WO2016161973A1 - 磁感应受体蛋白和其复合物及它们的用途 - Google Patents

磁感应受体蛋白和其复合物及它们的用途 Download PDF

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WO2016161973A1
WO2016161973A1 PCT/CN2016/078842 CN2016078842W WO2016161973A1 WO 2016161973 A1 WO2016161973 A1 WO 2016161973A1 CN 2016078842 W CN2016078842 W CN 2016078842W WO 2016161973 A1 WO2016161973 A1 WO 2016161973A1
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protein
seq
magr
protein complex
complex
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French (fr)
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谢灿
罗述金
覃思颖
银行
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Peking University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
    • H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
    • H01F1/42—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of organic or organo-metallic materials, e.g. graphene

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  • the invention relates to biology and its field of application. Specifically, the present invention relates to a protein which is magnetically self-researched and prepared for the first time, which is a magnetic induction receptor protein, and a homozygous protein complex of the magnetic induction receptor protein, and the magnetic induction receptor protein and light A hybrid protein complex formed by a sensing protein. The invention also provides the use of the protein or complex.
  • Iron is an essential trace element in living organisms, and proteins related to iron binding and transport have also received much attention and research.
  • Iron-binding proteins are iron-containing metal-binding proteins, one of which is called iron-sulfur protein, and its combined iron is in the form of an iron-sulfur cluster.
  • Iron-sulfur protein is a very important carrier of electron transport, mainly involving biochemical reactions such as respiratory chain and photosynthesis of plants.
  • Studies on iron-binding proteins and iron-sulfur proteins are extensive and in-depth, focusing on various aspects related to biochemical metabolism. The two most important iron-binding proteins in organisms are lactoferrin and transferrin.
  • transferrin which can carry about 3,000-5,000 iron atoms, is an important mechanism for storing iron in living things. Some studies have also shown that iron-loaded transferrin can be used as a nanoparticle for biomedical and other applications. However, to date, all iron-binding proteins that have been discovered do not have their own magnetic properties, that is, they are not "magnetic proteins.” For example, the transferrin having an extremely high iron-carrying ability can be attracted by a magnetic field under a magnetic field and has paramagnetic properties, but does not have its own magnetic properties. This protein has a reaction similar to that of an iron sphere under a magnetic field, but since it does not have its own magnetic properties, it cannot be used as a magnetic material such as a magnet used as a compass.
  • Protein or protein complexes that are themselves magnetic and/or photomagnetic have not been found in the art. Proteins or protein complexes which are themselves magnetic and/or photomagnetic are also not available in the art and are used in human production and life.
  • the present invention relates to a protein which is newly studied and prepared in modern biological research and which is magnetic and/or capable of forming a magnetic complex, namely a magnetic induction receptor protein, and a homozygous protein of the magnetic induction receptor protein. a complex, and a hybrid protein complex formed by the magnetically induced receptor protein and a light-sensitive protein.
  • the invention also provides the use of the magnetically induced receptor protein or complex.
  • the present invention provides an isolated magnetically induced receptor protein.
  • a magnetically induced receptor protein (or "Magnetoreceptor”, or “MagR”) is one of the first research, identification, and nomenclature of the inventors of the present invention in modern biological research, and its nucleic acid coding sequence is widely present in the genome of an organism. Highly conserved, it can form proteins with biomagnetic protein monomers or homozygous complexes or hybrid complexes.
  • the nucleic acid sequence of the magnetic induction receptor protein provided by the present invention exists in the genome of a large number of different species, and the encoded protein sequence is highly conserved and has a plurality of highly conserved domains, such as characteristic iron and/or sulfur binding. Domain.
  • the amino acid sequence of the magnetic induction receptor protein of the present invention comprises, in order from the N-terminus to the C-terminus, the following domains, in accordance with the amino acid position shown in Figure 13 of the present application: TVRA (SEQ ID NO: 17) at positions 10-13, RGCNGL (SEQ ID NO: 18) at positions 56-61, QDGV at position 80-83 (SEQ ID NO: 19), KKAQL at position 89-93 (SEQ ID NO: 20), positions 95-101 LLGTEMD (SEQ ID NO: 21) and NPNIKGTCGCGESF at positions 115-128 (SEQ ID NO: 22).
  • the amino acid sequence of the magnetic induction receptor protein of the present invention further comprises the following domains from the N-terminus to the C-terminus: R at the 17th position, R at the 23rd position, AL at the 25th to 26th positions, and 28-29 Bit LT, 32-33rd AV, 48th G, 51st V, 53rd V, 63rd Y, 67th Y, 71st K, 75th -76 bits of DE, 78th of V, 87th of I, 104th E, 106th to 107th KL, 110th to 111th EF, and 113th F.
  • the position of the amino acid shown in Figure 13 is the result of aligning a plurality of sequences, which is capable of corresponding to the actual position of the amino acid of most of the magnetically induced receptor proteins; when a new magnetically induced receptor protein is found Thereafter, those skilled in the art can easily know the position of the new protein corresponding to the amino acid position shown in Fig. 13 by comparison.
  • the magnetic induction receptor protein of the present invention or an active fragment or derivative thereof has an iron sulfur center of 2Fe2S.
  • the magnetically induced receptor protein of the present invention may be one of a family of magnetically induced receptor proteins.
  • the magnetically induced receptor protein of the invention may be derived from an animal, such as a vertebrate or an invertebrate.
  • An exemplary nucleic acid or protein sequence of MagR is obtained by the inventor of the present application from the domestic pigeon (Latin name Columba livia) and Monarch butterfly (or the American monarch butterfly, Latin name Danaus plexippus), the amino acid sequence of which is SEQ ID NO: 1 and SEQ ID NO: 2. These sequences were not disclosed prior to the present invention.
  • the magnetic induction receptor protein provided by the present invention also includes magnetic induction receptor proteins of other biological species, such as from Fruit fly (Dragon name Drosophila melanogaster), honey bee (Apis mellifera), and Amphioxus (Lancelet).
  • Branchiostoma floridae zebrafish (Zebrafish, Latin name Danio rerio), python (Burmese python, Latin name Python bivittatus), grasshopper (Zebra finch, Latin name Taeniopygia guttata), chicken (Chicken, Latin name Gallus gallus), whale (Minke whale, Latin name Balaenoptera acutorostrata), Bat (Little brown bat, Latin name Myotis lucifugus), Naked mole rat (Latin name Heterocephalus glaber), Mouse (Mouse, Latin name Mus musculus), Human (Human , Latin name Homo sapiens).
  • amino acid sequences of the above magnetic induction receptor proteins from Drosophila, honeybee, amphioxus, zebrafish, python, grass finch, chicken, whale, bat, naked mole, mouse and human are respectively SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12. SEQ ID NO: 13 and SEQ ID NO: 14.
  • an isolated magnetically-sensitive receptor protein or active fragment or derivative thereof having:
  • SEQ ID NO: 1 SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8.
  • SEQ ID NO: 9 amino acid sequence of SEQ ID NO: 10
  • SEQ ID NO: 11 amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14, or
  • SEQ ID NO: 1 SEQ ID NO: 2
  • SEQ ID NO: 3 SEQ ID NO: 4
  • SEQ ID NO: 5 SEQ ID NO: 6
  • SEQ ID NO: 7 SEQ ID NO: 8
  • SEQ ID NO: 9 SEQ ID NO: 10
  • SEQ ID NO: 11 SEQ ID NO: 12
  • SEQ ID NO: 13 or SEQ ID NO: 14 have at least 50%, 51%, 52 when compared.
  • the difference in residue positions therein is a conservative amino acid substitution.
  • conservative amino acid substitution refers to the replacement of an amino acid residue by another amino acid residue having a side chain R group of similar chemical nature (eg, charge or hydrophilicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein.
  • amino acid groups having side chains of similar chemical nature include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxy side chain: serine And threonine; 3) amide-containing side chain: asparagine and glutamine; 4) aromatic side chain: phenylalanine, tyrosine and tryptophan; 5) basic side chain: lysine , arginine and histidine; 6) acidic side chain: aspartic acid and glutamic acid; and 7) sulfur-containing side chain: cysteine and methionine.
  • Preferred conservative amino acid substitution combinations are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid - aspartic acid, and asparagine-glutamine.
  • an isolated magnetically-sensitive receptor protein or active fragment or derivative thereof having: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:
  • the amino acid substituted, deleted, or added is a conservative amino acid.
  • the magnetic induction receptor protein having the substitution, deletion, and addition provided by the present invention has magnetic induction receptor protein activity.
  • the isolated magnetically-induced receptor protein or active fragment or derivative thereof has magnetic properties and/or is capable of forming a protein complex having magnetic properties.
  • the isolated magnetically induced receptor protein or active fragment or derivative thereof has a domain that binds iron and/or sulfur.
  • the isolated magnetic induction receptor protein or active fragment or derivative thereof can form a magnetic homozygous protein complex, and/or can interact with Cryptochrome (Cry). A hybrid protein complex is formed.
  • the above homozygous protein complex or hybrid protein complex has a specific protein stereostructure, such as an elongated shape; in the homozygous protein complex or hybrid protein complex, A plurality of iron atoms, which also form an ordered arrangement in the inner near central axis region of the rod-like structure protein, thereby making the protein complex magnetic.
  • the invention provides a recombinant DNA construct comprising a nucleotide sequence encoding a magnetically induced receptor protein or an active fragment or derivative thereof as described above.
  • the nucleotide sequence encoding a magnetically-induced receptor protein or an active fragment or derivative thereof as described above is operably linked to at least one regulatory element, for example, in a eukaryotic or prokaryotic cell.
  • a functional promoter for example, in a eukaryotic or prokaryotic cell.
  • the present invention provides an isolated protein complex comprising two or more of the aforementioned magnetic induction receptor proteins of the present invention or active fragments or derivatives thereof, ie, homozygous protein complexes.
  • the protein complex comprises 4 or more of the magnetically induced receptor protein or an active fragment or derivative thereof, preferably 20 or more of the magnetically induced receptor protein or active fragment or derivative thereof Further, it is more preferably 20 to 24 of the magnetic induction receptor proteins or active fragments or derivatives thereof, and more preferably 20 of the magnetically induced receptor proteins or active fragments or derivatives thereof.
  • the three-dimensional structure of the protein complex is "long strip".
  • the three-dimensional structure of a protein refers to the spatial structure of a protein molecule.
  • the elongated three-dimensional structure of the protein may also be referred to as a "rod structure” or a "stick structure.”
  • the stereostructure of the protein can be observed and measured by an electron microscope.
  • the plurality of magnetically induced receptor proteins or active fragments or derivatives thereof are linearly polymerized and further aggregate to form a rod-like structure.
  • the plurality of magnetically-induced receptor proteins or active fragments or derivatives thereof are linearly polymerized to form long chains of two proteins (long-length proteins of equal length, ie, the same two proteins have the same single strand Number of protein monomers).
  • the long chains of the two proteins are combined in a helical form to form a protein complex of a rod-like structure.
  • a plurality of iron atoms are further contained in the protein complex of the present invention.
  • the plurality of iron atoms are located in an inner near central axis region of the rod-like structural protein complex.
  • each single magnetically-sensitive receptor protein forming the protein complex binds to an iron atom, and, for example, each magnetically-induced receptor protein monomer binds to an iron atom at the same or similar position, thereby being
  • the plurality of iron atoms are also arranged in an orderly arrangement in the inner near-axis axis region of the rod-like structure.
  • each of the magnetically-sensitive receptor proteins or active fragments or derivatives thereof in the protein complex contains a 2Fe2S iron-sulfur center, ie each magnetically-sensitive receptor protein contains 2 iron atoms and 2 A sulfur atom.
  • the protein complex contains about 20 magnetically induced receptor proteins, whereby the protein complex contains about 40 iron atoms.
  • the aforementioned protein complex may also incorporate a light sensing element.
  • the present invention also provides an isolated hybrid protein complex comprising: 1) the aforementioned magnetic induction receptor protein of the present invention or an active fragment or derivative thereof, and 2) a light-sensitive protein (eg, crypto-pigmented protein) Or an active fragment or derivative thereof.
  • a light-sensitive protein eg, crypto-pigmented protein
  • the light-sensing element is a part that can sense/transmit an optical signal, such as a molecule, a complex or a protein that can be bound to a protein, and the like, for example, a protein that can sense/transmit an optical signal.
  • an optical signal such as a molecule, a complex or a protein that can be bound to a protein, and the like, for example, a protein that can sense/transmit an optical signal.
  • proteins that can sense/transmit light signals have been discovered in the biological world. One of them is Cryptochrome (Cry) or an active fragment or derivative thereof.
  • every two magnetically induced receptor proteins bind to one cryptochromoprotein.
  • Cryptochrome is a kind of Flavoprotein capable of sensing blue light (400-500 nm) and near-ultraviolet light (320-400 nm) with a molecular weight of 70-80 KD.
  • the chromophore can be Flavin adenine dinucleotide (FAD) and pterin.
  • FAD Flavin adenine dinucleotide
  • Cry proteins and their coding genes are ubiquitous in plants, animals, and entire higher eukaryotes. Cry proteins may be involved in animal control of animal clock rhythms and circadian clocks.
  • the Cry family of proteins includes Cry, Cry1, Cry2, Cry3, and Cry4. In mammals and insects, its homologous gene encodes crypto-pigmented protein.
  • the cryptochromin protein in the protein complex of the invention, may be selected from the group consisting of Cry, Cry1, Cry2, Cry3 or Cry4. In still another aspect of the present invention, in the protein complex of the present invention, the cryptochromin protein has:
  • sequence identity amino acid sequence and has cryptochromin protein activity.
  • sequence identity can be calculated by the Clustal V method.
  • the difference in residue positions therein is a conservative amino acid substitution.
  • the cryptochrome protein in the isolated protein complex has: a substitution or deletion having one or more amino acids when compared to SEQ ID NO: 15 or SEQ ID NO: , added amino acid sequence.
  • the amino acid substituted, deleted, or added is a conservative amino acid.
  • the magnetic induction receptor protein having the substitution, deletion, and addition provided by the present invention has magnetic induction receptor protein activity.
  • the hybrid protein complex contains an iron sulfur atom.
  • the hybrid protein complex comprises 4 or more, preferably 20 or more of the magnetically induced receptor proteins or active fragments or derivatives thereof, more preferably 20-24, more preferably There are 20 such magnetically induced receptor proteins or active fragments or derivatives thereof.
  • the hybrid protein complex comprises 2 or more, preferably 10 or more of the cryptochromin proteins, further preferably 10-12, more Preferably, 10 of said cryptochromin proteins are used.
  • the aforementioned protein complex or hybrid protein complex of the invention is magnetic.
  • “Protein has magnetic properties” or “magnetic protein” means that a protein or protein complex exists as a main body of a magnetic material, and has magnetic properties itself.
  • the magnetic protein may have a magnet-like magnetic pole (north and north pole), can be pulled by an external magnetic field, and also has the property of attracting ferromagnetic substances such as iron, cobalt, and nickel. It is worth noting that the protein of the present invention has its own magnetic properties, and its magnetic properties are independent of the external magnetic field.
  • the aforementioned protein complex or hybrid protein complex of the invention can be prepared into crystals according to conventional methods in the art.
  • the invention also provides a method of producing the aforementioned protein complex of the invention comprising the steps of:
  • the magnetically induced receptor protein or an active fragment or derivative thereof is polymerized under conditions allowing formation of a protein complex to form a homologous polymer; the homopolymer is isolated.
  • the homopolymer is crystallized according to conventional techniques in the art.
  • the environment in which the magnetic induction receptor protein is produced has a bioavailable iron element and sulfur element.
  • the magnetically-sensitive receptor protein or an active fragment or derivative thereof expresses and forms a complex in a cell.
  • the cells may be cultured prokaryotic or eukaryotic animal cells, such as E. coli or mammalian cells.
  • the magnetically-sensitive receptor protein or an active fragment or derivative thereof contains iron and/or a sulfur atom during the cultivation of the cell, so that a bioavailable iron element is added to the culture solution and / or sulfur element (monomer or compound).
  • the method of producing the aforementioned protein complex of the invention can be carried out in the presence of a magnetic field.
  • the step in which the homologous polymer is separated can be carried out in the presence of a magnetic field.
  • the magnetic field is an applied magnetic field.
  • the isolated protein homologous polymer is carried out by affinity chromatography, such as by an affinity chromatography column; when the protein is separated by affinity chromatography, magnetic field conditions are provided.
  • the magnetic field strength of the magnetic field is greater than about 5 times the earth's magnetic field, such as about 10-100 times the earth's magnetic field.
  • a magnetic field strength of the magnetic field of more than about 100 times the earth's magnetic field can also obtain a good effect.
  • the present invention provides a method of producing the aforementioned hybrid protein complex of the present invention comprising the steps of:
  • a hybrid protein complex 1) the aforementioned magnetic induction receptor protein of the present invention or an active fragment or derivative thereof, and 2) the aforementioned photosensory protein (for example, cryptochromin) or an active fragment thereof Or the derivative is contacted and polymerized to form a hybrid polymer, wherein at least one of the proteins of 1) and 2) is isolated or recombinantly expressed; and the hybrid polymer is isolated.
  • the hybrid polymer is crystallized according to conventional techniques in the art.
  • the magnetically-sensitive receptor protein and the light-sensitive protein express and form a complex in the cell.
  • the cells may be cultured prokaryotic cells or eukaryotic animal cells, such as E. coli and mammalian cells.
  • the magnetically-sensitive receptor protein or active fragment thereof is cultured during the culture of the prokaryotic or eukaryotic animal cells
  • the derivative contains iron and/or a sulfur atom, so that a bioavailable iron element and/or a sulfur element (monomer or compound) is added to the culture solution.
  • the method of producing the aforementioned hybrid protein complex of the invention is carried out in the presence of a magnetic field.
  • the step in which the hybrid composite is separated is carried out in the presence of a magnetic field.
  • the magnetic field is an applied magnetic field.
  • the isolated hybrid protein complex is performed by affinity chromatography, such as by an affinity chromatography column; when the protein is separated by affinity chromatography, magnetic field conditions are provided.
  • the magnetic field strength of the magnetic field is greater than about 5 times the earth's magnetic field, such as about 10-100 times the earth's magnetic field.
  • a magnetic field strength of the magnetic field greater than about 100 times the earth magnetic field can also obtain a good effect.
  • a method of identifying a photomagnetic inductive composite formable by the aforementioned magnetically induced receptor protein of the invention comprising the steps of:
  • a magnetically related application of the aforementioned magnetically induced receptor protein and/or protein complex and/or hybrid protein complex of the invention for example, as a magnetic material or as a preparation thereof
  • the application of magnetic materials is highly sensitive to magnetic fields, and more particularly, it has intrinsic magnetism and can be used in various magnetic related applications. Magnetic material.
  • Magnetics-related applications are widely used, including but not limited to biomedical and medical devices such as magnetic biomaterials, purification of biomolecules, directed movement of intracellular substances, control of certain cellular life activities by magnetic fields, etc., magnetic recording media, super Applications such as nano-ring current superconductors, magnetorheological fluids, etc., electrical equipment such as electromagnetic conversion equipment, communication and navigation equipment such as applications in molecular gyroscopes.
  • polypeptide polypeptide
  • protein protein and peptide
  • peptide are used interchangeably herein to refer to a chain of amino acids wherein the amino acid residues are linked by peptide bonds or modified peptide bonds.
  • the amino acid chain can be any length greater than 2 amino acids.
  • polypeptide polypeptide
  • protein and “peptide” also include various modifications thereof. These modifications may be naturally occurring modified forms or chemically modified forms. Examples of modified forms include, but are not limited to, glycosylated forms, phosphorylated forms, ribosylated forms, acetylated forms, ubiquitinated forms, and the like.
  • Modifications also include intramolecular cross-linking and covalent attachment to various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, and the like. Additionally, modifications can also include cyclization, branching, and crosslinking. In addition, amino acids other than the 20 common amino acids encoded by the gene codon may also be included in the polypeptide.
  • Magnetic sensory receptor protein (or “Magnetoreceptor”, or “MagR”) is the inventor of the present application.
  • the first research, acquisition, identification, and nomenclature of a nucleic acid coding sequence in modern biological research is widely present in the genome of an organism and is highly conserved, forming a protein with a biomagnetic monomer or complex.
  • the nucleic acid sequence of the magnetically induced receptor protein provided by the present invention exists in the genome of a large number of different species, and the encoded protein sequence is highly conserved and has a plurality of highly conserved domains, such as a domain that binds iron and/or sulfur.
  • the magnetically induced receptor protein can form a homozygous protein complex having a specific morphological structure, having its own magnetic properties, and/or can form a hybrid protein complex with Cryptochrome (Cry).
  • “Protein family” refers to a series of proteins having homologous domains or sequences, evolutionarily related, functionally identical or similar species present in a biological species.
  • the protein family of magnetically induced receptor proteins includes a magnetic induction receptor protein of Columba livia and a magnetic induction receptor protein of Danaus plexippus, the amino acid sequences of which are SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Shown.
  • Drosophila melanogaster Drosophila melanogaster
  • Apis mellifera branchostoma floridae
  • Daneo rerio Python bivittatus
  • Taeniopygia guttata Other members of the protein family of magnetically induced receptor proteins include Drosophila melanogaster, Apis mellifera, Branchostoma floridae, Daneo rerio, Python bivittatus, and Taeniopygia guttata.
  • the magnetic induction receptor proteins of chicken (Gallus gallus), whale (Balaenoptera acutorostrata), bat (Myotis lucifugus), naked mole (Heterocephalus glaber), mouse (Mus musculus), human (Homo sapiens), and their amino acid sequences are as follows: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.
  • a homozygous protein complex of a magnetically induced receptor protein or a hybrid protein complex formed by a magnetically induced receptor protein and a cryptochromic protein has a specific protein steric structure, such as a long strip; in the homozygous protein complex or hybrid
  • the protein complex has a plurality of iron atoms which are also arranged in an ordered near central axis region of the rod-like structure protein, thereby making the protein complex magnetic.
  • a plurality of the magnetically induced receptor proteins undergo linear polymerization and further aggregate to form a rod-like structure.
  • the plurality of magnetic induction receptor proteins are linearly polymerized to form two long chains of proteins; the long chains of the two proteins are combined in a spiral form to form a protein complex of a rod-like structure.
  • the homozygous protein complex of the magnetic induction receptor protein or the hybrid protein complex formed by the magnetic induction receptor protein and the cryptochromic protein further comprises a plurality of iron atoms; the plurality of iron atoms are located in the rod-like structure protein complex Internal near central axis area.
  • each single magnetically-sensitive receptor protein forming the protein complex binds to an iron atom, and, for example, each magnetically-induced receptor protein monomer binds to an iron atom at the same or similar position, thereby being
  • the plurality of iron atoms are also arranged in an orderly arrangement in the inner near-axis axis region of the rod-like structure.
  • the central axis described herein has a generally understood meaning. "Internal near-central axis region" means a place inside the rod-like structure surrounding the central axis, in which a plurality of iron atoms are arranged to form a circular electron flow.
  • the "magnetic" of a substance refers to a property that is capable of generating a magnetic field and attracting a ferromagnetic substance such as a metal such as iron, nickel, or cobalt.
  • Protein is magnetic or “magnetic protein” means that the protein or protein complex itself is magnetic, exhibits a magnet-like magnetic pole (north and north pole), can be pulled by an external magnetic field, and also attracts iron, cobalt, nickel, etc.
  • the properties of ferromagnetic materials can be used as magnetic materials.
  • Magnetic refers to the phenomenon that magnetic properties (such as magnetic susceptibility, magnetocrystalline anisotropy, hysteresis loop, etc.) change after the material is exposed to light, including based on an induction of an optical signal and a magnetic field signal. The other is based on the action of light or magnetic field.
  • magnetic properties such as magnetic susceptibility, magnetocrystalline anisotropy, hysteresis loop, etc.
  • active fragment of a magnetically-sensitive receptor protein refers to a portion or fragment of a magnetically-sensitive receptor protein, a portion comprising a magnetically-induced receptor protein that maintains it in a manner similar to a full-length magnetically-sensitive receptor protein and has all or part of the activity of the magnetically-sensitive receptor protein, or one or more of a plurality of activities having the protein An active fragment, such as a domain fragment thereof.
  • a derivative of a magnetically-induced receptor protein means having a primary and/or tertiary structure similar to the native form of the magnetically-sensitive receptor protein of the present invention, but differing from the native form by one or more amino acids a protein (polypeptide) having a residue (eg, one or more amino acid substitutions, insertions, and/or deletions), or having/additionally other chemical groups or protein (polypeptide) groups with which it can bind; these derivatives have Or retain all or part of the activity of the magnetically induced receptor protein.
  • the bondable chemical group includes, for example, a post-translational derivatization or modification of a polypeptide, such as a PEGylation and/or a thiol group.
  • the bindable protein (polypeptide) group includes, for example, a His tag or an IgG protein or the like.
  • isolated is used to describe a substance (eg, a nucleic acid and/or a protein) that is different from a substance in its natural form or in a form found in the original cell or biological environment.
  • the substance is substantially free of components that normally accompany or react with the substance in a naturally occurring environment, or that the substance has been removed from the component.
  • isolated protein is a protein that is substantially separated from at least one component or other protein present in the natural source of the protein, or which, when chemically synthesized, does not substantially contain at least one chemical Body or other chemicals. When there are less than about 30%, 20%, 10% or 5% (by dry weight) of other proteins or other chemicals (also referred to herein as "contaminating proteins” or “contaminating chemicals"), in the protein
  • the protein in the formulation is “substantially separated” or “substantially free” from other proteins or other chemicals.
  • Isolated proteins can have several different physical forms.
  • the isolated protein can be present as a full length nascent or unprocessed polypeptide, or as a partially processed polypeptide, or as a processed polypeptide, or a combination thereof.
  • an isolated polypeptide can be a non-naturally occurring polypeptide.
  • an "isolated polypeptide” can be a "hybrid polypeptide”.
  • An "isolated polypeptide” can also be a polypeptide derived from a naturally occurring polypeptide by addition, deletion or substitution of an amino acid.
  • An isolated polypeptide may also be a "purified polypeptide", which is used herein to mean a substantially homogeneous preparation that does not substantially contain other cellular components, other polypeptides, viral materials or specific polypeptides of the culture medium, or When the polypeptide is chemically synthesized, it does not substantially contain a chemical precursor or a by-product related to chemical synthesis.
  • “Purified polypeptides” can be obtained from natural or recombinant host cells by standard purification techniques or by chemical synthesis, as will be apparent to the skilled artisan.
  • interaction means that two protein domains, fragments or intact proteins exhibit sufficient physical affinity to each other such that the two "interacting" protein domains, fragments or proteins are in each other Physically close.
  • the interaction may result from the formation of one or more chemical bonds that result in a continuous and stable proximity of the two interacting entities.
  • Interactions can also be based solely on physical affinity, which is equally effective in co-localizing two proteins. Examples of physical affinities and chemical bonds include, but are not limited to, forces caused by charge differences, hydrophobicity, hydrogen bonding, van der Waals forces, ionic forces, covalent bonds, and combinations thereof.
  • the proximity between interaction domains, fragments or proteins can be transient or permanent, reversible or irreversible.
  • interactions are exhibited by binding between interacting domains, fragments or proteins. Examples of interactions include specific interactions between antigens and antibodies, ligands and receptors, enzymes and substrates, and the like.
  • protein complex or “polypeptide complex” refers to a composite unit that is a combination of two or more proteins formed by interactions between proteins. Typically, two or more proteins are joined together by specific non-covalent binding affinity to form a "protein complex.”
  • Homozygoin complex refers to a polymer formed by polymerization (or “homogenization polymerization") of the same protein component.
  • Hybrid protein complex refers to a polymer formed by polymerization (or “heterologous polymerization”) of two or more different protein components.
  • isolated protein complex refers to a protein complex in a form different from that found in nature or found in the original cell or biological environment.
  • the protein complex is substantially free of components that normally accompany or react with the protein complex in a naturally occurring environment, or that the protein complex has been removed from the component.
  • An "isolated protein complex” can also be a protein complex not found in nature.
  • nucleic acid molecule is such that it is substantially separated from at least one other nucleic acid molecule present in the natural source of the nucleic acid, or when chemically synthesizing said nucleic acid molecule, it is substantially free of at least one chemical precursor Or other chemicals.
  • An "isolated" nucleic acid molecule can also be, for example, a nucleic acid molecule that is substantially free of at least one nucleotide sequence that naturally flank the nucleic acid molecule at the 5' and 3' ends in the source genomic DNA of the nucleic acid.
  • nucleic acid molecule when less than about 30%, 20%, 10% or 5% by dry weight of other nucleic acid molecules or other chemicals are present (also referred to herein as "contaminating nucleic acid molecules" or " When contaminating a chemical"), the nucleic acid molecule is “substantially separated” or “substantially free” from other nucleic acid molecules or other chemicals.
  • Sequence refers to the linear order in which monomers are present in a polymer, for example, the order of the amino acids in the polypeptide or the order of the nucleotides in the polynucleotide.
  • Nucleotide sequence refers to the arrangement of deoxyribonucleotide or ribonucleotide residues in a polymer in either single- or double-stranded form.
  • a nucleic acid sequence can be composed of natural nucleotides comprising: thymine, adenine, cytosine, guanine, and uracil; abbreviated as T, A, C, G, and U, respectively, and/or the natural A synthetic analog of a nucleotide.
  • Recombinant DNA construct refers to a combination of nucleic acid fragments that are not normally found together in nature.
  • recombinant DNA constructs may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences (such as promoters, etc.) and coding sequences that are derived from the same source but are arranged in a manner different from that normally found in nature.
  • the invention provides a method of preparing a protein complex of the invention.
  • the protein complex of the present invention can be prepared by a variety of methods.
  • the protein complex can be isolated directly from an animal tissue sample containing the protein complex, such as a human tissue sample.
  • the protein complex can also be isolated from a host cell that recombinantly expresses a member of the protein complex.
  • the protein complex can be constructed in vitro by combining individual members of the protein complex.
  • nucleic acids encoding MagR and Cry can be introduced into a suitable host cell.
  • the nucleic acid preferably in the form of DNA, is integrated into an expression vector to direct production of interacting protein members upon introduction into the host cell.
  • vectors can be used in the present invention.
  • an expression vector includes an expression cassette having a promoter operably linked to DNA encoding a member of the interacting protein.
  • the promoter may be a native promoter, a promoter found in naturally occurring cells responsible for expression of interacting protein members in the cell.
  • the expression cassette may be a chimeric expression cassette, i.e., a heterologous promoter having a native promoter that is not responsible for the expression of interacting protein members in naturally occurring cells.
  • the expression vector can also include a DNA origin of replication for replicating the vector in the host cell.
  • the expression vector comprises an origin of replication for amplifying the vector in, for example, E. coli, and a selection marker for selecting and maintaining only those host cells carrying the expression vector.
  • the expression cassette preferably also contains an inducible element whose function is to control transcription from DNA encoding members of the interacting protein.
  • Other regulatory sequences such as transcriptional enhancer sequences and translational regulatory sequences (eg, SD sequences), can also be operably included in the expression cassette.
  • Termination sequences e.g., from bovine growth hormone, SV40, and lacZ
  • the DNA fragments encoding the interacting protein members can be integrated into a single vector or into a different vector.
  • the expression vector can be introduced into a host cell by any technique known in the art, for example, by direct DNA transformation, microinjection, electroporation, viral infection, lipofection, gene gun, and the like.
  • the expression vector can be integrated into the chromosome of the host cell by conventional techniques such as selection of stable cell lines or site-specific recombination.
  • Vector constructs can be designed to be suitable for expression in a variety of host cells including, but not limited to, bacteria, yeast cells, plant cells, insect cells, and mammalian and human cells.
  • mammalian cells can be used as host cells for expressing fusion proteins and detecting protein-protein interactions.
  • virtually any mammalian cell can be used, including normal tissue cells, stable cell lines, and transformed tumor cells.
  • mammalian cell lines such as CHO cells, Jurkat T cells, NIH 3T3 cells, HEK-293 cells, CV-1 cells, COS-1 cells, HeLa cells, VERO cells, MDCK cells, WI38 cells, and the like are used.
  • Mammalian expression vectors are well known in the art and many are commercially available.
  • Suitable promoters for transcription of chimeric genes in mammalian cells include viral transcriptional promoters derived from the following viruses: adenovirus, simian virus 40 (SV40) (eg, early and late promoters of SV40), Rous sarcoma virus (RSV) and cytomegalovirus (CMV) (eg, CMV immediate early promoter), human immunodeficiency virus (HIV) (eg, long terminal repeat (LTR)), vaccinia virus (eg, 7.5K) Promoter) and herpes simplex virus (HSV) (eg, thymidine kinase promoter). Inducible promoters can also be used.
  • adenovirus eg, early and late promoters of SV40
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • HSV herpes simplex virus
  • Inducible promoters can also be used.
  • Suitable inducible promoters include, for example, the tetracycline response element (TRE) (see Gossen et al., Proc. Natl. Acad. Sci. USA, 89: 5547-5551 (1992)), metallothionein IIA promoter, ecdysone-responsive promoter and heat shock promoter.
  • TRE tetracycline response element
  • metallothionein IIA promoter metallothionein IIA promoter
  • ecdysone-responsive promoter ecdysone-responsive promoter
  • heat shock promoter ecdysone-responsive promoter
  • origins of replication for replication and maintenance of expression vectors in mammalian cells include, for example, the Epstein Barr origin of replication in the presence of Epstein Barr nuclear antigen (see Sugden et al, Mole. Cell.
  • SV40 origin of replication in the presence of the SV40 T antigen (which is present in COS-1 and COS-7 cells) (see Margolskee et al, Mole. Cell. Biol., 8: 2837 (1988) ).
  • Suitable selection markers include, but are not limited to, genes that confer resistance to neomycin, hygromycin, zeocin, and the like.
  • a number of commercially available mammalian expression vectors can be used in the present invention, including, for example, pCEP4, pcDNAI, pIND, pSecTag2, pVAX1, pcDNA3.1, and pBI-EGFP and pDisplay.
  • the vector can be introduced into mammalian cells using any known technique, such as calcium phosphate precipitation, lipofection, electroporation, and the like.
  • the bait vector and the prey vector can be co-transformed into the same cell, or two different cells can be introduced and subsequently fused together by cell fusion or other suitable technique.
  • a viral expression vector that allows the introduction of a recombinant gene into a cell by viral infection can also be used to express a fusion protein.
  • Viral expression vectors generally known in the art include viral vectors based on adenovirus, bovine papilloma virus, murine stem cell virus, and retrovirus.
  • Homologues and fragments of naturally interacting protein members can also be readily expressed using the recombinant methods described above.
  • a DNA fragment that is integrated into an expression vector can be selected such that it encodes only that protein fragment.
  • specific hybrid proteins can be expressed using recombinant DNA encoding a hybrid protein.
  • a homolog protein can be expressed from a DNA sequence encoding the homolog protein.
  • a DNA sequence encoding a homologous protein can be obtained by operating a sequence encoding a native protein using recombinant DNA technology. To this end, random or site-directed mutagenesis can be performed using techniques generally known in the art.
  • derivatives of the proteins of the invention are prepared by chemically linking certain moieties for modifying the protein to the amino acid side chains of the native protein.
  • the homologs and derivatives so produced can be tested to determine if they can interact with their intended ligand to form a protein complex. Testing can be performed, for example, by a yeast two-hybrid system or other methods known in the art for detecting protein-protein interactions.
  • the expressed protein can be purified by conventional biochemical and immunochemical methods well known to those skilled in the art.
  • Transfected eukaryotic or biological tissue samples can be homogenized and fractionated under suitable conditions for separation of different cellular components. Typically, cell lysates are run on a sucrose gradient (or other material that separates cellular components based on size and density).
  • the presence of the protein of interest is analyzed with a suitable antibody using methods well known to those skilled in the art, such as immunoblotting or immunoprecipitation.
  • the purified protein is then used for affinity chromatography: an extract from a cultured cell or homogenized tissue sample is loaded onto a column in a suitable buffer, and the protein or protein complex of interest can be combined with the components on the column. Binding; eluting non-binding proteins; then eluting the bound protein or protein complex using various methods, such as pH gradient or salt concentration gradient; finally, the eluted protein can be separated by two-dimensional gel electrophoresis, or by micro Sequencing was performed for identification. All of these methods are well known to those skilled in the art.
  • Purified target proteins or complexes can also be used to prepare antibodies in rabbits, mice, rats, chickens, goats, sheep, pigs, guinea pigs, cattle and horses. Methods for antibody production and characterization are well known to those skilled in the art.
  • Figure 1 Polyacrylamide gel electrophoresis analysis of the home pigeon MagR protein.
  • Figure 2 Polyacrylamide gel electrophoresis analysis of MagR-Cry protein complexes of domestic pigeons and Monarch butterfly.
  • Figure 3 A full-wavelength scan of a pigeon MagR protein sample.
  • Figure 4 Electron microscopic observation of the homozygous complex or hybrid protein complex formed by the pigeon MagR.
  • A cross-sectional structure of the homozygous complex formed by the pigeon MagR;
  • B longitudinal cross-section structure of the homozygous complex formed by the pigeon MagR;
  • C cross-sectional structure of the hybrid protein MagR-Cry4 hybrid protein complex ;
  • D Longitudinal section structure of the hybrid MagR-Cry4 hybrid protein complex.
  • Figure 5 Structure of the pigeon MagR homozygous complex.
  • A Electron microscopic morphology and structure of the longitudinal cross section of the pigeon MagR homozygous protein complex;
  • B Electron microscopic morphology and structure of the cross section of the pigeon MagR homozygous protein complex.
  • Figure 6 Structure diagram of the hybrid protein complex formed by the pigeons MagR and Cry. A: longitudinal section of the hybrid protein complex; B: cross section of the hybrid protein complex.
  • Figure 7 Electron microscopic observation of the crystal of the MagR homozygous complex of the pigeon. A: Electron micrograph; B: Statistical results.
  • Figure 8 Experimental and electrophoretic results of separation and purification of the pigeon MagR-Cry4 hybrid protein complex from cell lysate with iron powder.
  • Figure 9 Experimental results of simultaneous rotation of a family of MagR homozygous complex crystals in a rotating magnetic field.
  • Figure 10 Polyacrylamide gel electrophoresis analysis of Monrovia variegata MagR protein.
  • Figure 11 Results of simultaneous rotation experiments of crystals of the Monrovia Monroe MagR-Cry1 homozygous complex in a rotating magnetic field.
  • Figure 12 A full-wavelength scan of a domestic MagR mutant protein sample.
  • Figure 13 Sequence alignment of the MagR proteins of each species.
  • Figure 14 Negative control of room temperature magnetic measurement of the pigeon MagR-Cry4 hybrid protein complex in solution.
  • Figure 15 SQUID magnetic measurements of the pigeon MagR-Cry4 hybrid protein complex in solution at room temperature.
  • the reagents and instruments used are all commonly used reagents and instruments in the art, and can be purchased from chemical or biological products/preparation companies; the methods used in the following examples, such as PCR, affinity purification Molecular sieve gel chromatography, ion column exchange, etc. are all conventional methods in the art, and those skilled in the art can know these experiments without doubt according to the prior art such as "Molecular Cloning Experiment Guide” or according to the operation manual provided by the manufacturer. The operation process and get the corresponding results.
  • the homologous chicken MagR protein sequence (SEQ ID NO: 9) was used to search for homologous sequences in the pigeon genome, and the found homologous segments were extracted in the software sequencher and the chicken's MagR coding region sequence. The comparison revealed that there were a total of four exons. Among them, the sequences of exons 2, 3, and 4 have a good match with the chicken MagR sequence, but the exon 1 sequence of the MagR gene in the pigeon genome sequence is deleted. In order to obtain the complete sequence of the pigeon MagR, the following primers were designed according to the sequence of the exon 1 of the chicken MagR. And amplify the exon from the domestic pigeon genomic DNA:
  • Exon 1 primer F: ACGGACCGCTACCCAATAG (forward primer) (SEQ ID NO: 34), R: ACACTTTTTCAGCGGCTGTG (reverse primer) (SEQ ID NO: 35).
  • the MagR sequence was then amplified in the genomic DNA of the domestic pigeon to verify that the sequence was correct.
  • the 2-9th amino acid WSHPQFEK (SEQ ID NO: 26) is a Strep II affinity tag sequence; the 10-14th amino acid GGSTS (SEQ ID NO: 25) is a linker sequence; the underlined sequence is a domestic pigeon The protein sequence of MagR of (Columba livia), ie the amino acid sequence as shown in SEQ ID NO: 1.
  • positions 1-6 are NdeI cleavage sites, 7-30 positions are StrepII expression sequences, 31-45 positions are GGSTS (SEQ ID NO: 25) expression sequences, and 46-441 are domestic pigeons.
  • the MagR expression sequence, positions 442-444 are stop codons, and positions 445-450 are BamHI cleavage sites.
  • the gene was synthesized at Kingsray Biotechnology Co., Ltd.
  • the above plasmid containing the insert having the amino acid sequence encoding the pigeon MagR protein and having the Strep II affinity tag at the N-terminus was transformed into E. coli BL21 (DE3) (commercially available). Monoclonal inoculation was carried out into LB liquid medium to expand the culture, and the expression of the target protein was induced with 20 ⁇ M IPTG at 15 ° C, and collected after 20-24 hours.
  • the transformant expressing the MagR protein obtained above was resuspended in a buffer (20 mM Tris, 150 mM NaCl, pH 8.0, 10 mM mercaptoethanol and protease inhibitor), and then lysed by a sonicator (working for 4 s, pause for 8 s). , 99 cycles), after completion, high-speed centrifugation (17,000 rpm), and the supernatant was taken for affinity purification. After affinity purification by a Strep-Tactin affinity column (IBA), it was further purified by an anion exchange column (GE Healthcareg) or molecular sieve gel chromatography (GE Healthcareg) to obtain a high purity MagR protein. The entire purification process was carried out at 4 °C. The MagR protein obtained after purification showed a pronounced brownish yellow or tan color at a higher concentration, suggesting that the protein incorporates divalent or trivalent iron.
  • a buffer (20 mM Tris, 150 mM NaCl, pH
  • the collected MagR protein samples were subjected to denaturing polyacrylamide gel electrophoresis (SDS-PAGE) analysis.
  • SDS-PAGE denaturing polyacrylamide gel electrophoresis
  • the experimental results are shown in Figure 1.
  • the samples in each lane are: 1. Protein molecular weight standard; 2.
  • the purified MagR protein containing Strep II affinity tag has a molecular weight of about 15KDa.
  • Example 3 The homologous complex of dimers and multimers formed by the pigeon MagR protein
  • the housed MagR protein expressed and purified in Example 2 was further subjected to molecular sieve gel chromatography using a Superdex 200 10/300 GL purification column (GE Healthcare) after affinity purification by a Strep-Tactin affinity column (buffer: 20 mM Tris, 150 mM NaCl, pH 8.0, 10 mM mercaptoethanol), ie gel-filtration or Size-exclusion chromatography.
  • the molecular sieve gel chromatography of the sample showed a monomer peak, a dimer peak and a multimer peak, and samples of three peaks were respectively collected to obtain a purified protein. This shows that the purified MagR protein exists in three forms in molecular sieve gel chromatography.
  • the three peaks (elution volume: about 17.5 ml, 16.2 ml, 9.5 ml) have approximate molecular weights of 15 KDa, 30 KDa and greater than 200 KDa, which are related to the monomer, dimer and MagR protein.
  • the molecular weight of the polymer is consistent. Since the resolution of Superdex S200 is up to about 200 KDa, it is not possible to judge the molecular weight of the polymer and the number of monomers contained in the polymer.
  • Example 4 Expression and purification of hybrid protein complexes formed by the pigeon MagR protein and the domestic Cry4 protein
  • the 2-11th amino acid is a His-affinity tag sequence of 10 histidine; the 12-16th amino acid GGSTS (SEQ ID NO: 25) is a linker sequence; the underlined sequence is a Cry4 of a domestic pigeon.
  • Amino acid sequence ie, the amino acid sequence set forth in SEQ ID NO: 15.
  • positions 1-6 are NdeI cleavage sites
  • 7-36 positions are 10 ⁇ His expression sequences
  • positions 37-51 are GGSTS (SEQ ID NO: 25) expression sequences
  • positions 52-1542 are The domestic pigeon Cry4 expresses the sequence
  • the 1543-1545 position is the stop codon
  • the 1546-1551 position is the XhoI restriction site.
  • the gene was synthesized at Bomme Biotech Co., Ltd.
  • the plasmid having the gene encoding the domestic Cry4 protein and fused with the histidine tag (His-tag) is co-produced with the gene encoding the gene encoding the MagR protein of the pigeon and obtained by the StrepII-tagged plasmid obtained in the second step of the second embodiment. Transformation of E. coli BL21 (DE3).
  • the plasmid expressing MagR is kanamycin resistant, and the plasmid expressing Cry4 is ampicillin. Penicillin resistance, picking up E.
  • the sample of the collected pigeon MagR-Cry4 hybrid protein complex was subjected to denaturing polyacrylamide gel electrophoresis (SDS-PAGE) analysis.
  • the experimental results are shown in Fig. 2, in which the sample loaded from the left to the right lane 4 is the protein molecular weight standard, and the sample loaded in the fifth lane is the pigeon MagR-Cry4 hybrid protein complex.
  • RESULTS Due to the use of denaturing electrophoresis analysis, two protein samples were shown in lane 5, namely the MagR protein (molecular weight of approximately 15 kDa) containing the Strep II affinity tag and the Cry4 protein (His molecular weight of approximately 70 KDa) containing the His tag. ).
  • Example 5 Proof experiment of iron pigeon protein and MagR homozygous protein complex containing iron
  • a monomer peak sample of the pigeon MagR protein obtained in Example 3 was subjected to full-wavelength scanning using a spectrophotometer (Nanodrop 2000).
  • the results show that there are absorption peaks at the characteristic absorption sites (320 nm and 410 nm) of the Fe element.
  • the first absorption peak is the protein absorption peak of UV280.
  • the dimer and polymer samples formed by the MagR protein obtained in Example 3 were subjected to full-wavelength scanning using a spectrophotometer (Nanodrop 2000), and the results were similar to those of FIG.
  • Example 6 Electron microscopic image and structure of the pigeon MagR homozygous complex and the pigeon MagR-Cry4 hybrid protein complex
  • Protein samples of protein complexes were negatively stained with uranyl acetate or uranyl formate to prepare negatively stained electron microscopy samples of the protein.
  • the sample was placed under a transmission electron microscope and photographed, and its structure was analyzed according to a standard electron microscope structural analysis method, and the size (including protein length and width) was measured.
  • the electron microscopy structure analysis method can be referred to Ohi, M., et al. (2004), "Negative Staining and Image Classification-Powerful Tools in Modern Electron Microscopy.” Biol Proced Online 6: 23-34.
  • a and B are the electron microscope structures of the homozygous protein complex formed by the pigeon MagR, wherein A is a cross-sectional structure and B is a longitudinal cross-sectional structure.
  • C and D are electron microscope structures of the hybrid MagR-Cry4 hybrid protein complex, in which C is a cross-sectional structure and D is a longitudinal cross-sectional structure. Schematic diagrams of the organization and relative position of the MagR and/or Cry proteins in the complex are shown on the right and are labeled for measurement under electron microscopy. Size (in nanometers).
  • the pigeon MagR homozygous complex exhibits a long rod-like structure with a length of about 20-24 nanometers and a width of about 7-11 nanometers.
  • “Width” refers to the diameter measured when the cross-section of the rod-like structure is circular or circular-like under an electron microscope.
  • the results indicate that the complex has about 20 MagR proteins.
  • the domesticated MagR-Cry4 hybrid protein complex exhibits an elongated rod-like structure with a length of about 20-24 nanometers and a width of about 13-18 nanometers. Based on the homologous crystal structure and molecular model calculations, the results indicate that the multimer has about 20 MagR proteins and 10 Cry4 proteins.
  • FIG. 5 is a structural diagram of the pigeon MagR homozygous protein complex.
  • A shows the electron microscopic morphology and structure of the longitudinal section of the composite
  • B shows the electron microscopic morphology and structure of the cross section of the composite.
  • Each MagR homozygous protein complex comprises about 20 MagR proteins, which are ordered to form a linear double helix polymer, in which every 10 MagR protein monomers are connected end to end in a linear polymerization form to form two long chains of protein, two The long chains of bar proteins bind in a helical form, thereby forming a protein complex of elongated rod-like structures.
  • the dashed box in Figure 5 shows the structure of a MagR tetramer: the MagR homozygous complex rod-like structure as shown is composed of five such tetramers.
  • each MagR protein includes a 2Fe2S iron-sulfur center, that is, each MagR protein includes an iron binding site that combines two iron atoms.
  • the entire rod-like homozygous complex contains about 20 iron binding sites, combining about 40 iron atoms.
  • the plurality of iron atoms are located in an inner near central axis region of the protein complex of the rod-like structure.
  • the magnetic induction receptor protein monomer binds to the iron atom at the same or similar position, and is in the steric structure of the protein complex of the rod-like structure, thereby, in the protein complex of the linear polymerized rod-like structure of the magnetic induction receptor protein monomer, Multiple iron atoms also form a regular arrangement in the protein complex.
  • Figure 6 is a structural diagram of the hybrid MagR-Cry4 hybrid protein complex.
  • A shows the electron microscopic morphology (upper left), the tissue form (bottom left) and the structural schematic (right) of the longitudinal section of the hybrid protein complex
  • B shows the electron microscopic morphology of the cross section of the hybrid protein complex (top left), Organizational form (bottom left) and structure diagram (right).
  • the MagR-Cry4 hybrid protein complex about 10 photosensitive Cry4 proteins are entangled in the periphery of a rod-shaped linear double-helical polymer formed of a homozygous protein complex composed of about 20 MagRs, thereby forming a hybrid. Complex.
  • the entire rod-shaped hybrid protein complex contains about 20 iron binding sites, combining about 40 iron atoms.
  • the plurality of iron atoms are located in the inner near-axis region of the protein complex of the rod-like structure, and are also regularly arranged.
  • Example 7 Crystallization of the pigeon MagR protein complex
  • the protein sample is concentrated and then crystallized.
  • the protein crystallization method is carried out by a hanging drop method according to a standard experimental procedure as described in the textbook McPherson, A. (2009). Introduction to macromolecular crystallography. Hoboken, N.J., Wiley-Blackwell.
  • the protein solution is mixed with an equal volume of precipitant solution on a siliconized microscope cover slip to form a droplet; the coverslip is inverted over the cavity of the plate, and the desired precipitant solution is placed in the cavity, in the cover glass Before the sheet is placed, oil or grease is applied to the edges of the cavity to seal the cavity. Crystals formed by the MagR protein complex can be observed after about 2-7 days.
  • Figure 9 shows a crystal photograph of a homozygous complex formed by the MagR protein of the domestic pigeon of the present invention.
  • the first type is a black opaque cuboid-shaped crystal (see the crystal photograph given in the upper row in Figure 9; the crystallization conditions used are: 0.1 M HEPES, pH 7.5, 3.0 M sodium chloride), and the length is about 0.5 mm, showing that the iron element has been oxidized to a uniform trivalent in the crystal.
  • the second crystal is a pale yellow or yellow transparent crystal (see the crystal photograph given in the lower row of Fig. 9; the crystallization conditions used are: 0.1 M BIS-TRIS, pH 6.5, 3.0 M sodium chloride), shape Irregular or in the form of small pieces, indicating that iron may exist as a divalent. It was confirmed in the experiments of the following examples that both crystals exhibited very strong magnetic properties.
  • the morphology of the crystal of the hybrid complex formed by the MagR protein of the present invention and the Cry4 protein is similar to that of the crystal shown in the photograph of Fig. 9.
  • Example 8 Magnetic test of the pigeon MagR protein complex
  • Example 6 In the electron microscopy analysis sample described in Example 6, the protein sample of the pigeon MagR homozygous protein complex obtained by molecular sieve gel chromatography in Example 3 and the molecular sieve gel chromatography of Example 4 were used.
  • a negative dyed electron microscope sample was prepared from the protein sample of the purified MagR-Cry4 hybrid protein complex.
  • the electron magnetic field direction or the artificial magnetic field direction was marked on the electron microscope copper mesh sample.
  • the direction of all the stick-shaped protein complex particles is counted (the protein is a long bar-shaped shape whose direction refers to the longitudinal direction of the protein, ie, the axial direction of the long axis), according to the three shown in FIG.
  • Directional statistics (parallel to the direction of the Earth or the applied magnetic field, perpendicular to the Earth or the direction of the applied magnetic field, and the two directions cannot be attributed to the above two directions), calculate the proportion of the clasp protein complex in all directions.
  • Figure 7 shows the orientation of the MagR homozygous complex under magnetic field using an electron microscope. It shows that the MagR protein complex has its own magnetic properties and achieves a similar "protein compass" effect.
  • Figure 7A is an electron micrograph showing a protein complex particle parallel to the direction of the magnetic field (i.e., the same or a difference of 30 degrees or less from the direction of the magnetic field) with a dark colored square indicating a direction perpendicular to the magnetic field (ie, Protein complex particles that are the same in direction perpendicular to the magnetic field or differ by less than or equal to 30 degrees, and the circles show other directions between the two.
  • B of Fig. 7 shows the results of counting the number of particles in a large sample in the arrangement direction.
  • each experiment included approximately 1500 protein complex particles for statistical analysis, and the experiment was repeated three times, taking the mean and standard deviation plots.
  • the abscissa is the intensity of the magnetic field, in mT, and the earth's magnetic field is 0.04mT (based on the geographical position of Beijing).
  • the applied magnetic field used in the test is obtained by permanent magnets, and the magnetic field strength is measured by a magnetometer. The measurement is 1 mT.
  • the ordinate is a MagR protein complex arranged in all three proteins in three different directions (parallel to the Earth's magnetic field or the direction of the applied magnetic field, perpendicular to the Earth's magnetic field or the direction of the applied magnetic field, in other directions between the two) The proportion of the composite.
  • the pigeon MagR homozygous protein complex and the homegrown MagR-Cry4 hybrid protein complex can be rapidly separated and purified from the cell lysate by iron powder, indicating that they are all magnetic.
  • MagR-Cry4 hybrid protein complex Take the domesticated MagR-Cry4 hybrid protein complex as an example.
  • the specific method is shown in Figure 8.
  • the E. coli cells co-expressing MagR and Cry4 proteins in Example 4 were collected and buffered (20 mM Tris, 150 mM NaCl, pH 7). .5) Resuspend, sonicate the supernatant after ultrasonication, add commercially available nano-iron powder particles (Beaver Biotechnology Co., Ltd., #70301), incubate for 10 min at room temperature, and use standard TBS buffer (20 mM Tris, 150 mM).
  • the iron powder particles were washed with NaCl, pH 7.5, and then the iron powder was eluted with a protein electrophoresis loading buffer, and the eluate was subjected to denaturing polyacrylamide gel electrophoresis (SDS-PAGE) to analyze the protein component.
  • SDS-PAGE denaturing polyacrylamide gel electrophoresis
  • a sample of Escherichia coli cells co-expressing MagR and Cry4 protein in Example 4 was subjected to denaturing polyacrylamide gel electrophoresis by the above method, and showed a molecular weight of about 15 KDa.
  • Example 2 a sample of the E. coli cell expressing the pigeon MagR in Example 2 was subjected to denaturing polyacrylamide gel electrophoresis by the above method to exhibit a band having a molecular weight of about 15 kDa (MagR).
  • FIG. 9 is a graph showing the results of simultaneous rotation experiments of a family of MagR homozygous complex crystals in a rotating magnetic field. As shown in Fig. 9, when the magnetic field (the direction of the pointer in the circle is the direction of the magnetic field) is rotated, it can be observed that the protein crystal rotates almost completely synchronously along the magnetic field, like a compass composed of a magnet.
  • the protein crystal When a magnet is used to approach a protein crystal, the protein crystal will immediately approach. When the magnetic pole of the magnet is reversed, the protein crystal will immediately move away from and reverse the direction, and then close again, showing that the crystal formed by the protein is like a magnet, with its own magnetic and magnetic poles. .
  • a ferromagnetic substance such as an iron rod
  • a droplet of the crystal growth which contains a protein crystal
  • the protein crystal will rapidly approach the ferromagnetic substance, and even fly out of the droplet to approach the iron substance. It shows that protein crystals have very strong magnetic properties.
  • the magnetic measurement of the sample of the purified domestic pigeon MagR-Cry4 hybrid protein complex obtained in Example 4 was carried out in a solution state, and was reproducible. Very accurate magnetic measurement results.
  • the instrument used is a Superconducting Quantum Interference Device (SQUID), model MPMS-XL1.
  • Figure 14 is a negative comparison of the room temperature magnetic measurement of the pigeon MagR-Cry4 hybrid protein complex in solution, where a is the SQUID magnetic measurement of the sample tube (Holder) with the hybrid MagR-Cry4 hybrid protein complex. As a result, no hysteresis loop was shown; b is the SQUID magnetic measurement of the sample tube of the protein-loaded solution (ie, buffer) in the SQUID experiment, showing no hysteresis loop; c is the solution control, ie, the protein storage solution The SQUID magnetic measurement of the buffer (ie, buffer) shows no hysteresis loop. The container containing the protein or buffer used in the experiment was proved, and the buffer was free from magnetic contamination. The abscissa is the magnetic field strength and the ordinate is the magnetic moment.
  • a is the SQUID magnetic measurement of the hybrid MagR-Cry4 hybrid protein complex in solution at room temperature, showing a significant hysteresis loop;
  • b is the same housekeeping pigeon MagR-Cry4 hybrid protein complex After 40 hours of measurement, the hysteresis loop disappeared;
  • the left side of c is the SDS-PAGE result of the protein sample before measurement, the arrow indicates the position of Cry4 (top) and MagR protein (bottom), and the right side is 40 hours later.
  • SDS-PAGE analysis of the same tube sample showed that the Cry4 protein had been degraded into several small molecular weight bands, and MagR had completely degraded and disappeared.
  • the hybrid MagR-Cry4 hybrid protein complex has a significant hysteresis loop at room temperature in solution; after the protein degradation, the hysteresis loop disappears, which proves that the magnetic property measured by the SQUID experiment comes from the protein itself. Not a container (such as a sample tube), nor a solution (buffer).
  • the MagR protein sequence was then amplified in the genomic DNA of Monrovia, and the above sequence was verified to be correct.
  • the 2-9th amino acid WSHPQFEK (SEQ ID NO: 26) is a Strep II affinity tag sequence; the 10-14th amino acid GGSTS (SEQ ID NO: 25) is a linker sequence; the underlined sequence is a black vein
  • the protein sequence of MagR of the golden butterfly or the American monarch butterfly, Latin name Danaus plexippus, hereinafter also simply referred to as "butterfly", that is, the amino acid sequence shown in SEQ ID NO: 2.
  • positions 1-6 are NdeI cleavage sites, 7-30 positions are StrepII expression sequences, 31-45 positions are GGSTS (SEQ ID NO: 25) expression sequences, and positions 46-438 are black veins.
  • the Kingdo butterfly MagR expression sequence, 439-441 is the stop codon, and 442-447 is the BamHI restriction site.
  • the gene was synthesized at Bomme Biotech Co., Ltd.
  • the above plasmid containing an insert having an amino acid sequence encoding a butterfly MagR protein and having a Strep II affinity tag at the N-terminus was transformed into Escherichia coli BL21 (DE3) (commercially available). Monoclonal inoculation was carried out into LB liquid medium to expand the culture, and the expression of the target protein was induced with 20 ⁇ M IPTG at 15 ° C, and collected after 20-24 hours.
  • the transformant expressing the MagR protein obtained above was resuspended in a buffer (20 mM Tris, 150 mM NaCl, pH 8.0, 10 mM mercaptoethanol and protease inhibitor), and then lysed by a sonicator, and then centrifuged at high speed. The supernatant was taken for affinity purification. After affinity purification by Strep-Tactin affinity column, further purification is carried out by anion exchange column or molecular sieve gel chromatography to obtain a butterfly MagR protein with higher purity. The entire purification process was carried out at 4 °C.
  • the MagR protein obtained after purification can exhibit a distinct brownish yellow or tan color at a higher concentration, suggesting that the protein incorporates divalent or trivalent iron.
  • the collected MagR protein samples were subjected to denaturing polyacrylamide gel electrophoresis (SDS-PAGE) analysis.
  • SDS-PAGE denaturing polyacrylamide gel electrophoresis
  • the experimental results are shown in Fig. 10, wherein the samples of each lane are: 1. Protein molecular weight standard; 2.
  • the purified butterfly MagR protein containing Strep II affinity tag has a molecular weight of about 15 KDa.
  • Example 11 Experiment of the formation of homozygous complexes of Monrovia variegata MagR protein
  • Example 10 The butterfly MagR protein expressed and purified in Example 10 was subjected to molecular sieve gel chromatography using a Superdex 200 10/300 GL purification column (GE Healthcare) after affinity purification by a Strep-Tactin affinity column (experimental procedure reference) Example 3).
  • the molecular sieve gel chromatography of the sample showed a monomer peak, a dimer peak and a poly peak, and samples of three peaks were respectively collected to obtain a purified protein.
  • Example 12 Expression and purification of hybrid protein complexes formed by Monrovia variabilis MagR protein and Cry1 protein
  • the 2-11th amino acid is a 10 histidine His affinity tag sequence
  • the 12th-16th amino acid GGSTS (SEQ ID NO: 25) is a linker sequence
  • the underlined sequence is a butterfly Cry1 amino acid The sequence, ie the amino acid sequence set forth in SEQ ID NO: 16.
  • positions 1-6 are NdeI restriction sites
  • 7-36 are 10xHis expression sequences
  • positions 37-51 are GGSTS (SEQ ID NO: 25) expression sequences
  • 52-1653 are black veins.
  • the Cry1 expression sequence of the Kingdoptera, 1654-1656 is the stop codon
  • 1657-1662 is the BamHI restriction site.
  • the gene was synthesized at Bomme Biotech Co., Ltd.
  • the plasmid having the coding gene of the butterfly Cry1 protein and fused with the histidine tag (His-tag) was co-transformed into the large intestine with the plasmid encoding the butterfly MagR protein obtained in Step 2 of Example 10 and fused with the StrepII tag.
  • Bacillus BL21 (DE3) the plasmid expressing MagR is kanamycin resistant, the plasmid expressing Cry1 is ampicillin resistance, and two plasmids are picked in the double-antibody medium (kanamycin and ampicillin).
  • E. coli was inoculated into LB liquid medium to expand the culture, and the expression of the target protein was induced with 20 ⁇ M of IPTG at 15 ° C, and collected after 20-24 hours.
  • the above-obtained transformed bacteria co-expressing the butterfly MagR protein and the Cry1 protein were resuspended in a buffer (20 mM Tris, 150 mM NaCl, 10 mM imidazole, pH 8.0, 10 mM mercaptoethanol and protease inhibitor), and then subjected to a sonicator. After cleavage, high speed centrifugation is carried out, and the supernatant is taken for affinity purification. After affinity purification by Ni-NTA affinity column, it was purified by Strep-Tactin affinity column to obtain MagR-Cry1 hybrid protein complex formed by butterfly MagR protein and Cry1 protein.
  • the entire purification process was carried out at 4 ° C in the presence of an applied magnetic field.
  • the applied magnetic field is applied by: when the sample passes the affinity column, two identical strip magnets are respectively attached to the two sides of the affinity column, and the south pole and the north pole of the two identical strip magnets are respectively opposite to each other.
  • a uniformly stable magnetic field The results show that the applied magnetic field is applied during purification (the intensity of the applied magnetic field is 100 times that of the earth's magnetic field), and the yield of the purified protein is significantly increased; when the purified protein is eluted, the applied magnetic field is removed, and the protein elution efficiency is higher.
  • the collected samples of the butterfly MagR-Cry1 protein complex were subjected to denaturing polyacrylamide gel electrophoresis (SDS-PAGE) analysis.
  • the experimental results are shown in Fig. 2, in which the sample loaded from the left to the right lane 2 is the protein molecular weight standard, and the sample loaded in the third lane is the butterfly MagR-Cry1 hybrid protein complex.
  • Example 13 Proof of the Monarch butterfly MagR protein and the MagR homozygous protein complex containing iron Test
  • a monomer peak sample of the butterfly MagR protein obtained in Example 11 was subjected to full-wavelength scanning using a spectrophotometer (Nanodrop 2000). The result is similar to Figure 3.
  • a dimer or polymer sample formed of the butterfly MagR protein obtained in Example 11 was subjected to full-wavelength scanning using a spectrophotometer (Nanodrop 2000). The result is similar to Figure 3.
  • Example 14 Electron microscopic image and structure of Monrovia variabilis MagR homozygous protein complex and Monzona diamond MagR-Cry1 hybrid protein complex
  • the sample was negatively stained with uranyl acetate or uranyl formate, and the negative electron microscopy samples of the protein were prepared.
  • the sample was placed under a transmission electron microscope and photographed.
  • the structure was analyzed according to the standard electron microscope structure analysis method, and the size (including protein length and width) was measured. .
  • the results observed by electron microscopy showed that the butterfly MagR homozygous protein complex exhibited a long rod-shaped structure with a length of about 20-24 nm and a width of about 7-11 nm. Based on the homologous crystal structure and molecular model calculations, the results indicate that the complex has about 20 MagR proteins.
  • the results observed by electron microscopy showed that the butterfly MagR-Cry1 hybrid protein complex exhibited a rod shape with a length of about 20-24 nm and a width of about 13-18 nm wide. Based on the homologous crystal structure and molecular model calculations, the results indicate that the complex has about 20 MagR proteins and 10 Cry1 proteins.
  • each MagR homozygous complex comprises about 20 MagR proteins, which are ordered to form a linear double-helical polymer, in which every 10 MagR protein monomers are connected end to end in a linear polymerization form to form two
  • the protein has a long chain, and the long chains of the two proteins are combined in a spiral form, thereby forming a protein complex of a long rod-like structure.
  • Electron microscopy results also showed that in the butterfly MagR-Cry1 hybrid protein complex, about 10 photosensitive Cry1 protein windings were arranged on the periphery of a rod-shaped linear double-helical polymer formed of a protein complex composed of about 20 MagR. Thereby forming a hybrid protein complex.
  • the sample was concentrated and then crystallized.
  • the protein crystallization method is carried out by a hanging drop method according to a standard experimental procedure as described in the textbook McPherson, A. (2009). Introduction to macromolecular crystallography. Hoboken, N.J., Wiley-Blackwell.
  • the crystal form of the butterfly MagR homozygous complex and the crystal form of the butterfly MagR-Cry1 hybrid protein complex are similar to those of the crystal of the corresponding pigeon MagR homozygous complex or the crystal of the pigeon MagR-Cry4 hybrid protein complex.
  • Two types of crystals were prepared and observed separately: the first one is a black opaque cuboid-shaped crystal, suggesting that the iron element has been oxidized to a uniform trivalent in the crystal; the second crystal is light yellow or yellow transparent Crystals, irregular in shape or in the form of small pieces, suggest that iron may exist as a divalent.
  • Figure 11 shows A crystal photograph of the butterfly MagR-Cry1 hybrid protein complex shows that the crystal has a black opaque cuboid shape and a length of about 0.5 mm.
  • Example 8 The magnetic properties of the homozygous protein complex formed by the butterfly MagR protein and the butterfly MagR-Cry1 hybrid protein complex were tested in the same manner as in Example 8. The results showed that the magnetic test results of the butterfly MagR homozygous complex and the butterfly MagR-Cry1 hybrid protein complex were similar to those of the domestic pigeon (as observed in Example 8). This indicates that the butterfly MagR homozygous protein complex and the butterfly MagR-Cry1 hybrid protein complex are magnetic.
  • Figure 11 is a graph showing the results of simultaneous rotation experiments of a butterfly MagR homozygous complex crystal in a rotating magnetic field.
  • the experimental method is as described in (3) of Example 8.
  • Fig. 11 it can be observed that the crystal of the butterfly MagR-Cry1 homozygous protein complex is black, and synchronous rotation occurs in a rotating magnetic field.
  • the crystal of the butterfly MagR hybrid protein complex also exhibits a synchronous rotation in a rotating magnetic field.
  • the three cysteines C60, C124, C126 in the amino acid sequence of SEQ ID NO: 1 of the domestic pigeon MagR were tested to be mutated to alanine ( A, the ability of the protein obtained after the codon is GCC) to bind iron.
  • the Dove MagR protein and the MagR homozygous complex having the above C60A, C124A, C126A mutations were expressed and purified according to the methods and procedures described in Examples 2 and 3.
  • the mutated pigeon MagR protein undergoes molecular sieve gel chromatography and also exhibits monomer peaks, dimer peaks and polymer peaks. Samples of three peaks were collected separately. The resulting protein samples were subjected to full wavelength scanning using a spectrophotometer (Nanodrop 2000) according to the method described in Example 5.
  • the results show that the absorption curve of the MagR monomer protein formed by the above mutant protein (shown by the solid line) has no absorption peak at the characteristic absorption sites (320 nm and 410 nm) of the Fe element.
  • the absorption curves (shown by dashed lines) of the wild-type domestic pigeon MagR protein and the MagR homozygous complex as a control showed significant absorption peaks at 320 nm and 410 nm.
  • the absorption curve of the dimer or polymer of the pigeon MagR formed by the mutant protein obtained by molecular sieve gel chromatography and purification according to the aforementioned method is not absorbed at the characteristic absorption sites (320 nm and 410 nm) of the Fe element. peak.
  • the domestic pigeon MagR protein has a binding iron domain, which includes one or more of three sites C60, C124 and C126.
  • the obtained amino acid sequences were aligned by Clustal V alignment method.
  • Figure 13 is a result of sequence alignment of the above-described species MagR proteins.
  • the source species names from top to bottom and the serial numbers of the genes in NCBI are: (1) Drosophila melanogaster, gene sequence number NP_573062.1; (2) black vein gold spot Butterfly (Danaus plexippus); (3) Apis mellifera, gene sequence number XP_624993.1; (4) Brachiestoma floridae, gene sequence number XP_002589524.1; (5) zebrafish (Danio rerio) , the gene serial number is NP_001020349.1; (6) python bivittatus, gene serial number is XP_007429307.1; (7) domestic pigeon (Columba livia); (8) grass finch (Taeniopygia guttata), the genetic sequence number is XP_002194930.1; (9) Chicken (Gallus gallus), gene serial number XP_0000
  • the nucleic acid sequence of the magnetic induction receptor protein provided by the present invention exists in the genome of a large number of different species, and the encoded protein sequence is highly conserved and has a plurality of highly conserved domains, such as a domain that binds iron and/or sulfur, Three 100% conserved cysteine residues (C), indicated by the arrows in Figure 13, are positions that bind iron (or iron sulfur center).
  • Embodiments of the invention may employ conventional techniques of biotechnology, organic chemistry, inorganic chemistry, etc., unless otherwise indicated, and it is apparent that the invention may be practiced otherwise than as specifically described in the foregoing description and examples. Other aspects and modifications within the scope of the invention will be apparent to those skilled in the art. Many variations and modifications are possible in accordance with the teachings of the present invention and are therefore within the scope of the invention. All patents, patent applications, and scientific papers referred to herein are hereby incorporated by reference.

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Abstract

本发明提供了一种磁感应受体蛋白或其活性片段或衍生物、所述磁感应受体蛋白的纯合复合物以及磁感应受体蛋白与隐花色素蛋白的杂合复合物。本发明还提供了所述磁感应受体蛋白或其纯合复合物或其与隐花色素蛋白的杂合复合物的制备方法。本发明还提供了所述磁感应受体蛋白或其纯合复合物或其与隐花色素蛋白的杂合复合物的与磁性相关的应用。

Description

磁感应受体蛋白和其复合物及它们的用途 技术领域
本发明涉及生物学和其应用领域。具体地，本发明涉及一种科学上首次研究和制备得到的自身具有磁性的蛋白，即磁感应受体蛋白，以及所述磁感应受体蛋白的纯合蛋白复合物，以及所述磁感应受体蛋白和光感应蛋白形成的杂合蛋白复合物。本发明还提供了所述蛋白或复合物的用途。
背景技术
铁是生命体必需的微量元素，和铁结合、转运相关的蛋白质也得到了较多的关注和研究。铁结合蛋白是含铁的金属结合蛋白，其中有一个亚类称之为铁硫蛋白，其结合的铁是以铁硫中心(Iron-sulfur cluster)的形式存在。铁硫蛋白是非常重要的电子传递的载体，主要牵涉到呼吸链和植物的光合作用等生化反应过程。对铁结合蛋白和铁硫蛋白的研究非常广泛和深入，主要集中在和生化代谢相关的各个方面。生物中最重要的两个铁结合蛋白是乳铁蛋白和转铁蛋白，其中转铁蛋白构成的复合笼状结构，能装载大约3000-5000个铁原子，是生物中重要的储存铁的机制。一些研究也表明，可以利用装载铁原子的转铁蛋白作为纳米颗粒用于生物医学和其它领域的应用。然而，迄今为止，所有已经发现的铁结合蛋白都不具备自身的磁性，即都不是“磁性蛋白”。例如，上述载铁能力极高的转铁蛋白在磁场下能够被磁场吸引，具备顺磁的特性，但也不具备自身的磁性。该蛋白具有类似于铁球在磁场下的反应，但因为不具备自身的磁性，所以不能作为如同用作指南针的磁铁那样的磁性材料使用。
另外，动物的磁感应能力一直是生物学领域中最引人注目的未解之谜。很多年以来，人们已经知道许多动物在进行长距离迁徙时是通过感应地磁场来判断方向和导航的，例如黑脉金斑蝶、家鸽、蜜蜂、龙虾、海龟、鲨鱼、蝙蝠、牛羚羊以及众所周知的候鸟等等。除此之外，还有一些物种利用地磁场来引导自身的朝向和筑巢的走向，例如名为Tritonias diomedea的海洋软体动物、指南白蚁、裸鼹鼠等。然而，人们对动物体感应磁场的分子机理了解甚少。
动物能够通过某种分子机制(例如，通过某种特定的基因或者蛋白质，或者通过某条细胞信号通路)来感应地磁场的这一说法，从无人相信到被学界认可，经历了很长的一段时间。迄今为止，与动物体感应磁场有关的最关键的一个研究是隐花色素蛋白(Cryptochrome，简称为Cry)的发现和研究。2008年,在Gegear,R.J.,Casselman,A.,Waddell,S.& Reppert,S.M.等的Cryptochrome mediates light-dependent magnetosensitivity in Drosophila.Nature 454,1014-1018(2008)中描述了将模式动物果蝇的Cry基因敲除后，果蝇就失去了对磁场的感知能力。这一实验充分说明了动物对磁场的感知和利用磁场导航存在某种分子机制，并且这一过程由特定的基因和蛋白质参与。这表明，动物具有磁感应能力不是伪科学，而是由特定基因决定的，并证明Cry蛋白是动物磁感应信号通路中非常关键的一个环节。但是，该发现并不能确认Cry蛋白是直接感应磁场的受体蛋白。事实上，其后有很多该领域的相关文献也进一步说明了这一点，如Gould,J.L.的Magnetoreception.Curr Biol 20,R431-435 (2010)，其指出当前的几种假说都有缺陷，尤其是Cry作为磁感应蛋白的假说并不能解释蛋白质如何识别磁场的磁极，而Cry蛋白质在磁场下的反应并没有任何直接的实验证据支持。值得注意的是，Cry蛋白并不是一种含铁的蛋白质，因此Cry蛋白不能提供其具有感磁特性的让人信服的机理。
本领域尚未发现本身具有磁性和/或光磁性的蛋白或蛋白复合物。本领域亦未得到过本身具有磁性和/或光磁性的蛋白或蛋白复合物以及将其应用于人类生产生活中。
发明概述
本发明涉及一种在现代生物学研究中新研究和制备得到的自身具有磁性和/或能够形成具有磁性之复合物的蛋白，即磁感应受体蛋白，以及所述磁感应受体蛋白的纯合蛋白复合物，以及所述磁感应受体蛋白和光感应蛋白形成的杂合蛋白复合物。本发明还提供了所述磁感应受体蛋白或复合物的用途。
本发明提供了一种分离的磁感应受体蛋白。磁感应受体蛋白(或称“Magnetoreceptor”，或称“MagR”)是本申请发明人在现代生物学研究中首个研究、鉴定并命名的一种其核酸编码序列在生物体基因组中广泛存在并高度保守，可形成具有生物磁性的蛋白单体或纯合复合物或杂合复合物的蛋白质。本发明提供的磁感应受体蛋白的核酸序列在大量不同物种的基因组中都存在，其编码的蛋白序列高度保守，并具有多个高度保守的结构域，例如特征性的结合铁和/或硫的结构域。如按照本申请图13所示的氨基酸位置，本发明的磁感应受体蛋白的氨基酸序列从N端到C端依次包含以下结构域：第10-13位的TVRA(SEQ ID NO:17)、第56-61位的RGCNGL(SEQ ID NO：18)、第80-83位的QDGV(SEQ ID NO：19)、第89-93位的KKAQL(SEQ ID NO:20)、第95-101位的LLGTEMD(SEQ ID NO:21)以及第115-128位的NPNIKGTCGCGESF(SEQ ID NO:22)。
进一步地，本发明的磁感应受体蛋白的氨基酸序列从N端到C端依次还包含以下结构域：第17位的R、第23位的R、第25-26位的AL、第28-29位的LT、第32-33位的AV、第48位的G、第51位的V、第53位的V、第63位的Y、第67位的Y、第71位的K、第75-76位的DE、第78位的V、第87位的I、第104位的E、第106-107位的KL、第110-111位的EF和第113位的F。本领域技术人员应该明白，图13所示的氨基酸位置是将多条序列进行比对后所得的、能够对应大部分磁感应受体蛋白的氨基酸实际位置的结果；当有新的磁感应受体蛋白发现后，本领域技术人员能够很容易地通过比对的方式获知新蛋白中与图13所示的氨基酸位置相对应的位置。
更进一步地，本发明的磁感应受体蛋白或其活性片段或衍生物具有2Fe2S的铁硫中心。
本发明的磁感应受体蛋白可为磁感应受体蛋白家族中的其中一种。本发明的磁感应受体蛋白可来自动物，如脊椎动物或无脊椎动物。示例性的MagR的核酸或蛋白序列为本申请发明人从家鸽(拉丁名Columba livia)和黑脉金斑蝶(或称美洲帝王蝶，拉丁名Danaus plexippus)得到，其氨基酸序列分别如SEQ ID NO：1和SEQ ID NO：2所示。这些序列在本发明之前是未被公开的。
本发明提供的磁感应受体蛋白还包括其它生物物种的磁感应受体蛋白，例如来自果蝇(Fruit fly，拉丁名Drosophila melanogaster)，蜜蜂(Honey bee，拉丁名Apis mellifera)，文昌鱼(Lancelet，拉丁名Branchiostoma floridae)，斑马鱼(Zebrafish，拉丁名Danio rerio)，蟒蛇(Burmese python，拉丁名Python bivittatus)，草雀(Zebra finch，拉丁名Taeniopygia guttata)，鸡(Chicken，拉丁名Gallus gallus)，鲸鱼(Minke whale，拉丁名Balaenoptera acutorostrata)，蝙蝠(Little brown bat，拉丁名Myotis lucifugus)，裸鼹鼠(Naked mole rat，拉丁名Heterocephalus glaber)，小鼠(Mouse，拉丁名Mus musculus)，人(Human，拉丁名Homo sapiens)。上述来自果蝇、蜜蜂、文昌鱼、斑马鱼、蟒蛇、草雀、鸡、鲸鱼、蝙蝠、裸鼹鼠、小鼠和人的磁感应受体蛋白的氨基酸序列分别如SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13和SEQ ID NO：14所示。
在本发明的其中一个方面，提供了一种分离的磁感应受体蛋白或其活性片段或衍生物，其具有：
(1)如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13或SEQ ID NO：14所示的氨基酸序列，或
(2)在与SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13或SEQ ID NO：14进行比较时具有至少50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性的氨基酸序列。其中所述序列同一性可通过Clustal V比对方法计算得到。
优选地，其中不相同的残基位置的差异为保守性氨基酸取代。“保守性氨基酸取代”指其中氨基酸残基被另一种具有化学性质(例如：电荷或亲水性)类似之侧链R基的氨基酸残基取代。通常，保守性氨基酸取代实质上不会改变蛋白质的功能性质。具有相似化学性质之侧链的氨基酸基团实例包括：1)脂肪族侧链：甘氨酸、丙氨酸、缬氨酸、亮氨酸与异亮氨酸；2)脂肪族-羟基侧链：丝氨酸与苏氨酸；3)含酰胺侧链：天冬酰胺与谷氨酰胺；4)芳香族侧链：苯丙氨酸、酪氨酸与色氨酸；5)碱性侧链：赖氨酸、精氨酸与组氨酸；6)酸性侧链：天冬氨酸与谷氨酸；及7)含硫侧链：半胱氨酸与甲硫氨酸。优选的保守性氨基酸取代组合为：缬氨酸-亮氨酸-异亮氨酸、苯丙氨酸-酪氨酸、赖氨酸-精氨酸、丙氨酸-缬氨酸、谷氨酸-天冬氨酸、及天冬酰胺-谷氨酰胺。
在本发明的其中一个方面，提供了一种分离的磁感应受体蛋白或其活性片段或衍生物，其具有：在与SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO： 4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13或SEQ ID NO：14进行比较时具有一个或多个氨基酸的取代、缺失、添加的氨基酸序列。例如，其中取代、缺失、添加的氨基酸为保守性氨基酸。本发明提供的具有所述取代、缺失、添加的磁感应受体蛋白具有磁感应受体蛋白活性。
在本发明的其中又一个方面，上述分离的磁感应受体蛋白或其活性片段或衍生物具有磁性和/或能够形成具有磁性的蛋白复合物。在本发明的其中又一个方面，上述分离的磁感应受体蛋白或其活性片段或衍生物具有结合铁和/或硫的结构域。在本发明的其中又一个方面，上述分离的磁感应受体蛋白或其活性片段或衍生物能形成具有磁性的纯合蛋白复合物，和/或能与隐花色素蛋白(Cryptochrome，简称为Cry)形成杂合蛋白复合物。在本发明的其中又一个方面，上述纯合蛋白复合物或杂合蛋白复合物具有特定的蛋白立体结构，例如长条形；在所述纯合蛋白复合物或杂合蛋白复合物中，具有多个铁原子，其在棒状结构蛋白质的内部近中心轴区域也形成有序排列，由此使得所述蛋白复合物具有磁性。
在本发明的其中一个方面，本发明提供了一种重组DNA构建体，其包含编码如前所述的磁感应受体蛋白或其活性片段或衍生物的核苷酸序列。在所述构建体中，所述编码如前所述的磁感应受体蛋白或其活性片段或衍生物的核苷酸序列可操作地连接至少一个调控元件，例如可在真核细胞或原核细胞中有功能的启动子。
本发明提供了一种分离的蛋白复合物，其包含两个或两个以上前述的本发明的磁感应受体蛋白或其活性片段或衍生物，即纯合蛋白复合物。
在本发明的其中一个方面，所述蛋白复合物含有4个或以上所述磁感应受体蛋白或其活性片段或衍生物，优选为20个或以上所述磁感应受体蛋白或其活性片段或衍生物，进一步优选为20-24个所述磁感应受体蛋白或其活性片段或衍生物，更优选为20个所述磁感应受体蛋白或其活性片段或衍生物。
在本发明的其中一个方面，所述蛋白复合物的立体结构为“长条形”。蛋白质立体结构是指蛋白质分子的空间结构。长条形的蛋白立体结构也可称为“棒状结构”或“棍状结构”。蛋白质的立体结构可以通过电子显微镜观察和测量。在本发明的蛋白复合物中，所述多个磁感应受体蛋白或其活性片段或衍生物发生线性聚合，并进而聚集形成棒状结构。在本发明的其中一个方面，所述多个磁感应受体蛋白或其活性片段或衍生物发生线性聚合形成两条蛋白长链(等长的蛋白长链，即两条蛋白单链中含有相同个数的蛋白单体)。在本发明的其中又一个方面，所述两条蛋白长链以螺旋形式结合，形成棒状结构的蛋白复合物。在本发明的所述蛋白复合物中还包含多个铁原子。在本发明的其中一个方面，所述多个铁原子位于所述棒状结构蛋白复合物的内部近中心轴区域。例如，每个形成所述蛋白复合物的单个磁感应受体蛋白都结合铁原子，又例如，每个磁感应受体蛋白单体结合铁原子的位置都相同或相近，由此，在由磁感应受体蛋白单体线性聚合形成的棒状结构蛋白复合物中，所述多个铁原子在棒状结构的内部近中心轴区域也形成有序排列。
在本发明的其中一个方面，所述蛋白复合物中每个磁感应受体蛋白或其活性片段或衍生物含有1个2Fe2S的铁硫中心，即每个磁感应受体蛋白含有2个铁原子和2 个硫原子。在本发明的其中又一个方面，所述蛋白复合物含有约20个磁感应受体蛋白，由此，所述蛋白复合物含有约40个铁原子。
在本发明的其中一个方面，前述蛋白复合物还可结合有光感应元件。
因此，本发明还提供了一种分离的杂合蛋白复合物，其包含1)前述的本发明的磁感应受体蛋白或其活性片段或衍生物，以及2)光感应蛋白(例如隐花色素蛋白)或其活性片段或衍生物。
光感应元件是可感受/传递光信号的部分，如可结合到蛋白上的分子、复合物或蛋白等，例如可以是可感受/传递光信号的蛋白。在生物界已发现多种可感受/传递光信号的蛋白。其中一种是隐花色素蛋白(Cryptochrome，简称为Cry)或其活性片段或衍生物。在本发明的其中又一个方面，在所述杂合蛋白复合物中，每2个磁感应受体蛋白与1个隐花色素蛋白结合。
隐花色素蛋白(Cryptochrome，简称为Cry)是一类能够感受蓝光(400～500nm)和近紫外光(320～400nm)的黄素蛋白(Flavoprotein)，分子量为70～80KD，生色团可为黄素腺嘌呤二核苷酸(FAD)和蝶呤(pterin)。Cry蛋白及其编码基因普遍存在于植物、动物以及整个高等真核生物中。Cry蛋白在动物中可能与动物时钟节律和生物钟的控制有关。Cry蛋白家族包括Cry、Cry1、Cry2、Cry3和Cry4等。在哺乳动物、昆虫体内都有其同源基因编码隐花色素蛋白。
在本发明的其中一个方面，在本发明的蛋白复合物中，所述隐花色素蛋白可选自Cry、Cry1、Cry2、Cry3或Cry4。在本发明的其中又一个方面，在本发明的蛋白复合物中，所述隐花色素蛋白具有：
(1)如SEQ ID NO：15或SEQ ID NO：16所示的氨基酸序列，或
(2)在与SEQ ID NO：15或SEQ ID NO：16进行比较时具有至少50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性的氨基酸序列，并且具有隐花色素蛋白的活性。其中所述序列同一性可通过Clustal V比对方法计算得到。
优选地，其中不相同的残基位置的差异为保守性氨基酸取代。
在本发明的其中又一个方面，所述分离的蛋白复合物中的隐花色素蛋白具有：在与SEQ ID NO：15或SEQ ID NO：16进行比较时具有一个或多个氨基酸的取代、缺失、添加的氨基酸序列。例如，其中取代、缺失、添加的氨基酸为保守性氨基酸。本发明提供的具有所述取代、缺失、添加的磁感应受体蛋白具有磁感应受体蛋白活性。
在本发明的其中一个方面，所述杂合蛋白复合物中含有铁硫原子。
在本发明的其中一个方面，所述杂合蛋白复合物包含4个或以上、优选20个或以上所述磁感应受体蛋白或其活性片段或衍生物，进一步优选为20-24个、更优选为20个所述磁感应受体蛋白或其活性片段或衍生物。相应地，所述杂合蛋白复合物中包含2个或以上、优选10个或以上所述隐花色素蛋白，进一步优选为10-12个、更 优选为10个所述隐花色素蛋白。
在本发明的其中一个方面，前述本发明的蛋白复合物或杂合蛋白复合物具有磁性。“蛋白质具有磁性”或者“磁性蛋白质”是指蛋白质或者蛋白质复合物作为磁性材料的主体存在，自身具备磁性。所述磁性蛋白质可具有类似磁铁的磁极(南北极)，可以被外源的磁场牵引运动，也具备吸引铁、钴、镍等铁磁性物质的性质。值得指出的是，本发明所述的蛋白质具有自身的磁性，其磁性不依赖于外界磁场而存在。
在本发明的其中一个方面，前述的本发明蛋白复合物或杂合蛋白复合物可按照本领域的常规方法制备成为晶体。
本发明还提供了生产前述的本发明蛋白复合物的方法，其包含下述步骤：
在允许形成蛋白复合物的条件下，使所述磁感应受体蛋白或其活性片段或衍生物聚合，形成同源聚合物；分离该同源聚合物。
必要时，按照本领域的常规技术使该同源聚合物结晶。
在上述生产本发明的蛋白复合物的方法中，生产所述磁感应受体蛋白的环境中具有可生物利用的铁元素和硫元素。
在本发明的其中一个方面，在生产前述的本发明蛋白复合物的方法中，其中所述磁感应受体蛋白或其活性片段或衍生物在细胞中表达和形成复合物。所述细胞可以是培养的原核或者真核动物细胞，例如大肠杆菌或者哺乳动物细胞。在本发明的其中一个方面，在培养所述细胞过程中，所述磁感应受体蛋白或其活性片段或衍生物含有铁和/或硫原子，因此培养液中需加入可生物利用的铁元素和/或硫元素(单体或化合物)。
在本发明的其中一个方面，生产前述的本发明蛋白复合物的方法可在存在磁场的条件下进行。例如，其中分离同源聚合物的步骤可在存在磁场的条件下进行。
在本发明的其中又一个方面，所述磁场为外加磁场。在本发明的其中又一个方面，分离蛋白质同源聚合物通过亲和层析法进行，例如通过亲和层析柱；在采用亲和层析法分离所述蛋白质时，提供磁场条件。在本发明的其中一个方面，在生产前述的本发明蛋白复合物的方法中，所述磁场的磁场强度大于约5倍地球磁场，例如是地球磁场的约10-100倍。在生产前述的本发明蛋白复合物的方法中，所述磁场的磁场强度大于约100倍地球磁场也可以获得良好的效果。
同样地，本发明还提供了生产前述的本发明杂合蛋白复合物的方法，其包含下述步骤：
在允许形成杂合蛋白复合物的条件下，使1)前述的本发明的磁感应受体蛋白或其活性片段或衍生物与2)前述的光感应蛋白(例如隐花色素蛋白)或其活性片段或衍生物接触和聚合，形成杂合聚合物，其中1)和2)的蛋白中的至少一种是分离形式或被重组表达；和分离该杂合聚合物。
必要时，按照本领域的常规技术使该杂合聚合物结晶。
在本发明的其中一个方面，在生产前述的本发明杂合蛋白复合物的方法中，其中磁感应受体蛋白和光感应蛋白在细胞中表达和形成复合物。所述细胞可以是培养的原核细胞或者真核动物细胞，例如大肠杆菌和哺乳动物细胞。在本发明的其中一个方面，在培养所述原核或真核动物细胞过程中，所述磁感应受体蛋白或其活性片段 或衍生物含有铁和/或硫原子，因此培养液中需加入可生物利用的铁元素和/或硫元素(单体或化合物)。
在本发明的其中一个方面，生产前述的本发明杂合蛋白复合物的方法在存在磁场的条件下进行。例如，其中分离该杂合复合物的步骤在存在磁场的条件下进行。在本发明的其中又一个方面，所述磁场为外加磁场。在本发明的其中又一个方面，分离杂合蛋白复合物通过亲和层析法进行，例如通过亲和层析柱；在采用亲和层析法分离所述蛋白质时，可提供磁场条件。在本发明的其中一个方面，在生产前述的本发明杂合蛋白复合物的方法中，所述磁场的磁场强度大于约5倍地球磁场，例如是地球磁场的约10-100倍。在生产前述的本发明杂合蛋白复合物的方法中，所述磁场的磁场强度大于约100倍地球磁场也可以获得良好的效果。
在本发明的其中一个方面，提供了鉴别前述本发明磁感应受体蛋白可形成之光磁感应复合物的方法，其包含下述步骤：
a.在允许形成蛋白复合物的条件下，使前述的本发明的磁感应受体蛋白或其活性片段或衍生物与待测蛋白或衍生物接触；
b.测定所述磁感应受体蛋白或其活性片段或衍生物与待测蛋白或衍生物是否形成了复合物；
c.测定所形成的复合物内1)的蛋白与待测蛋白之间的相互作用。
在本发明的一个方面，提供了前述的本发明磁感应受体蛋白或蛋白复合物或杂合蛋白复合物的应用。
在本发明的其中一个方面，提供了前述的本发明磁感应受体蛋白和/或蛋白复合物和/或杂合蛋白复合物的与磁性相关的应用，例如，其作为磁性材料的应用或者制备这些磁性材料的应用。本发明的磁感应受体蛋白和/或蛋白复合物和/或杂合蛋白复合物具有对磁场的高度敏感，更特殊的是，其存在内在的磁性，可作为各种与磁性相关的应用中的磁性材料。与磁性相关的应用的领域非常广泛，包括但不限于生物医药和医疗器械如磁性生物材料、生物分子提纯、细胞内物质的定向移动、通过磁场控制某些细胞生命活动等，磁记录介质，超导应用如纳米环形电流超导体、磁流变流体等，电器设备如电磁转换设备，通讯和导航设备如分子陀螺仪中的应用等。
本文所引用的全部出版物都并入本文作为参考。除非另有定义，在本文中使用的全部技术和科学术语都具有与本发明所属领域的普通技术人员一般理解的相同的含义。
术语“多肽”、“蛋白”和“肽”在本文中互换地用于指氨基酸链，其中氨基酸残基通过肽键或修饰的肽键相连。氨基酸链可以是大于2个氨基酸的任意长度。除非另有说明，术语“多肽”、“蛋白”和“肽”也包括它们的各种修饰形式。这些修饰形式可以是天然存在的修饰形式或化学修饰形式。修饰形式的实例包括但不限于：糖基化形式、磷酸化形式、核糖基化形式、乙酰化形式、泛素化形式等。修饰也包括分子内交联和与各种部分(例如脂质、黄素、生物素、聚乙二醇或其衍生物等)的共价附着。另外，修饰也可以包括环化、分支和交联。此外，在多肽中也可以包含由基因密码子编码的20个常见氨基酸以外的氨基酸。
“磁感应受体蛋白”(或称“Magnetoreceptor”，或称“MagR”)是本申请发明人在 现代生物学研究中首个研究、得到、鉴定并命名的一种其核酸编码序列在生物体基因组中广泛存在并高度保守，可形成具有生物磁性单体或复合物的蛋白质。本发明提供的磁感应受体蛋白的核酸序列在大量不同物种的基因组中都存在，其编码的蛋白序列高度保守，并具有多个高度保守的结构域，如结合铁和/或硫的结构域。磁感应受体蛋白能形成具有特定形态结构、具有自身磁性的纯合蛋白复合物，和/或能与隐花色素蛋白(Cryptochrome，简称为Cry)形成杂合蛋白复合物。“蛋白家族”是指生物物种中存在的一系列具有同源性结构域或序列、进化上相关的、功能相同或相似的蛋白质。磁感应受体蛋白的蛋白家族中包括家鸽(Columba livia)磁感应受体蛋白和黑脉金斑蝶(Danaus plexippus)磁感应受体蛋白，其氨基酸序列分别如SEQ ID NO：1和SEQ ID NO：2所示。磁感应受体蛋白的蛋白家族的其它成员包括果蝇(Drosophila melanogaster)、蜜蜂(Apis mellifera)、文昌鱼(Branchiostoma floridae)、斑马鱼(Danio rerio)、蟒蛇(Python bivittatus)、草雀(Taeniopygia guttata)、鸡(Gallus gallus)、鲸鱼(Balaenoptera acutorostrata)、蝙蝠(Myotis lucifugus)、裸鼹鼠(Heterocephalus glaber)、小鼠(Mus musculus)、人(Homo sapiens)的磁感应受体蛋白，其氨基酸序列分别如SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13或SEQ ID NO：14所示。磁感应受体蛋白的纯合蛋白复合物或磁感应受体蛋白与隐花色素蛋白形成的杂合蛋白复合物具有特定的蛋白立体结构，例如长条形；在所述纯合蛋白复合物或杂合蛋白复合物中，具有多个铁原子，其在棒状结构蛋白质的内部近中心轴区域也形成有序排列，由此使得所述蛋白复合物具有磁性。例如，多个所述磁感应受体蛋白发生线性聚合，并进而聚集形成棒状结构。其中，多个所述磁感应受体蛋白线性聚合形成两条蛋白长链；所述两条蛋白长链以螺旋形式结合，形成棒状结构的蛋白复合物。磁感应受体蛋白的纯合蛋白复合物或磁感应受体蛋白与隐花色素蛋白形成的杂合蛋白复合物中还包含多个铁原子；所述多个铁原子位于所述棒状结构蛋白复合物的内部近中心轴区域。例如，每个形成所述蛋白复合物的单个磁感应受体蛋白都结合铁原子，又例如，每个磁感应受体蛋白单体结合铁原子的位置都相同或相近，由此，在由磁感应受体蛋白单体线性聚合形成的棒状结构蛋白复合物中，所述多个铁原子在棒状结构的内部近中心轴区域也形成有序排列。本文所述的中心轴具有通常理解的含义。“内部近中心轴区域”是指位于棒状结构内部、环绕中心轴的地方，多个铁原子在该区域排列可形成环状电子流。
物质的“磁性”是指能够产生磁场、具有吸引铁磁性物质(如铁、镍、钴等金属)的特性。
“蛋白质具有磁性”或者“磁性蛋白质”是指蛋白质或者蛋白质复合物自身具备磁性，体现出类似磁铁的磁极(南北极)，可以被外源的磁场牵引运动，也具备吸引铁、钴、镍等铁磁性物质的性质，可作为磁性材料。
“光磁性”是指物质受到光照后，磁学性质(如磁化率、磁晶各向异性、磁滞回线等)发生变化的现象，包括基于对光信号和磁场信号中的一种感应引发另一种基于光或磁场的作用。
本文使用的“磁感应受体蛋白的活性片段”是指磁感应受体蛋白的部分或片段，其 包含磁感应受体蛋白中维持它以类似于全长磁感应受体蛋白的方式并具有磁感应受体蛋白全部活性或部分活性的部分，或为具有该蛋白的多种活性中的某种或某几种活性的片段，例如其结构域片段。
本文使用的“磁感应受体蛋白的衍生物”是指，具有与本发明的磁感应受体蛋白的天然形式类似的一级和/或三级结构，但与所述天然形式相差一个或多个氨基酸残基(例如一个或多个氨基酸取代、插入和/或缺失)的蛋白(多肽)，或是具有/额外具有其它可以与之结合的化学基团或蛋白(多肽)基团；这些衍生物具有或保持磁感应受体蛋白的全部或部分活性。所述可结合的化学基团包括例如多肽的翻译后的衍生化或修饰，例如PEG化和/或巯基基团。所述可结合的蛋白(多肽)基团包括例如His标签或IgG蛋白等。
术语“分离的”用于描述这样的物质(例如核酸和/或蛋白质)，该物质不同于自然界中其天然存在或在原始细胞或生物环境中发现的形式的物质。该物质基本上不含在天然存在的环境中通常伴随该物质或与其反应的组分，或者说，该物质已从所述组分中移出。
“分离的蛋白”是这样的蛋白：它基本上与蛋白的天然来源中存在的至少一种组分或其它蛋白分离，或当化学合成所述蛋白时，它基本上不含有至少一种化学前体或其它化学药品。当存在小于约30％、20％、10％或5％(按干重计)的其它蛋白或其它化学药品(在本文中也称作“污染蛋白”或“污染化学药品”)时，在蛋白的制剂中蛋白“基本上分离于”或“基本上不含有”其它蛋白或其它化学药品。
分离的蛋白可以具有几种不同的物理形式。分离的蛋白可以作为全长新生的或未加工的多肽，或者作为部分加工的多肽，或者作为加工的多肽，或者它们的组合存在。
分离的多肽可以是非天然存在的多肽。例如，“分离的多肽”可以是“杂种多肽(hybrid peptide)”。“分离的多肽”也可以是通过氨基酸的添加、缺失或取代从天然存在的多肽衍生的多肽。分离的多肽也可以是“纯化的多肽”，后者在本文中用于指基本上均质的制剂中基本上不含有其它细胞组分、其它多肽、病毒材料或培养基的特定多肽，或当化学合成所述多肽时，基本上不含有化学前体或与化学合成有关的副产物。“纯化的多肽”可以通过标准的纯化技术或通过化学合成从天然或重组宿主细胞得到，如技术人员显而易见的。
如本文所使用的，术语“相互作用”是指，2个蛋白结构域、片段或完整蛋白彼此表现出足够的物理亲和力，以便使所述2个“相互作用”蛋白结构域、片段或蛋白彼此物理上接近。相互作用可以来自一个或多个化学键的形成，其导致2个相互作用实体的连续且稳定的接近。相互作用也可以仅仅基于物理亲和力，这在共同定位(co-localize)2个蛋白方面同样有效。物理亲和力和化学键的实例包括但不限于，由电荷差异造成的力、疏水性、氢键、范德华力、离子力、共价键以及它们的组合。相互作用域、片段或蛋白之间的接近状态可以是瞬时的或永久的，可逆的或不可逆的。在任何情况下，其与2个实体的天然随机运动造成的接触形成对照且可以区分开。通常，通过相互作用域、片段或蛋白之间的结合，表现出“相互作用”。相互作用的实例包括抗原和抗体、配体和受体、酶和底物等之间的特异性相互作用。
如本文所使用的，术语“蛋白复合物”或“多肽复合物”是指一种复合单位，它是通过蛋白之间的相互作用形成的2个或更多个蛋白的组合。通常，通过特异性非共价结合亲和力将2个或更多个蛋白结合到一起，形成“蛋白复合物”。“纯合蛋白复合物”是指由同一种蛋白质组分通过聚合(或称之为“同源聚合”)形成的多聚物。“杂合蛋白复合物”是指由两种或者多种不同的蛋白质组分通过聚合(或称之为“异源聚合”)形成的多聚物。
术语“分离的蛋白复合物”是指在不同于其在自然界中天然存在或在原始细胞或生物环境中发现的形式的蛋白复合物。该蛋白复合物基本上不含在天然存在的环境中通常伴随该蛋白复合物或与其反应的组分，或者说，该蛋白复合物已从所述组分中移出。“分离的蛋白复合物”也可以是未在自然界发现的蛋白复合物。
“分离的”核酸分子是这样的，它基本上与核酸的天然来源中存在的至少一种其它核酸分子分离，或当化学合成所述核酸分子时，它基本上不含有至少一种化学前体或其它化学药品。“分离的”核酸分子也可以是，例如，基本上不含有在该核酸的来源生物基因组DNA中在5'和3'末端天然侧接该核酸分子的至少一个核苷酸序列的核酸分子。在核酸分子的制剂中，当存在小于约30％、20％、10％或5％(按干重计)的其它核酸分子或其它化学药品(在本文中也称作“污染核酸分子”或“污染化学药品”)时，核酸分子“基本上分离于”或“基本上不含有”其它核酸分子或其它化学药品。
“序列”是指聚合物中单体出现的线性次序，例如，多肽中氨基酸的次序或多核苷酸中核苷酸的次序。“核苷酸序列”指单链或双链形式的聚合物中脱氧核糖核苷酸或核糖核苷酸残基的排列。核酸序列可以由包含下述碱基的天然核苷酸组成：胸腺嘧啶、腺嘌呤、胞嘧啶、鸟嘌呤和尿嘧啶；分别缩写为T、A、C、G和U，和/或所述天然核苷酸的合成类似物。
核苷酸序列或氨基酸序列比对和序列的同一性百分比可用设计用于鉴别同源序列的多种比较方法来确定，这些方法包括但不限于
Figure PCTCN2016078842-appb-000001
生物信息计算包(
Figure PCTCN2016078842-appb-000002
 Inc.，Madison，WI)的
Figure PCTCN2016078842-appb-000003
程序。除非另外说明，否则本文提供的序列多重比对用Clustal V比对方法(Higgins和Sharp，1989，CABIOS.5：151-153)采用默认参数(空位罚分＝10，空位长度罚分＝10)执行。用Clustal V方法进行成对比对和蛋白质序列的同一性百分比计算的默认参数为KTUPLE＝1、空位罚分＝3、窗口(WINDOW)＝5和DIAGONALS SAVED＝5。而对于核酸，这些参数为KTUPLE＝2，空位罚分＝5，窗口＝4和DIAGONALS SAVED＝4。用Clustal V程序比对序列后，可通过查看同一程序中的“序列距离”表来获得“同一性百分比”和“趋异”值。
“重组DNA构建体”指在自然界中通常不会一起存在的核酸片段的组合。因此，重组DNA构建体可包含源于不同来源的调控序列和编码序列，或源于相同来源但以不同于通常天然存在的方式排列的调控序列(如启动子等)和编码序列。
分子生物学和蛋白-蛋白相互作用领域的技术人员使用常规实验，可以在磁感应受体蛋白或其活性片段或衍生物中导入氨基酸序列变异。例如，本领域技术人员可以导入位点特异性的突变，例如将“CTT”密码子变成“ATT”密码子，从而造成天然多肽中的亮氨酸残基被替换为合成同源物中的异亮氨酸残基。
在另一个一般方面，本发明提供了制备本发明蛋白复合物的方法。本发明的蛋白复合物可以通过多种方法来制备。具体而言，蛋白复合物可以直接分离自含有该蛋白复合物的动物组织样品，例如人组织样品。蛋白复合物也可以分离自重组表达蛋白复合物成员的宿主细胞。或者，可以通过组合蛋白复合物的个别成员来体外构建蛋白复合物。
本领域技术人员会明白，为了表达蛋白或蛋白复合物的目的，在本发明中可以使用任何重组表达方法。例如，可以将编码MagR和Cry的核酸导入合适的宿主细胞中。通常，将核酸(优选地以DNA形式)整合入表达载体中，从而在导入宿主细胞后指导生产相互作用蛋白成员。许多类型的载体可以用于本发明。获知本公开内容的技术人员应了解构建用于本发明目的的表达载体的方法。
通常，表达载体包括具有启动子的表达盒，所述启动子可操作地连接到编码相互作用蛋白成员的DNA上。所述启动子可以是天然启动子，即在天然存在的细胞中发现的负责在该细胞中表达相互作用蛋白成员的启动子。或者，所述表达盒可以是嵌合表达盒，即，具有不是在天然存在的细胞中负责表达相互作用蛋白成员的天然启动子的异源启动子。表达载体还可以包括用于在宿主细胞中复制载体的DNA复制起点。优选地，表达载体包括用于在例如大肠杆菌中扩增载体的复制起点，和用于仅选择和维持携带表达载体的那些宿主细胞的选择标记。另外，表达盒优选地也含有诱导型元件，其功能是控制从编码相互作用蛋白成员的DNA的转录。其它调节序列例如转录增强子序列和翻译调节序列(例如,SD序列)也可以可操作地包含在表达盒中。终止序列(例如来自牛生长激素、SV40和lacZ)也可以可操作地与表达盒中编码蛋白成员的DNA连接。当需要在单个宿主细胞中表达2个或更多个相互作用蛋白成员时，编码相互作用蛋白成员的DNA片段可以整合入单个载体或不同载体中。
通过本领域已知的任意技术，例如，通过直接DNA转化、显微注射、电穿孔、病毒感染、脂转染、基因枪等，可以将表达载体导入宿主细胞中。或者可通过常规技术例如稳定细胞系的选择或位点特异性的重组，可以将表达载体整合入宿主细胞的染色体中。
可以将载体构建体设计成适合在各种宿主细胞中表达，宿主细胞包括但不限于细菌、酵母细胞、植物细胞、昆虫细胞和哺乳动物和人细胞。
其中，可以将哺乳动物细胞用作用于表达融合蛋白和检测蛋白-蛋白相互作用的宿主细胞。为此，实际上可以使用任何哺乳动物细胞，包括正常组织细胞、稳定的细胞系和转化的肿瘤细胞。方便地，使用哺乳动物细胞系，例如CHO细胞、Jurkat T细胞、NIH 3T3细胞、HEK-293细胞、CV-1细胞、COS-1细胞、HeLa细胞、VERO细胞、MDCK细胞、WI38细胞等。哺乳动物表达载体是本领域众所周知的，且许多可商业得到。用于在哺乳动物细胞中转录嵌合基因的合适启动子的实例包括源自下述病毒的病毒转录启动子：腺病毒、猿猴病毒40(SV40)(例如，SV40的早期和晚期启动子)、劳斯肉瘤病毒(RSV)和巨细胞病毒(CMV)(例如，CMV立即早期启动子)、人免疫缺陷病毒(HIV)(例如，长末端重复序列(LTR))、痘苗病毒(例如，7.5K启动子)和单纯疱疹病毒(HSV)(例如，胸苷激酶启动子)。也可以使用诱导型启动子。合适的诱导型启动子包括，例如，四环素响应元件(TRE)(参见Gossen等人,Proc.Natl.Acad. Sci.USA,89：5547-5551(1992))、金属硫蛋白IIA启动子、蜕皮激素-响应启动子和热激启动子。用于在哺乳动物细胞中复制和维持表达载体的合适复制起点包括，例如，在有Epstein Barr核抗原存在下的Epstein Barr复制起点(参见Sugden等人,Mole.Cell.Biol.,5：410-413(1985))和在有SV40 T抗原(它存在于COS-1和COS-7细胞中)存在下的SV40复制起点(参见Margolskee等人,Mole.Cell.Biol.,8：2837(1988))。合适的选择标记包括，但不限于，赋予对新霉素、潮霉素、zeocin等的抗性的基因。许多可商业得到的哺乳动物表达载体可以用于本发明，包括，例如，pCEP4、pcDNAI、pIND、pSecTag2、pVAX1、pcDNA3.1和pBI-EGFP和pDisplay。使用任何已知的技术，例如磷酸钙沉淀、脂转染、电穿孔等，可以将载体导入哺乳动物细胞。可以将诱饵载体和猎物载体共转化进相同细胞，或者，导入2个不同的细胞，随后通过细胞融合或其它合适的技术将它们融合到一起。
允许通过病毒感染将重组基因导入细胞中的病毒表达载体，也可以用于表达融合蛋白。本领域普遍已知的病毒表达载体包括基于腺病毒、牛乳头瘤病毒、鼠干细胞病毒和反转录病毒等的病毒载体。
使用上述的重组方法，也可以容易地表达天然相互作用蛋白成员的同源物和片段。例如，为了表达蛋白片段，可以选择整合入表达载体中的DNA片段，从而使它仅仅编码该蛋白片段。同样，使用编码杂种蛋白的重组DNA，可以表达特定的杂种蛋白。类似地，同源物蛋白可以从编码该同源物蛋白的DNA序列表达。通过使用重组DNA技术操作编码天然蛋白的序列，可以得到编码同源物蛋白的DNA序列。为此，可以使用本领域普遍已知的技术，进行随机的或定点诱变。
另外，通过使某些用于修饰蛋白的部分经由化学方式连接到天然蛋白的氨基酸侧链上，由此制备本发明的蛋白的衍生物。
如果需要，可以测试这样产生的同源物和衍生物，以确定它们是否能与它们预期的配体相互作用，以形成蛋白复合物。测试可以通过例如酵母双杂交系统或本领域已知的检测蛋白-蛋白相互作用的其它方法来进行。
通过本领域技术人员众所周知的常规生化和免疫化学方法，可以纯化表达的蛋白。转染的真核细胞或生物组织样品可以匀浆，并在将不同细胞组分进行分离的适宜条件下分级分离。通常，将细胞裂解物在蔗糖梯度(或基于大小和密度分离细胞组分的其它材料)上运行。使用本领域技术人员众所周知的方法，例如免疫印迹或免疫沉淀方法，用适宜的抗体分析目标蛋白的存在。
然后将纯化的蛋白用于亲和层析处理：将来自培养的细胞或匀浆的组织样品的提取物在适宜的缓冲液中装载到柱上，目的蛋白或蛋白复合物可与柱上的成分结合；洗脱非结合蛋白；然后使用各种方法，例如pH梯度或盐浓度梯度，洗脱结合蛋白或蛋白复合物；最后可以通过二维凝胶电泳，分离洗脱的蛋白，也可以通过微量测序进行鉴别。所有这些方法是本领域技术人员众所周知的。
纯化的目标蛋白或复合物，也可以用于在兔子、小鼠、大鼠、鸡、山羊、绵羊、猪、豚鼠、牛和马中制备抗体。用于抗体生成和表征的方法是本领域技术人员众所周知的。
附图说明
图1：家鸽MagR蛋白的聚丙烯酰胺凝胶电泳分析图。
图2：家鸽和黑脉金斑蝶的MagR-Cry蛋白复合物的聚丙烯酰胺凝胶电泳分析图。
图3：家鸽MagR蛋白样品全波长扫描结果图。
图4：家鸽MagR形成的纯合蛋白复合物或杂合蛋白复合物的电镜观察结果图。A：家鸽MagR形成的纯合蛋白复合物的横截面结构；B：家鸽MagR形成的纯合蛋白复合物的纵截面结构；C：家鸽MagR-Cry4杂合蛋白复合物的横截面结构；D：家鸽MagR-Cry4杂合蛋白复合物的纵截面结构。
图5：家鸽MagR纯合蛋白复合物的结构图。A：家鸽MagR纯合蛋白复合物的纵截面的电镜形态和结构示意；B：家鸽MagR纯合蛋白复合物的横截面的电镜形态和结构示意。
图6：家鸽MagR和Cry形成的杂合蛋白复合物的结构图。A：杂合蛋白复合物的纵截面；B：杂合蛋白复合物的横截面。
图7：家鸽MagR纯合蛋白复合物晶体受磁场影响的电镜观察结果图。A：电子显微镜照片；B：统计结果。
图8：用铁粉从细胞裂解液中分离和纯化家鸽MagR-Cry4杂合蛋白复合物的实验示意和电泳检测结果图。
图9：家鸽MagR纯合蛋白复合物晶体在旋转磁场中同步旋转实验结果图。
图10：黑脉金斑蝶MagR蛋白的聚丙烯酰胺凝胶电泳分析图。
图11：黑脉金斑蝶MagR-Cry1纯合蛋白复合物的晶体在旋转磁场中同步旋转实验结果图。
图12：家鸽MagR突变体蛋白样品全波长扫描结果图。
图13：各物种MagR蛋白的序列比对图。
图14：家鸽MagR-Cry4杂合蛋白复合物在溶液中的室温磁性测量的负对照。
图15：家鸽MagR-Cry4杂合蛋白复合物在溶液中在室温下的SQUID磁性测量结果。
具体实施方式
以下实施例中，如无特殊说明，所使用的试剂、仪器都是本领域常用试剂、仪器，可以从化学或生物制品/制剂公司购买；以下实施例中使用的方法，如PCR、亲和纯化、分子筛凝胶层析、离子柱交换等都是本领域常规方法，本领域技术人员根据如《分子克隆实验指南》等现有技术或根据生产商提供的操作手册可以毫无疑义地知道这些实验的操作过程并获得相应结果。
实施例1：家鸽MagR蛋白的获得
利用同源性较高的鸡MagR蛋白质序列(SEQ ID NO:9)在家鸽基因组中通过blast寻找同源序列，并将找到的同源区段提取出来在软件sequencher中与鸡的MagR编码区序列进行比对，得知一共四个外显子。其中，外显子2、3、4的序列和鸡的MagR序列有较好的匹配，但家鸽基因组序列中MagR基因的外显子1序列缺失。为了获得完整的鸽子MagR的序列，根据鸡的MagR的外显子1的序列设计如下引物， 并从家鸽基因组DNA中扩增该外显子：
外显子1引物：F:ACGGACCGCTACCCAATAG(正向引物)(SEQ ID NO:34)，R:ACACTTTTTCAGCGGCTGTG(反向引物)(SEQ ID NO:35)。
将PCR得到的外显子1的序列和通过上述基因组匹配获得的外显子2、3、4的序列合并，获得家鸽MagR蛋白的编码区全序列：
ATGGCCTCGTCGGCCTCGTCCGTGGTGAGGGCCACGGTGCGCGCCGTCAGCAAGCGGAAGATCCAGGCTACGCGCGCCGCCCTCACCCTGACCCCATCAGCTGTTCAGAAGATAAAAGAGCTTCTTAAAGATAAACCTGAGCATGTAGGCGTGAAAGTAGGTGTTCGCACAAGAGGATGCAATGGACTTTCTTACACATTAGAATATACAAAATCAAAAGGAGACTCTGATGAAGAAGTAGTTCAAGATGGGGTTAGAGTGTTTATTGAGAAGAAGGCACAGCTGACGCTTTTAGGCACTGAAATGGACTATGTAGAAGACAAACTGTCCAGTGAATTTGTCTTCAATAATCCAAACATCAAAGGAACATGTGGCTGTGGAGAAAGCTTTAACATCTGA(SEQ ID NO:36)。
其编码的氨基酸序列如SEQ ID NO:1所示，命名为clMagR。
之后在家鸽基因组DNA中扩增MagR序列，验证上述序列正确。
实施例2：家鸽MagR蛋白的表达和纯化
1.人工合成编码以下氨基酸序列的插入片段(表达框)：
MWSHPQFEKGGSTSMASSASSVVRATVRAVSKRKIQATRAALTLTPSAVQKIKEL LKDKPEHVGVKVGVRTRGCNGLSYTLEYTKSKGDSDEEVVQDGVRVFIEKKAQLTLL GTEMDYVEDKLSSEFVFNNPNIKGTCGCGESFNI(SEQ ID NO:23)；
插入片段序列：
CATATGTGGAGCCACCCCCAGTTCGAAAAGGGCGGTTCCACTAGTATGGCTTC TTCTGCTTCTTCTGTTGTTCGTGCTACCGTTCGTGCTGTTTCTAAACGTAAAATCCA GGCTACCCGTGCTGCTCTGACCCTGACCCCGTCTGCTGTTCAGAAAATCAAAGAAC TGCTGAAAGACAAACCGGAACACGTTGGTGTTAAAGTTGGTGTTCGTACCCGTGGT TGCAACGGTCTGTCTTACACCCTGGAATACACCAAATCTAAAGGTGACTCTGACGA AGAAGTTGTTCAGGACGGTGTTCGTGTTTTCATCGAAAAAAAAGCTCAGCTGACCC TGCTGGGTACTGAAATGGACTACGTTGAAGACAAACTGTCTTCTGAATTCGTTTTC AACAACCCGAACATCAAAGGTACTTGCGGTTGCGGTGAATCTTTCAACATCTAAGGATCC(SEQ ID NO:24)；
其中，第2-9个氨基酸WSHPQFEK(SEQ ID NO:26)为Strep II亲和标签序列；第10-14个氨基酸GGSTS(SEQ ID NO:25)为链接区序列；带下划线的序列为家鸽(Columba livia)的MagR的蛋白质序列，即如SEQ ID NO：1所示的氨基酸序列。
核苷酸序列中，第1-6位是NdeI酶切位点，7-30位是StrepII表达序列，31-45位是GGSTS(SEQ ID NO:25)表达序列，46-441位是家鸽MagR表达序列，442-444位是终止密码子，445-450位是BamHI酶切位点。该基因在金斯瑞生物科技有限公司进行合成所得。
2.将上述合成的编码家鸽MagR蛋白的氨基酸序列且N端具有Strep II亲和标签的插入片段通过位点NdeI和BamHI克隆到pET-21a(+)质粒(Novagen)。
3.将上述包含具有编码家鸽MagR蛋白的氨基酸序列且N端具有Strep II亲和标签的插入片段的质粒转化大肠杆菌BL21(DE3)(商购)。挑取单克隆接种至LB液体培养基中扩大培养，15℃下用20μM的IPTG诱导目的蛋白的表达，20-24小时后收菌。
4.将上述得到的表达了MagR蛋白的转化菌用缓冲液(20mM Tris，150mM NaCl，pH8.0，10mM巯基乙醇以及蛋白酶抑制剂)重悬后，用超声破碎仪裂解(工作4s，暂停8s，99个循环)，完成后进行高速离心(17000rpm)，取上清液进行亲和纯化。通过Strep-Tactin亲和柱(IBA公司)亲和纯化后，进行阴离子交换柱(GE Healthcareg公司)或者分子筛凝胶层析(GE Healthcareg公司)进一步纯化，得到纯度较高的家鸽MagR蛋白。纯化全过程在4℃进行。纯化后得到的MagR蛋白在浓度较高时呈现明显的棕黄色或棕褐色，这提示蛋白质中结合了二价或三价的铁。
5.对收集的MagR蛋白样品进行变性聚丙烯酰胺凝胶电泳(SDS-PAGE)分析。实验结果如图1所示，其中各泳道的样品为：1、蛋白分子量标准；2、纯化的含有Strep II亲和标签的家鸽MagR蛋白，分子量约为15KDa。
实施例3：家鸽MagR蛋白形成二聚体和多聚体的纯合复合物
将实施例2中表达和纯化的家鸽MagR蛋白，在通过Strep-Tactin亲和柱亲和纯化后进一步用Superdex 200 10/300 GL纯化柱(GE Healthcare)进行分子筛凝胶层析(缓冲液：20mM Tris，150mM NaCl，pH8.0，10mM巯基乙醇)，即凝胶过滤(Gel-filtration)或凝胶排阻层析(Size-exclusion chromatography)。样品的分子筛凝胶层析呈现出单体峰、二聚体峰和多聚体峰，分别收集三个峰的样品，获得纯化蛋白。这显示，纯化的MagR蛋白在分子筛凝胶层析中以三种形式存在。与蛋白质分子量标准进行比较可知，三个峰(洗脱体积大约为：17.5ml、16.2ml、9.5ml)的大致分子量为15KDa、30KDa和大于200KDa，这与MagR蛋白的单体、二聚体和多聚体的分子量相吻合。由于Superdex S200的分辨率最高为约200KDa，所以，尚无法判断多聚体的分子量和多聚体所含单体个数。
实施例4：家鸽MagR蛋白和家鸽Cry4蛋白形成的杂合蛋白复合物的表达和纯化
1.人工合成编码以下氨基酸序列的插入片段(表达框)：
MHHHHHHHHHHGGSTSMPHRTIHLFRKGLRLHDNPTLLAALESSETIYPVYVLDR RFLASAMHIGALRWHFLLQSLEDLHKNLSRLGARLLVIQGEYESVLRDHVQKWNITQ VTLDAEMEPFYKEMEANIRRLGAELGFEVLSRVGHSLYDTKRILDLNGGSPPLTYKRF LHILSQLGDPEVPVRNLTAEDFQRCMSPEPGLAERYRVPVPADLEIPPQSLSPWTGGET EGLRRLEQHLTDQGWVANFTKPRTIPNSLLPSTTGLSPYFSMGCLSVRTFFQRLSNIYA QAKHHSLPPVSLQGQLLWREFFYTVASATQNFTQMAGNPICLQIHWYEDAERLHKWK TAQTGFPWIDAIMTQLRQEGWIHHLARHAVACFLTRGDLWISWEEGMKVFEELLLDA DYSINAGNWMWLSASAFFHHYTRIFCPVRFGKRTDPEGQYIRKYLPVLKNFPTKYIYE PWTASEEEQRQAGCIIGRDYPFPMVNHKEASDRNLQLMRRVREEQRGTAQLTR(SEQ ID NO:27)。
插入片段的序列：
CATATGCATCATCATCATCATCACCATCACCATCACGGCGGTTCCACTAGTAT GCCGCACCGTACCATCCACCTGTTCCGTAAAGGTCTGCGTCTGCACGACAACCCGA CCCTGCTGGCTGCTCTGGAATCTTCTGAAACCATCTACCCGGTTTACGTTCTGGACC GTCGTTTCCTGGCTTCTGCTATGCACATCGGTGCTCTGCGTTGGCACTTCCTGCTGC AGTCTCTGGAAGACCTGCACAAAAACCTGTCTCGTCTGGGTGCTCGTCTGCTGGTT ATCCAGGGTGAATACGAATCTGTTCTGCGTGACCACGTTCAGAAATGGAACATCA CCCAGGTTACCCTGGACGCTGAAATGGAACCGTTCTACAAAGAAATGGAAGCTAA CATCCGTCGTCTGGGTGCTGAACTGGGTTTCGAAGTTCTGTCTCGTGTTGGTCACTC TCTGTACGACACCAAACGTATCCTGGACCTGAACGGTGGTTCTCCGCCGCTGACCT ACAAACGTTTCCTGCACATCCTGTCTCAGCTGGGTGACCCGGAAGTTCCGGTTCGT AACCTGACCGCTGAAGACTTCCAGCGTTGCATGTCTCCGGAACCGGGTCTGGCTGA ACGTTACCGTGTTCCGGTTCCGGCTGACCTGGAAATCCCGCCGCAGTCTCTGTCTC CGTGGACCGGTGGTGAAACCGAAGGTCTGCGTCGTCTGGAACAGCACCTGACCGA CCAGGGTTGGGTTGCTAACTTCACCAAACCGCGTACCATCCCGAACTCTCTGCTGC CGTCTACCACCGGTCTGTCTCCGTACTTCTCTATGGGTTGCCTGTCTGTTCGTACCT TCTTCCAGCGTCTGTCTAACATCTACGCTCAGGCTAAACACCACTCTCTGCCGCCG GTTTCTCTGCAGGGTCAGCTGCTGTGGCGTGAATTCTTCTACACCGTTGCTTCTGCT ACCCAGAACTTCACCCAGATGGCTGGTAACCCGATCTGCCTGCAGATCCACTGGTA CGAAGACGCTGAACGTCTGCACAAATGGAAAACCGCTCAGACCGGTTTCCCGTGG ATCGACGCTATCATGACCCAGCTGCGTCAGGAAGGTTGGATCCACCACCTGGCTCG TCACGCTGTTGCTTGCTTCCTGACCCGTGGTGACCTGTGGATCTCTTGGGAAGAAG GTATGAAAGTTTTCGAAGAACTGCTGCTGGACGCTGACTACTCTATCAACGCTGGT AACTGGATGTGGCTGTCTGCTTCTGCTTTCTTCCACCACTACACCCGTATCTTCTGC CCGGTTCGTTTCGGTAAACGTACCGACCCGGAAGGTCAGTACATCCGTAAATACCT GCCGGTTCTGAAAAACTTCCCGACCAAATACATCTACGAACCGTGGACCGCTTCTG AAGAAGAACAGCGTCAGGCTGGTTGCATCATCGGTCGTGACTACCCGTTCCCGAT GGTTAACCACAAAGAAGCTTCTGACCGTAACCTGCAGCTGATGCGTCGTGTTCGTG AAGAACAGCGTGGTACCGCTCAGCTGACCCGTTAACTCGAG(SEQ ID NO:28)。
其中，第2-11个氨基酸为10个组氨酸的His亲和标签序列；第12-16个氨基酸GGSTS(SEQ ID NO:25)为链接区序列；带下划线的序列为家鸽的Cry4的氨基酸序列，即如SEQ ID NO：15所示的氨基酸序列。
核苷酸序列中，第1-6位是NdeI酶切位点，7-36位是10×His表达序列，37-51位是GGSTS(SEQ ID NO:25)表达序列，52-1542位是家鸽Cry4表达序列，1543-1545位是终止密码子，1546-1551位是XhoI酶切位点。该基因在博迈德生物科技有限公司进行合成所得。
2.将上述合成的编码家鸽Cry4蛋白的氨基酸序列并且N端具有His亲和标签的插入片段通过NdeI和XhoI位点克隆到pET-21a(+)质粒(Novagen)。
3.将前述具有家鸽Cry4蛋白的编码基因并融合了组氨酸标签(His-tag)的质粒与实施例2步骤2得到的具有家鸽MagR蛋白的编码基因并融合了StrepII标签的质粒共同转化大肠杆菌BL21(DE3)。表达MagR的质粒为卡那霉素抗性，表达Cry4的质粒为氨苄 青霉素抗性，在双抗培养基(卡那霉素和氨苄青霉素)中挑取含有两种质粒的大肠杆菌单克隆接种至LB液体培养基中扩大培养，15℃下用20μM的IPTG诱导目的蛋白的表达，20-24小时后收菌。
4.将上述得到的共表达了家鸽MagR蛋白和Cry4蛋白的转化菌用缓冲液(20mM Tris，150mM NaCl，10mM咪唑，pH8.0，10mM巯基乙醇以及蛋白酶抑制剂)重悬后，用超声破碎仪裂解，完成后进行高速离心，取上清液进行亲和纯化。通过Ni-NTA亲和柱(QIAGEN公司)亲和纯化后，再通过Strep-Tactin亲和柱(IBA公司)纯化，得到家鸽MagR蛋白和Cry4蛋白形成的MagR-Cry4杂合蛋白复合物。进一步通过阴离子交换柱和分子筛凝胶层析(GE Healthcare公司)纯化，得到纯度较高的杂合蛋白复合物。纯化全过程在4℃和存在外加磁场的条件下进行。外加磁场通过下述方法施加：当样品过亲和柱时，将两个相同的条形磁铁分别绑在亲和柱的两侧，该两个相同的条形磁铁的南极和北极分别相对，提供一个均匀稳定的磁场。结果显示，在纯化时施加地球磁场100倍的外加磁场，纯化蛋白的产率有显著增加；在洗脱纯化蛋白时，撤去磁场，蛋白质洗脱效率更高。
5.对收集的家鸽MagR-Cry4杂合蛋白复合物样品进行变性聚丙烯酰胺凝胶电泳(SDS-PAGE)分析。实验结果如图2所示，其中从左向右第4泳道加载的样品为蛋白质分子量标准，第5泳道加载的样品为家鸽MagR-Cry4杂合蛋白复合物。结果：由于使用变性电泳分析，泳道5中显示出两种蛋白质样品，即含有Strep II亲和标签的家鸽MagR蛋白(分子量约为15KDa)以及含有His标签的家鸽Cry4蛋白(分子量约为70KDa)。
实施例5：家鸽MagR蛋白以及MagR纯合蛋白复合物含有铁的证明实验
将实施例3得到的家鸽MagR蛋白的单体峰样品用分光光度计(Nanodrop 2000)进行全波长扫描。
如图3所示，结果显示在Fe元素的特征吸收位置(320nm和410nm)有吸收峰。第一个吸收峰为UV280的蛋白质吸收峰。
将实施例3中得到的家鸽MagR蛋白形成的二聚物和多聚物样品分别用分光光度计(Nanodrop 2000)进行全波长扫描，结果与图3相似。
实施例6：家鸽MagR纯合蛋白复合物和家鸽MagR-Cry4杂合蛋白复合物的电子显微镜图像和结构
将实施例3中经分子筛凝胶层析纯化后获得的家鸽MagR纯合蛋白复合物的多聚体蛋白样品以及实施例4中经分子筛凝胶层析纯化后的家鸽MagR-Cry4杂合蛋白复合物的蛋白样品采用乙酸铀或甲酸铀负染，制备蛋白质的负染电镜样品。将样品放置于透射电镜下观察并拍照，按照标准的电镜结构分析方法解析其结构，并测量大小(包括蛋白长度和宽度)。所述电镜结构分析方法可参照Ohi,M.,et al.(2004)，"Negative Staining and Image Classification-Powerful Tools in Modern Electron Microscopy."Biol Proced Online 6:23-34。
结果如图4所示。其中A和B是家鸽MagR形成的纯合蛋白复合物的电镜结构，其中A为横截面结构，B为纵截面结构。C和D为家鸽MagR-Cry4杂合蛋白复合物的电子显微镜结构，其中C为横截面结构，D为纵截面结构。MagR和/或Cry蛋白在复合物中的组织形式和相对位置的示意图如右侧所示，并标出了在电镜下测量的 大小尺寸(单位为纳米)。
如图4的A和B显示，家鸽MagR纯合蛋白复合物呈现为长条形的棒状结构，长度为约20-24纳米长，宽度为约7-11纳米。“宽度”是指电镜下该棒状结构的横截面为圆形或类似圆形的形状时测量到的直径。根据同源晶体结构和分子模型计算，结果表明该复合物具有约20个MagR蛋白。如图4的C和D显示，家鸽MagR-Cry4杂合蛋白复合物呈现为长条形的棒状结构，长度为约20-24纳米长，宽度为约13-18纳米。根据同源晶体结构和分子模型计算，结果表明该多聚体具有约20个MagR蛋白和10个Cry4蛋白。
图5为家鸽MagR纯合蛋白复合物的结构图。图5中，A显示复合物的纵截面的电镜形态和结构示意，B显示复合物的横截面的电镜形态和结构示意。每一个MagR纯合蛋白复合物包括约20个MagR蛋白，有序排列形成线性双螺旋聚合体，其中每10个MagR蛋白单体以线性聚合的形式首尾相接，形成两条蛋白长链，两条蛋白长链以螺旋形式结合，由此形成长条形棒状结构的蛋白复合物。图5中的虚线框显示出一个MagR四聚体的结构：如图所示的MagR纯合蛋白复合物棒状结构由5个该四聚体构成。另外，每一个MagR蛋白包括一个2Fe2S的铁硫中心，也即每一个MagR蛋白包括一个铁结合位点，结合了2个铁原子。整个棒状的纯合蛋白复合物包含约20个铁结合位点，结合了约40个铁原子。所述多个铁原子位于棒状结构的蛋白复合物的内部近中心轴区域。磁感应受体蛋白单体结合铁原子的位置相同或相近，处于棒状结构的蛋白复合物的立体结构的内部，由此，在磁感应受体蛋白单体线性聚合棒状结构的蛋白复合物中，所述多个铁原子也在蛋白复合物中形成有规律的排列。
图6为家鸽MagR-Cry4杂合蛋白复合物的结构图。图6中，A显示杂合蛋白复合物的纵截面的电镜形态(左上)、组织形式(左下)和结构示意(右)，B显示杂合蛋白复合物的横截面的电镜形态(左上)、组织形式(左下)和结构示意(右)。在MagR-Cry4杂合蛋白复合物中，在由约20个MagR组成的纯合蛋白复合物所形成的棒状线性双螺旋聚合体的外围，有约10个感光的Cry4蛋白缠绕排列，从而形成杂合复合物。同样地，整个棒状的杂合蛋白复合物包含约20个铁结合位点，结合了约40个铁原子。所述多个铁原子位于棒状结构的蛋白复合物的内部近中心轴区域，也形成有规律的排列。
实施例7：家鸽MagR蛋白复合物结晶
将实施例3中经分子筛凝胶层析纯化后获得的家鸽MagR纯合蛋白复合体的蛋白样品以及实施例4中经分子筛凝胶层析纯化后的家鸽MagR-Cry4杂合蛋白复合物的蛋白样品浓缩，然后进行结晶。蛋白质结晶方法按照如教科书McPherson,A.(2009).Introduction to macromolecular crystallography.Hoboken,N.J.,Wiley-Blackwell记载的标准实验流程采用悬滴法进行。在硅化过的显微镜盖玻片上将蛋白质溶液与等体积的沉淀剂溶液混合形成液滴；盖玻片被倒置在盘子的空穴上方，空穴中放置了所需的沉淀剂溶液，在盖玻片放置前，在空穴的边缘涂抹油或脂以密封空穴。大约2-7天后可以观察到MagR蛋白复合物形成的晶体。
图9显示了本发明的家鸽MagR蛋白形成的纯合复合物的晶体照片。
结果：共观察到两种类型的晶体：
第一种是黑色不透明的长方体形状的晶体(参见图9中上排给出的晶体照片；所使用的结晶条件为：0.1M HEPES，pH7.5，3.0M氯化钠)，其长度约为0.5mm，显示铁元素在晶体中已被氧化成均一的三价。第二种晶体为浅黄色或黄色的透明晶体(参见图9中下排给出的晶体照片；所使用的结晶条件为：0.1M BIS-TRIS，pH6.5，3.0M氯化钠)，形状不规则或呈小片状，显示铁元素可能以二价存在。在以下实施例的实验中证明，两种晶体都表现出非常强的磁性。
本发明的家鸽MagR蛋白与Cry4蛋白形成的杂合复合物的晶体的形态与图9的照片显示的晶体相似。
实施例8：家鸽MagR蛋白复合物的磁性测试
家鸽MagR纯合蛋白复合物以及家鸽MagR-Cry4杂合蛋白复合物的磁性测试通过以下几个实验来进行：
(1)电子显微镜下的磁性测试：
在实施例6中描述的电镜分析制样中，将实施例3中经分子筛凝胶层析纯化后获得的家鸽MagR纯合蛋白复合物的蛋白样品以及实施例4中经分子筛凝胶层析纯化后的家鸽MagR-Cry4杂合蛋白复合物的蛋白样品制备负染电镜样品，此时在电镜铜网样品上标记制样时的地球磁场方向或者人工外加的磁场方向，在电镜得到的蛋白颗粒的图像中，统计所有的棍状的蛋白复合物颗粒的方向(蛋白为长条形棒状，其方向是指该蛋白长度方向，即长轴的轴向)，按照图7所示的三个方向统计(平行于地球或者外加磁场方向的，垂直于地球或者外加磁场方向的，以及在两者之间不能归于上述两个方向的)，计算各个方向的棍状蛋白复合物所占的比例。
结果如图7所示。图7为利用电子显微镜对MagR纯合蛋白复合物在磁场下的排列方向进行研究，显示MagR蛋白复合物具有自身磁性，达到了类似“蛋白质指南针”的效果。图7的A为电子显微镜照片，其中用深色方框标示平行于磁场方向(即与磁场方向相同或相差小于等于30度)的蛋白质复合物颗粒，浅色方框标示垂直于磁场方向(即与磁场垂直的方向相同或相差小于等于30度)的蛋白质复合物颗粒，圆圈显示介于两者之间的其它方向。图7的B显示对于大样本的颗粒数目按排列方向进行统计的结果。每次实验包括大约1500个蛋白复合物颗粒进行统计分析，实验重复三次，取平均值和标准差作图。图7的B中横坐标为磁场的强度，以mT为单位，地球磁场为0.04mT(以北京的地理位置为准)，试验中所采用的外加的磁场通过永磁铁获得，磁场强度经过磁力计测量为1mT。纵坐标为沿着三个不同的方向(平行于地球磁场或者外加磁场的方向，垂直于地球磁场或者外加磁场的方向，介于两者之间的其它方向)排列的MagR蛋白复合物在所有蛋白复合物中所占的比例。结果显示，家鸽磁感应受体蛋白MagR形成的纯合蛋白复合物有将近或超过一半的蛋白质颗粒顺应(平行于)地球磁场或者外加人工磁场的方向。增加磁场强度，平行于磁场方向的蛋白质颗粒所占比例将随之上升，其它两个方向的比例将相应地下降。这充分说明即使在微观条件下的纳米尺度的蛋白质颗粒也呈现了非常明显的磁性，具备与指南针类似的物理性质。蛋白质颗粒在磁场中的方向不是像宏观世界的指南针一样100％地顺应磁场方向分布，是因为在微观的纳米尺度下，分子热运动和溶液中的布朗运动严重影响了蛋白质颗粒的取向，使得微观粒子(如本实验中的纳米尺度下的蛋白质颗粒)的取向只遵循统计物理学的规律。
本发明的家鸽MagR蛋白与Cry4蛋白形成的杂合复合物的晶体受磁场影响的实验和电镜观察结果与图7的照片显示的相似。
(2)利用蛋白质的磁性对蛋白质进行分离和纯化：
家鸽MagR纯合蛋白复合物和家鸽MagR-Cry4杂合蛋白复合物可用铁粉从细胞裂解液中快速分离和纯化出来，表明它们均具有磁性。
以家鸽MagR-Cry4杂合蛋白复合物为例，具体方法如图8所示：将实施例4中共表达MagR与Cry4蛋白质的大肠杆菌细胞收集起来，用缓冲液(20mM Tris，150mM NaCl，pH7.5)重悬，超声破碎后取上清液，向其加入市售的纳米铁粉颗粒(海狸生物科技有限公司，#70301)，室温孵育10min后，用标准TBS缓冲液(20mM Tris，150mM NaCl，pH7.5)清洗铁粉颗粒，然后用蛋白电泳上样缓冲液对铁粉进行洗脱，对洗脱液进行变性聚丙烯酰胺凝胶电泳(SDS-PAGE)，分析蛋白成分。
结果显示，经上述方法得到的纳米铁粉洗脱液中含有对应的蛋白。如图8所示，在实施例4中共表达MagR与Cry4蛋白质的大肠杆菌细胞的样品经上述方法得到的纳米铁粉洗脱液经变性聚丙烯酰胺凝胶电泳后显示出同时含有分子量约为15KDa的条带(MagR)和约为70KDa的条带(Cry4)。
另外，在实施例2中表达家鸽MagR的大肠杆菌细胞的样品经上述方法得到的纳米铁粉洗脱液经变性聚丙烯酰胺凝胶电泳后显示出分子量约为15KDa的条带(MagR)。
(3)蛋白质晶体的磁性检测：
将实施例7中获得的MagR纯合蛋白复合物的晶体或MagR-Cry4杂合蛋白复合物的晶体置于光学显微镜下观察，在显微镜的载物台上安装旋转的磁场，保持均匀的磁场强度(10高斯)。将晶体生长的液滴(其中含有蛋白质晶体)置于旋转磁场的中心。图9为家鸽MagR纯合蛋白复合物晶体在旋转磁场中同步旋转实验结果图。如图9所示，在磁场(圆圈中指针方向为磁场方向)旋转的时候，可以观察到蛋白质晶体几乎完全同步地沿着磁场旋转，如同由磁铁构成的指南针一样。当用磁铁去接近蛋白质晶体时，蛋白质晶体会立即靠近，当颠倒磁铁的磁极时，蛋白质晶体会即刻远离并颠倒方向，然后重新靠近，显示蛋白质形成的晶体如同磁铁一样，具有自身的磁性和磁极。
本发明的家鸽MagR蛋白与Cry4蛋白形成的杂合复合物的晶体在旋转磁场中同步旋转实验的结果与图9的照片显示的相似。
另外，当用铁磁性物质(如铁棒)接近前述晶体生长的液滴(其中含有蛋白质晶体)时，蛋白质晶体会向该铁磁性物质迅速靠近，甚至飞出液滴去接近该铁性物质，显示蛋白质晶体具备非常强的磁性。
(4)蛋白质的磁性测量
为了在物理学上证明蛋白质是否具有内在的磁性，在溶液状态下，对实施例4中获得的纯化的家鸽MagR-Cry4杂合蛋白复合物的样品进行物理学的磁性测定，得到了可以重复的非常精确的磁测量结果。所用仪器是超导量子干涉磁强计((Superconducting Quantum Interference Device，SQUID)，型号为MPMS-XL1。所有测量均在室温下进行，为标准的SQUID测量流程，具体为：将纯化的家鸽MagR-Cry4杂合蛋白复合物保存在缓冲液中(20mM Tris,pH7.5,150mM NaCl)，蛋白质浓度为3.8mg/ml；负对照为：(1)对蛋白质样品的样品管(holder)进行测量，确认没有磁滞回线后才用于后续的蛋白质磁 性测量；(2)对缓冲液的样品管(holder)进行测量，确认没有磁滞回线后才用于后续的缓冲液磁性测量作为基准；(3)对缓冲液在同样条件下进行测量。最终，蛋白质的测量结果以缓冲液测量结果修正。
测量结果如图14和图15所示。图14为家鸽MagR-Cry4杂合蛋白复合物在溶液中的室温磁性测量之负对照，其中，a为装有家鸽MagR-Cry4杂合蛋白复合物的样品管(Holder)的SQUID磁性测量结果，显示无磁滞回线；b为SQUID实验中装载蛋白质储存的溶液(即缓冲液)的样品管的SQUID磁性测量结果，显示无磁滞回线；c为溶液对照，即蛋白质储存的溶液(即缓冲液)的SQUID磁性测量结果，显示无磁滞回线。证明了实验过程中使用的装载蛋白质或者缓冲液的容器，以及缓冲液均无磁性污染。横坐标为磁场强度，纵坐标为磁矩。
图15中，a为家鸽MagR-Cry4杂合蛋白质复合物在溶液中在室温下的SQUID磁性测量结果，显示有明显的磁滞回线；b为同一管家鸽MagR-Cry4杂合蛋白质复合物在测量持续40小时以后，磁滞回线消失；c中左侧为测量前的蛋白样品SDS-PAGE结果，箭头标示Cry4(上)和MagR蛋白质(下)的位置，右侧为40小时后磁滞回线消失后，对同一管样品的SDS-PAGE分析，显示Cry4蛋白已经降解为几条小分子量的条带，MagR已经完全降解消失。可以看出家鸽MagR-Cry4杂合蛋白复合物在溶液中室温下具有明显的磁滞回线；而蛋白质降解后，磁滞回线消失，证明了SQUID实验测到的磁性来自于蛋白质本身，而不是容器(如样品管)，也不是来自于溶液(缓冲液)。
实施例9：黑脉金斑蝶MagR蛋白的获得
使用果蝇MagR蛋白序列(SEQ ID NO:3)在美洲帝王蝶基因组中用blast寻找同源序列，并将找到的同源区域提取出来在sequencher中与果蝇MagR编码区序列进行比对，获得5个外显子区域位置。将得到的5个外显子序列提取出来、拼接、翻译，得到的蛋白序列与其他物种MagR序列一致性较高，且含有保守区，因此认定为黑脉金斑蝶MagR蛋白序列，具体序列如SEQ ID NO:2所示，其编码区核苷酸序列为：
ATGTCTACTAAAACTATAGCAAGTGCAACTGTTAGGGCAGTAAAAAAGCGTCTGCTACCATCCAGAGCTGCTCTAGTTTTGACTTCTTCAGCCGTAAATAAAGTTAAGGAAATAATGGCCAAGGAAGAAGGCAAGGGTTATATAGGATTGAAAGTTGGTGTGCGGCAAAGAGGTTGCAATGGATTGTCATATACCTTAGATTATGCAACATCAAAAGGGAAACTTGACGAAGAAGTAAAACAGGATGGAGTCACTATAATTATTGACAAAAAAGCACAGTTGACCTTGTTGGGTACTGAAATGGATTTTGTCGAAGATAAGCTGTCAGCGGAATTTGTGTTTAACAATCCGAATATAAAAGGCACTTGTGGATGTGGAGAATCTTTCAGTATATAA(SEQ ID NO:37)。
之后在黑脉金斑蝶的基因组DNA中扩增MagR蛋白序列，验证上述序列正确。
实施例10：黑脉金斑蝶MagR蛋白的表达和纯化
1.人工合成编码以下氨基酸序列的插入片段(表达框)：
MWSHPQFEKGGSTSMSTKTIASATVRAVKKRLLPSRAALVLTSSAVNKVKEIMA KEEGKGYIGLKVGVRQRGCNGLSYTLDYATSKGKLDEEVKQDGVTIIIDKKAQLTLLG TEMDFVEDKLSAEFVFNNPNIKGTCGCGESFSI(SEQ ID NO:29)；
插入片段的序列：
CATATGTGGAGCCACCCCCAGTTCGAAAAGGGCGGTTCCACTAGTATGTCTAC TAAAACTATAGCAAGTGCAACTGTTAGGGCAGTAAAAAAGCGTCTGCTACCATCC AGAGCTGCTCTAGTTTTGACTTCTTCAGCCGTAAATAAAGTTAAGGAAATAATGGC CAAGGAAGAAGGCAAGGGTTATATAGGATTGAAAGTTGGTGTGCGGCAAAGAGG TTGCAATGGATTGTCATATACCTTAGATTATGCAACATCAAAAGGGAAACTTGACG AAGAAGTAAAACAGGATGGAGTCACTATAATTATTGACAAAAAAGCACAGTTGAC CTTGTTGGGTACTGAAATGGATTTTGTCGAAGATAAGCTGTCAGCGGAATTTGTGT TTAACAATCCGAATATAAAAGGCACTTGTGGATGTGGAGAATCTTTCAGTATATAAGGATCC(SEQ ID NO:30)。
其中，第2-9个氨基酸WSHPQFEK(SEQ ID NO:26)为Strep II亲和标签序列；第10-14个氨基酸GGSTS(SEQ ID NO:25)为链接区序列；带下划线的序列为黑脉金斑蝶(或称美洲帝王蝶，拉丁名Danaus plexippus，以下也可简称为“蝴蝶”)的MagR的蛋白质序列，即如SEQ ID NO：2所示的氨基酸序列。
核苷酸序列中，第1-6位是NdeI酶切位点，7-30位是StrepII表达序列，31-45位是GGSTS(SEQ ID NO:25)表达序列，46-438位是黑脉金斑蝶MagR表达序列，439-441位是终止密码子，442-447位是BamHI酶切位点。该基因在博迈德生物科技有限公司进行合成所得。
2.将上述合成的编码蝴蝶MagR蛋白的氨基酸序列并且N端具有Strep II亲和标签的插入片段通过NdeI和BamHI位点克隆到pET-21a(+)质粒(Novagen)。
3.将上述包含具有编码蝴蝶MagR蛋白的氨基酸序列且N端具有Strep II亲和标签的插入片段的质粒转化大肠杆菌BL21(DE3)(商购)。挑取单克隆接种至LB液体培养基中扩大培养，15℃下用20μM的IPTG诱导目的蛋白的表达，20-24小时后收菌。
4.将上述得到的表达了MagR蛋白的转化菌用缓冲液(20mM Tris，150mM NaCl，pH8.0，10mM巯基乙醇以及蛋白酶抑制剂)重悬后，用超声破碎仪裂解，完成后进行高速离心，取上清液进行亲和纯化。通过Strep-Tactin亲和柱亲和纯化后，进行阴离子交换柱或者分子筛凝胶层析进一步纯化，得到纯度较高的蝴蝶MagR蛋白。纯化全过程在4℃进行。纯化后得到的MagR蛋白在浓度较高时可呈现明显的棕黄色或棕褐色，这提示蛋白质中结合了二价或三价的铁。
5.对收集的MagR蛋白样品进行变性聚丙烯酰胺凝胶电泳(SDS-PAGE)分析。实验结果如图10所示，其中各泳道的样品为：1、蛋白质分子量标准；2、纯化的含有Strep II亲和标签的蝴蝶MagR蛋白，分子量约为15KDa。
实施例11：黑脉金斑蝶MagR蛋白形成纯合复合物的实验
将实施例10中表达和纯化的蝴蝶MagR蛋白，在通过Strep-Tactin亲和柱亲和纯化后进一步用Superdex 200 10/300 GL纯化柱(GE Healthcare)进行分子筛凝胶层析(实验过程参考实施例3)。
样品的分子筛凝胶层析呈现出单体峰、二聚体峰和多聚峰，分别收集三个峰的样品，获得纯化蛋白。这显示，纯化的MagR蛋白在分子筛凝胶层析中以三种形式存在。与蛋白质分子量标准进行比较可知，三个峰的大致分子量为15KDa、30KDa和大于200KDa，这与MagR蛋白的单体、二聚体和多聚体的分子量相吻合。由于 Superdex S200的分辨率最高为约200KDa，所以，尚无法判断多聚体的分子量范围和多聚体所含单体个数。
实施例12：黑脉金斑蝶MagR蛋白和Cry1蛋白形成的杂合蛋白复合物的表达和纯化
1.人工合成编码以下氨基酸序列的核苷酸插入片段(表达框)：
MHHHHHHHHHHGGSTSMLGGNVIWFRHGLRLHDNPSLHSALEDASSPFFPIFIFD GETAGTKMVGYNRMRYLLEALNDLDQQFRKYGGKLLMIKGRPDLIFRRLWEEFGIRT LCFEQDCEPIWRPRDASVRALCRDIGVSCREHVAHTLWNPDTVIKANGGIPPLTYQMF LHTVEIIGNPPRPVDDVDLNGVNFGSLPESFYREFVVFDKAPKPEDLGVFLENEDIRMIR WVGGETAALKQMQERLAVEYETFCRGSYLPTHGNPDLLGPPISLSPALRFGCLSVRRF YWSLQDLFQQVHQGRLASTQFITGQLIWREYFYTMSVNNPNYAQMSGNPICLDIPWK EPENDELQRWKEGRTGFPFVDAAMRQLRTEGWLHHVVRNTVASFLTRGTLWLSWEH GLQHFLKYLLDADWSVCAGNWMWVSSSAFEALLDSGECACPVRLGRRLEPTGHYVR RYVPELARMPGEYIYEPWRAPLEVQEAAGCVIGRDYPAPVVDHTAAAARNRANMQE LRRLLEKAPPHCCPSSEDEVRQFMWLGDDSQPELTTT(SEQ ID NO:31)；
插入片段的序列：
CATATGCATCATCATCATCATCACCATCACCATCACGGCGGTTCCACTAGTAT GCTTGGTGGTAATGTCATTTGGTTCCGTCACGGTCTCCGTCTCCACGACAACCCTTC GCTTCACAGCGCTCTGGAAGATGCAAGCTCACCGTTCTTCCCTATATTCATATTTG ATGGAGAGACAGCTGGTACAAAGATGGTGGGCTACAATCGTATGCGATACCTGCT GGAGGCGCTGAACGATTTGGACCAGCAGTTCAGGAAGTACGGCGGGAAACTGCTC ATGATTAAGGGGAGACCTGATTTAATATTCAGGAGGCTGTGGGAGGAATTTGGTA TACGTACGCTATGCTTCGAGCAGGACTGTGAGCCAATATGGCGTCCGCGCGACGC GAGCGTGCGTGCTCTGTGCCGCGACATAGGCGTGTCGTGCCGCGAGCACGTCGCA CACACGCTGTGGAACCCGGACACAGTCATCAAGGCCAATGGAGGAATACCGCCGC TTACGTACCAGATGTTCCTGCATACAGTTGAAATCATCGGTAATCCTCCGCGTCCC GTAGACGACGTCGACCTGAACGGCGTCAACTTTGGCTCGCTGCCTGAGAGCTTCTA CAGGGAATTCGTTGTCTTTGATAAGGCTCCAAAACCAGAAGATCTGGGTGTGTTCC TGGAAAACGAAGATATTCGTATGATTCGCTGGGTGGGAGGAGAGACAGCGGCCTT GAAGCAGATGCAGGAGAGATTGGCTGTGGAGTACGAGACATTCTGCAGGGGTTCT TATTTGCCGACCCATGGCAACCCCGACCTCCTTGGACCGCCGATATCTCTGAGTCC AGCCTTGCGCTTCGGATGTCTGTCTGTCCGTCGCTTCTACTGGAGTCTCCAGGACCT GTTCCAGCAGGTGCATCAGGGACGCCTGGCTTCCACTCAGTTTATCACTGGTCAGT TAATATGGCGGGAGTATTTCTACACGATGAGCGTCAATAACCCCAACTACGCCCA AATGTCGGGGAATCCTATCTGCCTGGACATACCGTGGAAGGAACCGGAAAATGAC GAGTTACAGAGATGGAAGGAGGGTCGTACGGGGTTCCCATTCGTGGACGCGGCCA TGCGCCAGCTGCGTACGGAGGGCTGGTTGCATCACGTTGTTCGGAACACCGTGGCC TCGTTCCTCACCCGCGGGACCCTGTGGCTGTCCTGGGAACACGGGCTGCAGCACTT CCTCAAGTATCTGCTGGATGCTGATTGGTCGGTGTGTGCGGGTAACTGGATGTGGG TGTCGTCCAGTGCGTTCGAGGCCTTGTTGGACTCTGGCGAGTGCGCGTGTCCCGTC AGACTGGGCCGAAGACTGGAGCCCACTGGCCATTATGTACGGAGATACGTACCAG AACTGGCTCGGATGCCCGGAGAGTACATTTACGAGCCGTGGCGTGCCCCGCTCGA GGTGCAGGAGGCTGCGGGCTGTGTCATAGGTCGAGACTACCCCGCGCCGGTCGTC GACCACACAGCTGCGGCCGCCAGGAACAGGGCCAACATGCAGGAGCTGCGCCGCC TGTTGGAGAAAGCTCCTCCTCACTGCTGTCCGTCATCTGAAGACGAGGTGCGGCAG TTCATGTGGCTTGGAGACGACTCGCAGCCTGAGCTCACCACCACATGAGGATCC(SEQ ID NO:32)。
其中，第2-11个氨基酸为10个组氨酸的His亲和标签序列；第12-16个氨基酸GGSTS(SEQ ID NO:25)为链接区序列；带下划线的序列为蝴蝶的Cry1的氨基酸序列，即如SEQ ID NO：16所示的氨基酸序列。
核苷酸序列中，第1-6位是NdeI酶切位点，7-36位是10xHis表达序列，37-51位是GGSTS(SEQ ID NO:25)表达序列，52-1653位是黑脉金斑蝶Cry1表达序列，1654-1656位是终止密码子，1657-1662位是BamHI酶切位点。该基因在博迈德生物科技有限公司进行合成所得。
2.将上述合成的编码蝴蝶Cry1的氨基酸序列并且N端具有His亲和标签的插入片段通过NdeI和BamHI位点克隆到pET-21a(+)质粒(Novagen)。
3.将前述具有蝴蝶Cry1蛋白的编码基因并融合了组氨酸标签(His-tag)的质粒与实施例10步骤2得到的具有蝴蝶MagR蛋白的编码基因并融合了StrepII标签的质粒共同转化大肠杆菌BL21(DE3)，表达MagR的质粒为卡那霉素抗性，表达Cry1的质粒为氨苄青霉素抗性，在双抗培养基(卡那霉素和氨苄青霉素)中挑取含有两种质粒的大肠杆菌单克隆接种至LB液体培养基中扩大培养，15℃下用20μM的IPTG诱导目的蛋白的表达，20-24小时后收菌。
4.将上述得到的共表达了蝴蝶MagR蛋白和Cry1蛋白的转化菌用缓冲液(20mMTris，150mM NaCl，10mM咪唑，pH8.0，10mM巯基乙醇以及蛋白酶抑制剂)重悬后，用超声破碎仪裂解，完成后进行高速离心，取上清液进行亲和纯化。通过Ni-NTA亲和柱亲和纯化后，再通过Strep-Tactin亲和柱纯化，得到蝴蝶MagR蛋白和Cry1蛋白形成的MagR-Cry1杂合蛋白复合物。进一步通过阴离子交换柱和分子筛凝胶层析纯化，得到纯度较高的杂合蛋白复合物。纯化全过程在4℃和存在外加磁场的条件下进行。外加磁场通过下述方法施加：当样品过亲和柱时，将两个相同的条形磁铁分别绑在亲和柱的两侧，该两个相同的条形磁铁的南极和北极分别相对，提供一个均匀稳定的磁场。结果显示，在纯化时施加外加磁场(外加磁场的强度是地球磁场的100倍)，纯化蛋白的产率有显著增加；在洗脱纯化蛋白时，撤去外加磁场，蛋白质洗脱效率更高。
5.对收集的蝴蝶MagR-Cry1蛋白复合物样品进行变性聚丙烯酰胺凝胶电泳(SDS-PAGE)分析。实验结果如图2所示，其中从左向右第2泳道加载的样品为蛋白质分子量标准，第3泳道加载的样品为蝴蝶MagR-Cry1杂合蛋白复合物。结果：由于使用变性电泳分析，泳道3中显示出两种蛋白质样品，即含有Strep II亲和标签的蝴蝶MagR蛋白(分子量约为15KDa)以及含有His标签的蝴蝶Cry1蛋白(分子量约为70KDa)。
实施例13：黑脉金斑蝶MagR蛋白以及MagR纯合蛋白复合物含有铁的证明实 验
将实施例11得到的蝴蝶MagR蛋白的单体峰样品用分光光度计(Nanodrop 2000)进行全波长扫描。结果与图3相似。
将实施例11中得到的蝴蝶MagR蛋白形成的二聚物或多聚物样品，用分光光度计(Nanodrop 2000)进行全波长扫描。结果与图3相似。
实施例14：黑脉金斑蝶MagR纯合蛋白复合物和黑脉金斑蝶MagR-Cry1杂合蛋白复合物的电子显微镜图像和结构
将实施例11中经分子筛凝胶层析纯化后获得的蝴蝶MagR纯合蛋白复合物的蛋白样品以及实施例12中经分子筛凝胶层析纯化后的蝴蝶MagR-Cry1杂合蛋白复合物的蛋白样品采用乙酸铀或者甲酸铀负染，制备蛋白质的负染电镜样品，将样品放置于透射电镜下观察并拍照，按照标准的电镜结构分析方法解析其结构，并测量大小(包括蛋白长度和宽度)。
电镜结果显示，蝴蝶MagR纯合蛋白复合物和蝴蝶MagR-Cry1杂合蛋白复合物的结构形式与实施例6图4中观测到的家鸽MagR纯合蛋白复合物和MagR-Cry4杂合蛋白复合物的结构形式相同。
电镜观测到的结果显示，蝴蝶MagR纯合蛋白复合物呈现为长条形的棒状结构，长度为约20-24纳米长，宽度为约7-11纳米。根据同源晶体结构和分子模型计算，结果表明该复合物具有约20个MagR蛋白。电镜观测到的结果显示，蝴蝶MagR-Cry1杂合蛋白复合物呈现为棒状，长度为约20-24纳米长，宽度为约13-18纳米宽。根据同源晶体结构和分子模型计算，结果表明该复合物具有约20个MagR蛋白和10个Cry1蛋白。
电镜结果还显示，每一个MagR纯合蛋白复合物包括约20个MagR蛋白质，有序排列形成线性双螺旋聚合体，其中每10个MagR蛋白单体以线性聚合的形式首尾相接，形成两条蛋白长链，两条蛋白长链以螺旋形式结合，由此形成长条形棒状结构的蛋白复合物。
电镜结果还显示，在蝴蝶MagR-Cry1杂合蛋白复合物中，在由约20个MagR组成的蛋白复合物所形成的棒状线性双螺旋聚合体的外围，有约10个感光的Cry1蛋白质缠绕排列，从而形成杂合蛋白复合物。
实施例15：黑脉金斑蝶MagR蛋白复合物结晶
将实施例11中经分子筛凝胶层析纯化后获得的蝴蝶MagR纯合蛋白复合物的蛋白样品以及实施例12中经分子筛凝胶层析纯化后的蝴蝶MagR-Cry1杂合蛋白复合物的蛋白样品浓缩，然后进行结晶。蛋白质结晶方法按照如教科书McPherson,A.(2009).Introduction to macromolecular crystallography.Hoboken,N.J.,Wiley-Blackwell记载的标准实验流程采用悬滴法进行。
蝴蝶MagR纯合蛋白复合物的晶体和蝴蝶MagR-Cry1杂合蛋白复合物的晶体的形态与对应的家鸽MagR纯合蛋白复合物的晶体或家鸽MagR-Cry4杂合蛋白复合物的晶体类似，分别制备和观察到两种类型的晶体：第一种是黑色不透明的长方体形状的晶体，提示铁元素在晶体中已被氧化成均一的三价；第二种晶体为浅黄色或黄色的透明晶体，形状不规则或呈小片状，提示铁元素可能以二价存在。图11示出了 蝴蝶MagR-Cry1杂合蛋白复合物的晶体照片，可见该晶体具有黑色不透明的长方体形状，长度约为0.5mm。
实施例16：黑脉金斑蝶MagR蛋白复合物的磁性测试
采用与实施例8中同样的方法对蝴蝶MagR蛋白形成的纯合蛋白复合物以及蝴蝶MagR-Cry1杂合蛋白复合物的磁性进行测试。结果显示，蝴蝶MagR纯合蛋白复合物和蝴蝶MagR-Cry1杂合蛋白复合物的磁性测试结果与家鸽的测试结果(如实施例8中观察到的)类似。这表明，蝴蝶MagR纯合蛋白复合物和蝴蝶MagR-Cry1杂合蛋白复合物具有磁性。
图11为蝴蝶MagR纯合蛋白复合物晶体在旋转磁场中同步旋转实验结果图。实验方法如实施例8的(3)所描述。如图11所示，可以观察到蝴蝶MagR-Cry1纯合蛋白复合物的晶体为黑色，并且在旋转磁场中发生同步旋转。同样，蝴蝶MagR杂合蛋白复合物的晶体在旋转磁场中也出现同步旋转的现象。
实施例17：家鸽MagR蛋白以及MagR纯合蛋白复合物具有结合铁的保守结构域的证明实验
根据实施例2、3和5描述的方法和步骤，测试将家鸽MagR的如SEQ ID NO：1所示的氨基酸序列中的三个半胱氨酸C60、C124、C126突变为丙氨酸(A，密码子为GCC)后得到的蛋白结合铁的能力。
即将实施例2步骤1中人工合成的插入片段所编码的氨基酸序列改变为：
MWSHPQFEKGGGTSMASSASSVVRATVRAVSKRKIQATRAALTLTPSAVQKIKELLKDKPEHVGVKVGVRTRGANGLSYTLEYTKSKGDSDEEVVQDGVRVFIEKKAQLTLLGTEMDYVEDKLSSEFVFNNPNIKGTAGAGESFNI(SEQ ID NO:33)。
根据实施例2和3描述的方法和步骤表达和纯化具有上述C60A、C124A、C126A突变的家鸽MagR蛋白以及MagR纯合蛋白复合物。突变的家鸽MagR蛋白经过分子筛凝胶层析，也呈现出单体峰、二聚体峰和多聚体峰。分别收集三个峰的样品。将得到的蛋白样品分别根据实施例5描述的方法用分光光度计(Nanodrop 2000)进行全波长扫描。
如图12所示，结果显示上述突变蛋白形成的家鸽MagR单体蛋白的吸收曲线(实线所示)在Fe元素的特征吸收位置(320nm和410nm)没有吸收峰。作为对照的野生型的家鸽MagR蛋白以及MagR纯合蛋白复合物的吸收曲线(虚线所示)则显示在320nm和410nm有明显的吸收峰。类似的，根据前述方法经过分子筛凝胶层析和纯化后得到的突变蛋白形成的家鸽MagR的二聚物或多聚物的吸收曲线在Fe元素的特征吸收位置(320nm和410nm)也没有吸收峰。
结果表明，家鸽MagR蛋白具有结合铁的结构域，其包括C60、C124、C126三个位点中的一个或多个。
实施例18：各物种MagR蛋白的序列比对
测序或根据NCBI数据库获得果蝇(Drosophila melanogaster)、黑脉金斑蝶(Danaus plexippus)、蜜蜂(Apis mellifera)、文昌鱼(Branchiostoma floridae)、斑马鱼(Danio rerio)、蟒蛇(Python bivittatus)、家鸽(Columba livia)、草雀(Taeniopygia guttata)、鸡(Gallus gallus)、鲸鱼(Balaenoptera acutorostrata)、蝙蝠(Myotis lucifugus)、裸鼹鼠(Heterocephalus glaber)、 小鼠(Mus musculus)、人(Homo sapiens)的磁感应受体蛋白的氨基酸序列，其分别如SEQ ID NO：3、SEQ ID NO：2、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：1、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13或SEQ ID NO：14所示。
用Clustal V比对方法对获得的氨基酸序列进行比对。
图13是上述各物种MagR蛋白的序列比对的结果。其中从上至下的来源物种名称以及基因在NCBI的序列号(如果有的话)分别为：(1)果蝇(Drosophila melanogaster)，基因序列号为NP_573062.1；(2)黑脉金斑蝶(Danaus plexippus)；(3)蜜蜂(Apis mellifera)，基因序列号为XP_624993.1；(4)文昌鱼(Branchiostoma floridae)，基因序列号为XP_002589524.1；(5)斑马鱼(Danio rerio)，基因序列号为NP_001020349.1；(6)蟒蛇(Python bivittatus)，基因序列号为XP_007429307.1；(7)家鸽(Columba livia)；(8)草雀(Taeniopygia guttata)，基因序列号为XP_002194930.1；(9)鸡(Gallus gallus)，基因序列号为XP_003643055.1；(10)鲸鱼(Balaenoptera acutorostrata)；(11)蝙蝠(Myotis lucifugus)，基因序列号为XP_006102189.1；(12)裸鼹鼠(Heterocephalus glaber)，基因序列号为XP_004879403.1；(13)小鼠(Mus musculus)，基因序列号为NP_081197.1；(14)人(Homo sapiens)，基因序列号为NP_112202.2。其中(2)黑脉金斑蝶、(7)家鸽和(10)鲸鱼的序列为本申请发明人测序获得。
结果显示，本申请发明人首次研究、制得、鉴定并命名的磁感应受体蛋白的核酸编码序列在生物体基因组中广泛存在。本发明提供的磁感应受体蛋白的核酸序列在大量不同物种的基因组中都存在，其编码的蛋白序列高度保守，并具有多个高度保守的结构域，如结合铁和/或硫的结构域，在图13中箭头所指的三个100％保守的半胱氨酸残基(C)，其为结合铁(或铁硫中心)的位置。
除非另外指出，本发明的实施方式可使用生物技术、有机化学、无机化学等的常规技术，显然除在上述说明和实施例中所特别描述之外，还可以别的方式实现本发明。其它在本发明范围内的方面与改进将对本发明所属领域的技术人员是显而易见的。根据本发明的教导，许多改变和变化是可行的，因此其在本发明的范围之内。本文所提到的所有专利、专利申请与科技论文均据此通过引用并入本文中。

Claims (15)

  1. 一种分离的磁感应受体蛋白或其活性片段或衍生物，优选地，所述磁感应受体蛋白来自脊椎动物或无脊椎动物；进一步优选地，所述脊椎动物为家鸽、斑马鱼、蟒蛇、草雀、鸡、鲸鱼、蝙蝠、裸鼹鼠、小鼠或人，所述无脊椎动物为果蝇、文昌鱼、蜜蜂或黑脉金斑蝶。
  2. 权利要求1所述的磁感应受体蛋白或其活性片段或衍生物，其中，参照图13所示的氨基酸位置，所述磁感应受体蛋白或其活性片段或衍生物的氨基酸序列从N端到C端依次包含以下结构域：第10-13位的TVRA、第56-61位的RGCNGL、第80-83位的QDGV、第89-93位的KKAQL、第95-101位的LLGTEMD以及第115-128位的NPNIKGTCGCGESF。
  3. 权利要求2所述的磁感应受体蛋白或其活性片段或衍生物，还包含以下结构域：第17位的R、第23位的R、第25-26位的AL、第28-29位的LT、第32-33位的AV、第48位的G、第51位的V、第53位的V、第63位的Y、第67位的Y、第71位的K、第75-76位的DE、第78位的V、第87位的I、第104位的E、第106-107位的KL、第110-111位的EF和第113位的F。
  4. 权利要求1-3中任一项所述的磁感应受体蛋白或其活性片段或衍生物，其具有2Fe2S的铁硫中心。
  5. 权利要求1-4中任一项所述的磁感应受体蛋白或其活性片段或衍生物，其中所述磁感应受体蛋白或其活性片段或衍生物具有：
    (1)如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13或SEQ ID NO：14所示的氨基酸序列；或
    (2)在与SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13或SEQ ID NO：14进行比较时具有至少75％的序列同一性，优选具有至少90％的序列同一性，最优选具有至少95％的序列同一性的氨基酸序列；或
    (3)在与SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13或SEQ ID NO：14进行比较时具有一个或多个氨基酸的取代、缺失、添加的氨基酸序列。
  6. 一种分离的蛋白复合物，包含两个或两个以上如权利要求1-5中任一项所述的磁感应受体蛋白或其活性片段或衍生物。
  7. 权利要求6所述的蛋白复合物，其中所述蛋白复合物包含4个或以上所述磁感应受体蛋白或其活性片段或衍生物，优选为20-24个所述磁感应受体蛋白或其活性片段或衍生物，更优选为20个所述磁感应受体蛋白或其活性片段或衍生物。
  8. 权利要求7所述的蛋白复合物，其中，所包含的多个磁感应受体蛋白或其活性片 段或衍生物线性聚合，形成两条蛋白长链并以螺旋形式结合，形成棒状结构的复合物。
  9. 权利要求8所述的蛋白复合物，其具有多个铁原子，多个铁原子在棒状结构的内部近中心轴区域形成有序排列。
  10. 生产权利要求6-9中任一项所述的蛋白复合物的方法，其包含下述步骤：
    a.在允许形成蛋白复合物的条件下，使所述磁感应受体蛋白或其活性片段或衍生物聚合，形成同源聚合物；
    b.分离该同源聚合物；优选地，该步骤在存在磁场的条件下进行。
  11. 一种分离的杂合蛋白复合物，其包含1)如权利要求1-5中任一项所述的磁感应受体蛋白或其活性片段或衍生物，以及2)光感应蛋白或其活性片段或衍生物，每2个所述磁感应受体蛋白或其活性片段或衍生物与1个光感应蛋白或其活性片段或衍生物结合。
  12. 权利要求11所述的杂合蛋白复合物，其中，所述光感应蛋白为隐花色素蛋白；
    优选地，所述隐花色素蛋白具有：
    (1)如SEQ ID NO：15或SEQ ID NO：16所示的氨基酸序列；或
    (2)在与SEQ ID NO：15或SEQ ID NO：16进行比较时具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列，并且具有隐花色素蛋白的活性；或
    (3)在与SEQ ID NO：15或SEQ ID NO：16进行比较时具有一个或多个氨基酸的取代、缺失、添加的氨基酸序列。
  13. 权利要求11-12中任一项所述的杂合蛋白复合物，其中所述杂合蛋白复合物包含4个或以上所述磁感应受体蛋白或其活性片段或衍生物，优选包含20-24个所述磁感应受体蛋白或其活性片段或衍生物，更优选包含20个所述磁感应受体蛋白或其活性片段或衍生物。
  14. 一种生产权利要求11-13中任一项所述的杂合蛋白复合物的方法，包括以下步骤：
    a.在允许形成蛋白复合物的条件下，使1)如权利要求1-5中任一项所述的磁感应受体蛋白或其活性片段或衍生物与2)光感应蛋白或其活性片段或衍生物接触，形成杂合聚合物，其中1)和2)的蛋白中的至少一种是分离形式或被重组表达；
    b.分离该杂合聚合物；优选地，该步骤在存在磁场的条件下进行。
  15. 权利要求1-5中任一项所述的磁感应受体蛋白或其活性片段或衍生物或权利要求6-9中任一项所述的蛋白复合物或权利要求11-13中任一项所述的杂合蛋白复合物作为磁性材料或在制备磁性材料中的应用。
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