protein concentration of the Sephadex G-50 fractions was determined with a BCA Total protein assay kit (Pierce) according to manufacturers protocol. The distinct structural features of the metal binding proteins of the present invention were then identified in the following studies.
Metal Binding Protein Characterization Studies
Chromatographic and molecular weight studies were performed to ascertain structural features of the metal binding proteins. All protocols used were as described previously in B. Harpham, “Isolation of Metal Binding Proteins From Artemia”, Master's Thesis, California State University, Long Beach Library, 1998. Using anion exchange and reverse phase chromatography techniques well known in the art and described, for example, in B. Harpham “Isolation of Metal Binding Proteins From Artemia”, supra, metal binding proteins from Artemia were purified and determined to have molecular weights and amino acid sequence length unexpectedly lower than other known metal binding proteins. Under SDS-PAGE conditions, Artemia metal binding proteins have molecular weight of about 5.8 kDa as compared to 6-7 kDa for metal binding proteins from other mammalian species. Protein analysis of Artemia metal binding proteins indicate a sequence length of 48 amino acids. The Artemia MT amino acid sequence was unexpectedly and significantly shorter in length than other known metal binding proteins, which range in length from 60 to 68 amino acid residues.
EXAMPLE 2
Cloning and Sequencing of a Gene Encoding Artemia Metal Binding Protein
Total RNA was isolated from 48 hour nauplii (the larval stage of Artemia) using the RNAzol method. Forty-eight hour nauplii samples were prepared as described above in Example 1. The PolyTract Procedure (Promega, WI) was then used to isolate mRNA from the total RNA samples. cDNA was generated from the mRNA using SuperScript and 3′ RACE Kit procedures (Cat #18373, Gibco/BRL, WI) and then subjected to the following synthesis reaction.










cDNA synthesis reaction:


















Artemia mRNA
25 μl (500 ng)



DEPC H2O
30 μl



10 μM AP
 5 μl









The above mixture was incubated for 10 min at 70° C., then placed on ice for 1-2 min. Volatilized liquid was collected by centrifugation for 10 sec at 10,000 rpm. The following were then added to the above RNA cocktail to produce a cDNA solution:















10x PCR Buffer
10 μl



 25 mM MgCl2
10 μl



 10 mM dNTP
 5 μl



0.1 mM DTT
10 μl









The above resulting cDNA solution was then mixed and incubated at 42° C. for 5 min. Five (5) μL of Superscript II RT was added and the mixture incubated at 42° C. for 50 min for cDNA synthesis. The reverse transcription reaction was terminated by incubating the solution for 15 min at 70° C.; 5 μL of RNase was then added and the solution incubated for 20 min at 37° C. The final solution containing Artemia cDNA was then stored at −20° C. until used for PCR amplification as described below.
The initial PCR Primer Sequences used were as follows: the 5′ primer (N-terminal side) was designated “MT-Not I” (SEQ ID NO. 5) and the 3′ primer (C-terminal side) was designated “dT-Spe I” (SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, or SEQ ID NO. 9)







SEQ ID NO. 5


5′-ACC TAT GCG GCC GCA AAT GGA CTG CTG CAA GAA C-3′


 


SEQ ID NO. 6


5′-GCA CCA ACT AGT GCC TTT TTT TTT TTT TTT A-3′


 


SEQ ID NO. 7


5′-GCA CCA ACT AGT GCC TTT TTT TTT TTT TTT C-3′


 


SEQ ID NO. 8


5′-GCA CCA ACT AGT GCC TTT TTT TTT TTT TTT G-3′.






The above 5′ and 3′ primers were then used in the following amplification cocktail.










PCR Reaction Cocktail:


















10X PCR Buffer
5 μl



25 mM MgCl2
3 μl



10 mM dNTP
1 μl



10 μM dT-Spel
1 μl



10 μM MT-Not I
1 μl









To the above PCR Reaction Cocktail, a Gem 50 wax bead was added to the tube and the tube incubated at 80° C. for 2-3 minutes. Upon hardening of the wax at room temperature for 10-15 min, the following were layered on top of the hardened wax:















Sterile H2O
36.5 μl



Artemia cDNA mixture
  2 μl



Taq Polymerase
 0.5 μl









This final mixture was then subjected to the following PCR amplification program.
PCR Program:
Initial denaturation for 3 min at 95° C., followed by 29 cycles of:
94° C. for 1 min
49° C. for 1 min
72° C. for 1 min
Then one cycle of:
72° C. for 10 min
Then holding the mixture at 4° C.
Once amplified, the PCR product was verified for successful amplification on a 1.2% agarose gel. The PCR product was then purified for subsequent cloning using Qiagen QIAquick Gel Extraction (Qiagen, CA). The following primers which contain modifying restriction sites incorporated into their sequence were used to amplify and subclone the purified PCR product containing brine shrimp Artemia metal binding protein gene sequences.

    MT Nco I (5′ primer containing an Nde I site):









SEQ ID NO. 9


5′-GCT ACA CAT ATG TCC ATG GAC TGC TGC AAG AAC-3′






    MT Sal I (3′ primer containing Sal I site):









SEQ ID NO. 10


5′-ACG AAC GTC GAC GCC TTT TTT TTT TTT TTT A-3′






Using the MT Nco I and MT Sal I primers, with an annealing temperature of 72° C. for 1 min, the Artemia MT nucleotide sequence was amplified and then subsequently cloned into TOPO CR2.1 using TA cloning and then subcloned into the pGEM3 vector's Eco R1 site. Once cloned, the cloned metal binding protein gene can then be easily modified or further processed for use in expression, production or other methods requiring use of an isolated nucleic acid sequence encoding a metal binding protein.
The entire coding sequence for MT gene was then determined using a Applied Biosystems DNA sequencer. Sequence comparison studies of the MT gene from Artemia indicate it to have unexpectedly different sequence as compared to other known metal binding protein genes. When the Artemia MT gene sequence was aligned with that of equine and human MT, homology was observed at the locations of the metal-binding cysteine residues. The ability of the exemplary metal binding protein of the present invention to bind heavy metals was then confirmed in the following studies.
EXAMPLE 3
Polymer Membranes for Toxic Metal Removal from Water
Metallothionein was extracted from Artemia embryos as described above. The protein extract (80 mL) was placed in a boiling water bath for 15 minutes. The solution was centrifuged at 30,000×g (16,000 rpm in a SA600 rotor) for 30 minutes at 4° C. The supernatant containing the metallothionein was transferred to a clean tube containing 60 μL of 109Cd (Amersham Biosciences). The solution was mixed well and allowed to stand at room temperature for five minutes. This allows for exchange of the radioactive cadmium onto the metallothionein and provides us with a method for detecting the protein during its purification. The solution was then applied to a 100×4.8 cm G-50 molecular exclusion column and eluted with nitrogen saturated 50 mM Tris, pH 8.0. Fifteen milliliter fractions were collected into tubes containing 25 μL of 1M DTT. The fractions with peak metal binding activity were pooled and stored at 4° C. The solution is referred to as MT. (See FIG. 1)
Metal Binding at Neutral pH
Pall Biodyne membranes (Biodyne A and Biodyne B, 0.45 μm, Lot numbers 002245 and 035241, respectively) were used as a solid support for these experiments. A 1 cm2 piece of membrane was placed in a 10 mL Millipore glass frit filtering unit. Ten milliliters of MT was passed through the membrane under vacuum at a flow rate of approximately 100 mL/minute (FIG. 3). The flow through was collected for protein analysis. Next, 10 mL of a solution of cadmium (0.1 μg/mL of CdCl2 and 10 μL 109Cd in 50 mL of water) was passed through the membrane under vacuum (FIG. 4). The membrane was then washed twice, each with 10 mL of PBS. Five milliliters of the pooled eluate was analyzed for radioactivity. The membrane was removed from the filtering unit, place in a 12×75 mm centrifuge tube and analyzed for radioactivity in an LKB gamma counter. As a control, the procedure was repeated with a second membrane that had not been treated with MT. This membrane is referred to as the “blank.” The results are shown below in Table 1.










TABLE 1






Sample
MT Membrane
Blank








Biodyne A
152,876
3768



Biodyne B
158,762
1774









The results demonstrate that membrane-bound MT is capable of removing cadmium (as 109Cd) from a solution of the metal passed through the membrane. Membranes without MT remove little, if any, metal from the solution.
Metal Binding at Varying pH
The next series of experiments were to determine the effect of extremes of pH on the metal binding activity of the protein on the membrane. A fresh sample of MT was prepared for these studies. The solution of cadmium used for these experiments was prepared as follows: 2 μL of 109Cd was added to 1 mL of an aqueous solution of CdCl2 (1 ppm). Then 100 μL of this radioactive cadmium solution was added to 10 mL of each of the following solution: PBS, 10 mM glycine, 150 mM NaCl, pH 3.0, and 10 mM H2CO3/HCO3, 150 mM NaCl, pH 10.1. Only the Biodyne A membrane was used for this study. Membranes not treated with MT and washed with PBS containing radioactive cadmium served as the control. Membranes were placed in the Millipore filtering unit and processed as follows:

    
    
        Membrane #1 (blank) was washed with 5 mL of PBS containing radioactive cadmium. The membrane was then washed twice with 10 mL of non-radioactive, metal-free PBS.
        Membrane #2 was washed first with 10 mL of MT solution and then 5 mL of PBS containing radioactive cadmium. The membrane was then washed twice with 10 mL of non-radioactive, metal-free PBS.
        Membrane #3 was washed with 10 mL of MT solution and then 5 mL of 10 mM H2CO3/HCO3, 150 mM NaCl, pH 10.1, containing radioactive cadmium. The membrane was then washed twice with 10 mL of non-radioactive, metal-free 10 mM H2CO3/HCO3, 150 mM NaCl, pH 10.1.
        Membrane #4 was washed with 10 mL of MT solution and then 5 mL of 10 mM glycine, 150 mM NaCl, pH 2.0, containing radioactive cadmium. The membrane was then washed twice with 10 mL of non-radioactive, metal-free 10 mM glycine, 150 mM NaCl, pH 2.0.
    
    


Each membrane was analyzed for radioactivity as described above, The results are shown below in Table 2.










TABLE 2






Sample

CPM



















Membrane 1
(blank)
174



Membrane 2
pH 7.5
33380



Membrane 3
pH 10.1
6890



Membrane 4
pH 2.0
651









This experiment demonstrates that the membrane-bound MT is capable of binding metal at pHs ranging from 7.5 to 10.1 but not at a pH of 2. Once metal is bound to the MT, it can be recovered by exposing the membrane to acid (pH=2). These experiments were conducted by adding all the solutions directly to the membrane. To evaluate effects of pre-equilibrating the membranes with buffer prior to addition of MT, i.e., is the efficiency of metal binding effected, membranes (Biodyne B) were processed as follows:

    
    
        Membrane #1 (blank) was washed with 5 mL of PBS containing radioactive cadmium. The membrane was then washed twice with 10 mL of non-radioactive, metal-free PBS.
        Membrane #2 was washed first with 10 mL of MT solution and then 5 mL of PBS containing radioactive cadmium. The membrane was then washed twice with 10 mL of non-radioactive, metal-free PBS.
        Membrane #3 was pre-washed with 10 mL of metal-free 10 mM H2CO3/HCO3, 150 mM NaCl, pH 10.1, then washed with 10 mL of MT solution and then 5 mL of 10 mM H2CO3/HCO3, 150 mM NaCl, pH 10.1, containing radioactive cadmium. Finally, the membrane was washed twice with 10 mL of non-radioactive, metal-free 10 mM H2CO3/HCO3, 150 mM NaCl, pH 10.1.
    
    


The results are shown below in Table 3.









TABLE 3






Sample
CPM


















Membrane #1
190



Membrane #2
4218



Membrane #3
7431









Equilibrating the membrane at pH 10.1 results in better efficiency of protein binding to the membrane.
Specificity of MT Metal Binding
Binding affinity/specificity was measured against bovine serum albumin, a protein containing several cysteine residues and known to bind heavy metals. The Biodyne A membrane was used for this experiment. The concentration of MT solution was found to be approximately 7 μg/mL. The concentration of the flow through is equivalent to the starting material indicating that the amount bound to the membrane is in ng (nanograms), thus indicating that the metal binding capacity of the protein is significant. Therefore, 7 μg/mL and 100 μg/mL solutions of BSA were made in D-PBS using the 2 mg/mL BSA standard from Pierce Chemical, Inc. The cadmium binding solution was prepared as follows: 1.5 mL of aqueous 1 ppm CdCl2 was mixed with 3 μL of 109Cd. The solution is stored at 4° C. The assay was run as follows:
Membrane #1 (blank) was washed with 5 mL of PBS containing radioactive cadmium. The membrane was then washed twice with 10 mL of non-radioactive, metal free PBS.
Membrane #2 was washed first with 5 mL of MT solution and then 5 mL of PBS containing radioactive cadmium. The membrane was then washed twice with 10 mL of non-radioactive, metal free PBS.
Membrane #3 was washed with 5 mL of BSA solution (7 μg/mL) and then 5 mL of PBS containing radioactive cadmium. The membrane was then washed twice with 10 mL of non-radioactive, metal-free PBS.
Membrane #4 was washed with 10 mL of BSA solution (100 μg/mL) and then 5 mL of PBS containing radioactive cadmium. The membrane was then washed twice with 10 mL of non-radioactive, metal-free PBS.
The results of these experiments are shown below in Table 4.










TABLE 4






Sample

CPM








Membrane 1
No MT
174



Membrane 2
MT (5 mL)
1171 



Membrane 3
BSA (5 mL of 7 μg/mL)
 77



Membrane 4
BSA (10 mL of 100 μg/mL)
 151*





*this membrane was tested a different day where the MT binding activity was greater than 3000 CPM.






Under these experimental conditions, BSA does not remove metal from aqueous solutions, even when using a 10-fold higher concentration of BSA than MT to prepare the membrane. This experiment demonstrates the utility of membrane bound MT for remediation of metal from water or other aqueous substrates (FIG. 5).
Effect of Temperature on Metal Binding Activity.
These binding experiments were performed with Biodyne A membranes.
Membrane #1 (blank) was washed with 5 mL of PBS containing radioactive cadmium. The membrane was then washed twice with 10 mL of non-radioactive, metal-free PBS.
Membrane #2 was washed with 10 mL of MT solution and then 5 mL of PBS containing radioactive cadmium pre-warmed to 60° C. The membrane was then washed twice with 10 mL of non-radioactive, metal-free PBS pre-warmed to 60° C.
Membrane #3 was washed with 10 mL of MT solution and then 5 mL of PBS containing radioactive cadmium cooled to 4° C. The membrane was then washed twice with 10 mL of non-radioactive, metal free PBS cooled to 4° C.
The results of these experiments are shown below in Table 5.









TABLE 5






Sample
CPM


















Membrane #1
139



Membrane #2
3886



Membrane #3
2672









EXAMPLE 4
Comparison of Rabbit and Artemia MT
Metal remediation with the MT proteins of the present invention can be accomplished using metallothionein proteins from a variety of sources. Rabbit liver MT was obtained as a lyophilized protein (Sigma) and solubilized in 400 μL of 50 mM Tris, pH 8.0, 0.001 M DTT to a final concentration of 2.5 mg/mL (rabbit MT stock solution). Artemia MT was purified as described supra in Example 1.
Membranes were prepared having bound Artemia MT or rabbit liver MT by passing an MT-containing solution through the membrane, as described supra in Example 1. Three membranes, a blank, a membrane bound with Artemia MT and a membrane bound with rabbit liver MT, were then placed in a 13 mm fritted glass filtering unit and 10 mL of a metal binding solution (a stock solution of 9000 cpm of 109Cd/25 μL of solution diluted to 75 μL/10 mL PBS to form the metal binding solution) was passed through the membrane under vacuum. The membrane was then washed three times in PBS, and the membrane-bound radioactivity was measured in a Packard gamma counter. In a second experiment, a larger quantity of Artemia MT was bound to the membrane. The results of these two experiments are found in Tables 6 and 7.










TABLE 6






Sample

CPM








Membrane 1
Blank
351



Membrane 2
Artemia (20 mL bound to the membrane
685



Membrane 3
Rabbit (25 μL of a 2.5 mg/mL solution
985



















TABLE 7






Sample

CPM








Membrane 1
Blank
231



Membrane 2
Artemia (25 mL bound to the membrane
980









Membrane-bound metallothionein, regardless of source, provides removal of metals from aqueous solutions. In addition, the metal binding activity is a function of the amount of protein applied to the membrane and increasing the amount of MT protein on the membrane results in increased metal binding activity by the membrane.
EXAMPLE 5
Binding of Thimerosal to Metallothionein
The binding of thimerosal (or the ethyl mercury from thimerosal) to MT was verified by incubating metallothionein with thimerosal (FIG. 10) and then fractionating the thimerosal/metallothionein complex from unbound thimerosal by molecular exclusion chromatography.
Rabbit liver MT (Sigma) was solubilized in 10 mM Tris, pH 8.0 to a final concentration of 1 mg/mL. Recombinant Artemia MT was produced in bacteria and extracted by sonication in 10 mM Tris, pH 8.0, 0.1 mM DTT, and 0.5 mM of PMSF. The bacterial extract was placed in boiling water for 10 minutes. The resulting precipitate was collected by centrifugation. The supernatant containing Artemia MT was incubated with 109Cd and fractionated on a G-50 molecular exclusion column with 50 mM Tris, pH 8.0. Peak metal binding fractions were collected, pooled, and fractionated by FPLC on a Superdex column using Dulbecco's Phosphate Buffered Saline, pH 7.5, with 0.1 mM DTT as elution buffer (D-PBS). The concentration of the purified Artemia MT was 206 μg/mL. Thimerosal (Sigma) was solubilzed to a final concentration of 10 mg/mL in D-PBS. Five milliliter polyacrylamide desalting columns were used to fractionate MT bound thimerosal from unbound thimerosal. Metallothionein, and molecules bound thereto, elutes in the void volume (vo) of the column, well ahead of the smaller, unbound thimerosal. In order to calibrate the column, i.e., determine the vo, purified Artemia MT (or rabbit MT) was radiolabeled with 109Cd. The columns were first washed with 10.5 mL of PBS, followed by 1.0 mL of PBS/5 mM EDTA, and then 14 mL of D-PBS. Four hundred microliters of the labeled Artemia MT was applied to the surface of a desalting column and allowed to enter the gel by gravity. The protein was eluted from the column with D-PBS. One-half mL fractions were collected from the column and analyzed for radioactivity on a Packard Gamma Counter. The MT eluted from the column (the vo) in fractions four and five. The procedure was repeated with thimerosal to determine where unbound thimerosal would elute from the column. Two micrograms of thimerosal was added to 400 μL of D-PBS. The entire sample was fractionated on a desalting column exactly as described above. Thimerosal was measured by assaying for mercury. The thimerosal eluted from the column in fractions 8 through 10. The results are depicted in FIG. 11.
Next, rabbit liver MT was diluted to 200 μg/mL with 10 mM Tris, pH 8.0. One microgram of thimerosal was pre-incubated with 400 μl of either the rabbit liver MT or the recombinant Artemia MT. The mixtures were then fractionated on a polyacrylamide desalting column as described above and the fractions were assayed for mercury. The results of the chromatography is depicted in FIG. 12. Therefore, MT is capable of binding thimerosal in solvents and conditions used to manufacture/store vaccines, i.e., PBS, pH 7.5.
Regardless of the source of MT, nearly 50% of the measurable thimerosal was bound to the MT. There was no detectable mercury in the solution of MT, i.e., the only source of the metal was from the thimerosal. In order to show specificity of the MT for thimerosal and the high binding affinity between the two, the experiment was repeated exactly as described above except for substituting with bovine serum albumin (BSA) for the MT. Bovine serum albumin is a known metal binding protein. Elution of BSA from the column was monitored by measuring the absorbance of each fraction at 220 nm. The results of this experiment are shown in FIG. 13. Thimerosal did not bind to BSA in appreciable amounts under these conditions.
EXAMPLE 6
Thimerosal Binding to Membrane-bound Metallothionein
Experiments were conducted to demonstrate that preformed MT/thimerosal complexes could be removed from a solution by filtration though a membrane filter and that MT bound to a membrane filter could be used to remove thimerosal from a solution.
Rabbit liver MT was used for these experiments. The protein was solubilized in 10 mM Tris, pH 8.0, to a final concentration of 1 mg/mL. Millipore Immobilon—Psq PVDF transfer membrane was used as the solid support for these experiments (0.2 μm). The membrane was cut into 1 cm2 pieces and the individual membrane squares were wetted just prior to use by placing them in 100% methanol for 3 seconds, then immersing them in water for two minutes, and finally equilibrating them in 10 mM Tris, pH 8.0 for three minutes. Three different membrane samples were used.
Membrane I—A 1 cm2 piece of membrane was placed in a 10 mL Millipore glass frit filtering unit. Five milliliters of a solution of thimerosal (200 ppb) was passed through the membrane under vacuum. The flow through was collected and analyzed for mercury. This assay served as the control to monitor the efficiency of thimerosal removal from a solution in the assays listed below.
Membrane II—A 1 cm2 piece of membrane was placed in a 10 mL Millipore glass frit filtering unit. Five milliliters of a solution of rabbit liver MT (0.04 mg/mL, 200 μg total protein in 10 mM Tris, pH 8.0) was passed through the membrane under vacuum. This was followed by passing 5 mL of a solution of 200 PPB thimerosal in 10 mM Tris, pH 8.0 through the membrane under vacuum. The flow through was collected and analyzed for mercury. This assay was designed to show that MT immobilized on a permeable membrane was capable of removing thimerosal from a solution passing through the membrane.
Membrane III—A 1 cm2 piece of membrane was placed in a 10 mL Millipore glass frit filtering unit. One microliter (1 μl) of thimerosal (1 μg/μl in 10 mM Tris, pH 8.0) was mixed with 200 μg of MT (200 μl of the 1 mg/mL stock solution). The solution was diluted to a final volume of 5 mL with 10 mM Tris, pH 8.0 to give a final thimerosal concentration of 200 PPB. The MT/thimerosal mixture was then passed through the membrane. The flow through was collected and analyzed for mercury. This assay was designed to show that thimerosal could be removed from a solution by first reacting a thimerosal-containing solution with MT to generate MT/thimerosal complexes and then removing the complexes from the solution by passing the solution through a permeable membrane with a high binding affinity for protein. The results of this experiment are shown in Table 8 below.










TABLE 8







Thimerosal
% Thimerosal



Membrane
(PPB)
removed



















Thimerosal Solution
148
—



Membrane I (No MT)
138
 6.7%



Membrane II (MT)
94
36.5%



Membrane III (MT/Thimerosal)
127
14.2%









The results of this experiment demonstrate that MT bound to a permeable membrane is capable of removing thimerosal from a solution passing through the membrane. Additionally, thimerosal can be removed from a solution by first adding MT to the solution containing thimerosal to form MT/thimerosal complexes and then collecting the complexes by passing the solution through a permeable membrane or some other device form for capture of the MT/thimerosal complexes.
In closing it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principals of the invention. Other modifications may be employed which are within the scope of the invention and accordingly, the present invention is not limited to that precisely as shown and described in the present specification.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on those embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of limits” the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.
Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are individually incorporated by reference herein in their entirety.
In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

## Claims

1. A system for the removal of thimerosal from a volume of medication, the system comprising:
a container containing a volume of a thimerosal-containing medication comprising a plurality of doses of said medication;
a solid support having associated therewith at least one substantially purified metallothionein protein, or a portion thereof;
passing said thimerosal containing-medication through said solid support into dosing vials, wherein said at least one substantially purified metallothionein protein binds said thimerosal from said medication resulting in a substantially thimerosal-free medication in said dosing vials.

2. The system of claim 1 wherein said medication is a vaccine, immunogenic composition, liquid pharmaceutical composition, colloidal pharmaceutical composition, suspension pharmaceutical composition, aerosol, or dry powder.

3. The system of claim 1 wherein said solid support is a filter, membrane, nanoparticles, beads, solid support particulates, or polymer coating.

4. The system of claim 3 wherein said at least one substantially pure metallothionein protein is associated with a plurality of beads or nanoparticles.

5. The system of claim 1 wherein said solid support comprises a biocompatible polymer.

6. The system of claim 5 wherein said biocompatible polymer is a fluorinated polymer, polyolefin, polystyrene, substituted polystyrene, polysulfone, polyester, polyacrylate, polycarbonate; vinyl polymer, copolymer of butadiene and styrene, fluorinated ethylene-propylene copolymer, ethylenechlorotrifluoroethylene copolymer, nylon, or mixtures thereof.

7. The system of claim 3 wherein said solid support is a filter.

8. The system of claim 1 wherein said at least one substantially purified metallothionein protein, or a portion thereof, is from a mammal, fish, mollusk, echinoderm, crustacean, reptile, nematode, grain, plant, yeast, or fungus.

9. The system according to claim 8 wherein said crustacean is brine shrimp (Artemia).

10. The system of claim 1 wherein said metallothionein protein has an amino acid sequence of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 21, or SEQ ID NO. 23.

11. A method of removing thimerosal from a medication to be administered to a subject comprising:
contacting a volume of a thimerosal-containing medication with at least one substantially purified metallothionein protein associated with a support; and
separating the support having thimerosal bound thereto from the thimerosal free medication; and portioning the thimerosal-free medication into dosing vials.