Patent Application
20110091956
The present invention relates to a biomolecule-immobilized carrier and a method for immobilizing a biomolecule to carrier, particularly to a biomolecule- and/or membrane-binding molecule-immobilized magnetic microparticle, a probe-immobilized carrier using said biomolecule and/or said membrane-binding molecule as a probe and a separating agent for a biomolecule.
Since a magnetic microparticle can be magnetically collected, conventional researches focus on a technology for immobilizing a biomolecule, such as enzyme and antibody, to the magnetic microparticle. For example, Japanese Unexamined Patent Application Publications No. 1998-075780 and 1999-243951 disclose a magnetic microparticle to which an enzyme is directly immobilized by a conventional chemical method (Patent Documents 1 and 2). Meanwhile, Japanese Unexamined Patent Application Publication No. 2005-289822 discloses a membrane-fusion magnetic microparticle (magnetosome) derived from a magnetic bacterium to which a peptide is anchored (Patent Document 3), and Japanese Unexamined Patent Application Publication No. 2006-075103 discloses a membrane-fusion magnetosome derived from a magnetic bacterium obtained by expressing a fusion protein composed of an anchor protein MMs13 and an IgG-binding peptide in vivo (Patent Document 4).
Meanwhile, some polypeptides having cellular adhesiveness are conventionally known, e.g. syndecan-binding laminin-derived polypeptide as extracellular sugar chain (Non-Patent Document 1), Arg-Gly-Asp (RDG) tripeptide sequence derived from fibronectin (Non-Patent Document 2) as integrin ligand sequence, or a polypeptide present on a C terminal side of α1,3-fucosyltransferase and α1,4-fucosyltransferase derived from Helicobacter pylori (Non-Patent Document 3 and 4) as polypeptide having cell membrane adhesiveness. It is also known that the polypeptide present on a C terminal side of α1,3/α1,4-fucosyltransferase derived from Helicobacter pylori is provided with an α-helix structure, thereby providing a property of binding to a cell membrane (Non-Patent Document 3).
Patent Document 1: Japanese Unexamined Patent Application Publication No. 1998-075780
Patent Document 2: Japanese Unexamined Patent Application Publication No. 1999-243951
Patent Document 3: Japanese Unexamined Patent Application Publication No. 2005-289822
Patent Document 4: Japanese Unexamined Patent Application Publication No. 2006-075103
Non-Patent Document 1: Pierschbacher, M. et al. Nature, 309, 303-303, 1984
Non-Patent Document 2: Mayumi Michizuki, Yuichi Kadoya, Motoyoshi Nomizu, Tanpakushitu kakusan koso (Protein nucleic acid enzyme), 50, 374-382, 2005
Non-Patent Document 3: J. Biol. Chem., 278, 21893-21900, 2003
Non-Patent Document 4: J. Biol. Chem., 282, 9973-9982, 2007
However, a magnetic microparticle disclosed in Patent Documents 1 and 2 is not given any specific properties on a repeated use thereof, other than a possible enzyme deactivation in an immobilizing reaction due to use of a chemical method, resulting in less reliable durability and biomolecule activity persistence. In addition, the magnetic microparticle causes a serious difficulty in controlling molecular orientation.
A magnetic microparticle disclosed in Patent Documents 3 and 4 is an extremely hydrophobic transmembrane peptide used as an anchor protein. Furthermore, Patent Document 3 discloses no successful technologies for utilizing a magnetic microparticle from a biomolecule such as enzyme as a useful protein anchored onto the magnetic microparticle, and Patent Document 4 discloses problems of encapsulation and flocculation due to expression of transmembrane peptide and target protein as a fusion protein. To solve these problems, it is necessary to prevent flocculation by treating a magnetic microparticle for solubilization using a surfactant of high concentration, but resultant deactivation of an enzyme, etc. by the surfactant can make a use of the magnetic microparticle difficult with an activity of the enzyme, etc. maintained.
To solve these problems, it is, therefore, one object of the present invention to provide a biomolecule-immobilized carrier, comprising immobilizing a biomolecule having a membrane-binding peptide containing a C terminal polypeptide of fucosyltransferase derived from Helicobacter pylori or a membrane-binding molecule having the membrane-binding peptide and various biomolecules and a method for immobilizing a biomolecule to a carrier, particularly a biomolecule-immobilized magnetic microparticle, a probe-immobilized carrier using the biomolecule and/or the membrane-binding molecule used as a probe, and a separating agent for a biomolecule to which the biomolecule-immobilized magnetic microparticle is applied.
The inventors successfully completed this invention by finding out a property of the above-mentioned known C terminal polypeptide of α1,3/α1,4-fucosyltransferase derived from Helicobacter pylori to firmly bind to an organic membrane containing a phospholipid (e.g. biomembrane and biomimetic membrane) as well and an ability of an α-helix structure to control molecular orientation according to a property of the C terminal polypeptide of α1,3/α1,4-fucosyltransferase derived from Helicobacter pylori to firmly bind to a cell membrane, a property to be solubilized in an aqueous solvent due to high charge and a property to have an α-helix structure when binding to a cell membrane.
Specifically, the present invention comprises the following items:
[1] A biomolecule-immobilized carrier, comprising binding a biomolecule having a membrane-binding peptide containing either an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence encoded by a base sequence represented by SEQ ID NO: 2 and/or a membrane-binding molecule having said membrane-binding peptide and the biomolecule to a carrier coated with an organic membrane containing a phospholipid;
[2] The biomolecule-immobilized carrier according to item [1], wherein said carrier is a magnetic microparticle, or a probe-immobilized carrier using a biomolecule having said membrane-binding peptide and/or said membrane-binding molecule as a probe;
[3] The biomolecule-immobilized carrier according to item [1] or [2], wherein said biomolecule is an enzyme or a sugar chain;
[4] The biomolecule-immobilized carrier according to item [3], wherein said enzyme is glycosyl transferase or glycosidase;
[5] A separating agent for a biomolecule, comprising binding a biomolecule having a membrane-binding peptide containing either an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence encoded by a base sequence represented by SEQ ID NO: 2 and/or a membrane-binding molecule having said membrane-binding peptide and the biomolecule to a magnetic microparticle coated with an organic membrane containing a phospholipid; and
[6] A method for immobilizing a biomolecule to a carrier which is a method for directly or indirectly immobilizing a biomolecule to a carrier with bioactivity maintained, comprising a phospholipid modifying step for modifying a fixed ligand to be immobilized to the carrier to a phospholipid, a carrier coating step for forming and coating an organic membrane on a surface of the carrier using a phospholipid which modifies said fixed ligand, and a binding step for binding a biomolecule having a membrane-binding peptide containing either an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence encoded by a base sequence represented by SEQ ID NO: 2 and/or a biomolecule to which said membrane-binding peptide is fused or bound as a linker to said organic membrane.
The present invention can provide a biomolecule-immobilized carrier capable of controlling molecular orientation with biomolecule activity maintained due to excellent durability and being readily handled due to solubilization of a membrane-binding molecule, particularly a biomolecule-immobilized magnetic microparticle, a probe-immobilized carrier using a biomolecule having said membrane-binding peptide and/or said membrane-binding molecule as a probe, and a separating agent for a biomolecule to which the biomolecule-immobilized magnetic microparticle is applied, in addition to a method for immobilizing a biomolecule to a carrier.
A biomolecule-immobilized carrier and a method for immobilizing a biomolecule to a carrier according to the present invention will be described in detail.
A biomolecule-immobilized carrier according to the present invention comprises binding a biomolecule having a membrane-binding peptide containing either an amino acid sequence encoded by an amino acid sequence represented by SEQ ID NO: 1 or a base sequence represented by SEQ ID NO: 2 and/or a membrane-binding molecule having said membrane-binding peptide and the biomolecule to a carrier coated with an organic membrane containing a phospholipid.
The membrane-binding peptide containing either an amino acid sequence encoded by amino acid sequence represented by the SEQ ID NO: 1 or a base sequence represented by SEQ ID NO: 2 is provided with a property of reversibility, i.e. a random structure in an aqueous solvent to be solubilized, but an α-helix structure in neutrality and in the presence of a phospholipid. Specifically, the membrane-binding peptide, which is, under neutrality conditions, of an α-helix structure to bind to an organic membrane, will be of a random structure under alkaline conditions (pH as approximately 10) to desorb from the organic membrane. According to the inventors, etc., it is also known that the membrane-binding peptide cannot be provided with an α-helix structure again only by lowering pH to make the membrane-binding peptide neutral, but it can be provided with an α-helix structure again in the presence of a phospholipid.
Conformational analysis of a membrane-binding peptide is not particularly limited in method, but in an embodiment according to the present invention, conformational analysis is performed by a method for measuring circular dichroism (CD).
“Bind(ing)” in this invention generally means that A is bound to B, or A binds to B, and two objects make contacts on the surface and the state is kept. In this invention, this term is interchangeably used as “immobilize (immobilizing)” or “immobilization.” Specifically, it means that other than a case in which a membrane-binding portion of a membrane-binding peptide and a surface of an organic membrane make contacts, a membrane-binding portion of a membrane-binding peptide and an organic membrane in this invention remain touched in such a way that the membrane-binding portion gets into or through the organic membrane.
“Having a membrane-binding peptide” in this invention means not only integration of a membrane-binding peptide into a membrane-binding portion having a membrane-binding ability, but also complete conversion of a membrane-binding peptide to a membrane-binding portion. In addition, except for a case in which a biomolecule naturally has a membrane-binding peptide such as fucosyltransferase derived from Helicobacter pylori, a biomolecule might have a membrane-binding peptide by making a biomolecule fuse or bind to a membrane-binding peptide, as shown in a membrane-binding molecule comprising fusing or binding a membrane-binding peptide to e.g. maltose-linked protein-fused galactose transferase (MBP-GalT), O-linked glycopeptide or N-acetylgalactosamine transferase 2 (ppGalNAcT2).
A biomolecule in this invention is a molecule which naturally has a membrane-binding peptide or a molecule which can fuse or bind to a membrane-binding peptide, and it is not particularly limited as long as the molecule is synthesized, metabolized and stored in vivo, e.g. DNA, RNA, PNA, aptamer, gene, chromosome, peptide, enzyme, receptor, sugar compound (monosaccharide, oligosaccharide, polysaccharide, sugar chain complex, glycoprotein, glycolipid, their derivative, etc), lipid (fatty acid, phospholipid, glycolipid, glyceride, etc), antibody, antigen, hapten, hormone, virus, natural low-molecule (hormone molecule, antibiotic, toxic substance, vitamins, bioactive substance, secondary metabolite, etc), and synthetic low molecule (synthetic substance of natural low molecule, their derivative, etc). In an embodiment according to the present invention, an enzyme and a sugar chain are used as a favorable biomolecule, and a glycosyl transferase and a glycosidase are used as a favorable enzyme.
A membrane-binding molecule in this invention includes a membrane-binding peptide and a biomolecule, particularly a molecule in which these two individual substances are fused or bound. This type of biomolecule might include a membrane-binding peptide as one substance.
A membrane-binding molecule in this invention may contain some or no other substances as long as a membrane-binding ability of a membrane-binding peptide and functions of a biomolecule are not impaired. In an embodiment according to the present invention, there might exist between a membrane-binding peptide and a biomolecule a repeated sequence composed of 7 residues (Asp-Asp-Leu-Arg-Val-Asn-Tyr) called Heptad Repeat and a sequence composed of 5 residues (Leu-Pro-Glu-Thr-Gly).
A membrane-binding molecule in this invention can be prepared by fusing or binding a membrane-binding peptide and a biomolecule, using genetic recombinant techniques, enzyme reaction, linker, etc. For example, in cases where the biomolecule is a protein, a membrane-binding peptide and a biomolecule can be fused and expressed as a membrane-binding molecule to prepare a membrane-binding molecule using genetic recombinant techniques, and a membrane-binding peptide and a protein can be fused or bound to prepare a membrane-binding molecule using an enzyme, etc. For example, sortase A (SrtA) enzyme, which binds a peptide having an amino acid sequence LPETG (Leu-Pro-Glu-Thr-Gly) and a peptide having an amino acid sequence GGG (Gly-Gly-Gly; triglycine), can be used to prepare a membrane-binding molecule.
For example, in cases where a biomolecule is a sugar chain, a consensus sequence is linked to a sequence encoding a membrane-binding peptide to add a sugar chain thereto by genetic recombinant techniques to express genes in cells having a sugar chain-adding ability such as CHO cell, COS cell and yeast cell, thereby preparing a membrane-binding peptide as a glycopeptide. Also, a membrane-binding peptide can be prepared by fusing or binding a membrane-binding peptide and a sugar chain, using a favorable linker.
A biomolecule and a membrane-binding molecule in this invention are, under alkaline conditions, released from a carrier coated with an organic membrane containing a phospholipid, and can be immobilized again in neutrality and in the presence of an phospholipid. Even with a repeated use thereof, adhesiveness of a molecule of a membrane-binding peptide, enzyme activity and sugar chain function can be maintained.
In cases where a biomolecule and a membrane-binding molecule are obtained by purification, a purification method thereof is not particularly limited as long as it can be selected by those skilled in the art accordingly, and it can be any method such as each type of chromatography and mass spectrometry. Gel filtration column chromatography, reversed-phase high-performance liquid chromatography (RP-HPLC) and Matrix Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometer (MALDI-TOF-MS) are used in an embodiment according to the present invention.
A phospholipid in this invention is one having two or more hydrophobic groups composed of phosphate group and acyl group and/or alkyl group in a molecule, e.g. phosphatidyl choline (lecithin), phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl inositol-4-phosphoric acid, phosphatidyl inositol 4,5-diphosphoric acid, phosphatidyl serine, phosphatidylglycerol, diphoshatidylglycerol, phosphatidic acid, sphingomyelin or a mixture containing one or two of these derivatives, etc. Phosphatidyl choline is used as a favorable phospholipid in an embodiment according to the present invention.
A fatty acid comprising a phospholipid in this invention is not particularly limited, but it can be a saturated or unsaturated fatty acid such as caprylic acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, arachic acid, palmitoleic acid, oleic acid, linoleic acid, α- and γ-linoleic acid, erucic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and tetracosa tetraenoic acid.
Herein, “organic membrane containing a phospholipid” in this invention means, for example, not only an organic membrane formed by linking a fixed ligand to be immobilized to a carrier which is modified (bound) to a phospholipid to the carrier (biomimetic membrane), but also e.g. an organic membrane formed by forming and adsorbing a polymer containing a phospholipid (biomimetic membrane) and an organic membrane formed by directly linking a phospholipid to a carrier (biomimetic membrane). Specifically, this means a case in which a phospholipid is not only partially, but also completely comprises an organic membrane (biomimetic membrane).
In cases where said fixed ligand is modified to a phospholipid, the fixed ligand can be selected according to a phospholipid and a carrier, e.g. thiol group, epoxy group, tosyl group, activated ester, amino group and bromoacetamide, etc which can be modified (linked) to a terminus of acyl group and/or alkyl group of a phospholipid. When a polymer containing said phospholipid is used, it can be selected according to a carrier, e.g. methacrylate polymer in which phosphocholine group is dispersed. In an embodiment according to the present invention, phosphatidyl choline is used as a favorable phospholipid, and thiol group which can be modified (bound) to a terminus of alkyl group of phosphatidyl choline is used as a favorable fixed ligand, which is subsequently linked a carrier to form an organic membrane (biomimetic membrane).
A carrier in this invention can be selected according to a purpose of use as long as it can be coated with an organic membrane containing a phospholipid, e.g. organic polymer compound in which polymerizable monomers such as magnetic microparticle, styrenic polymerizable monomer, acrylic polymerizable monomer, methacrylic polymerizable monomer and vinyl polymerizable monomer are polymerized, metal oxide, metal, semiconductor compound, agarose, sepharose, wooden plate, glass plate, silicon substrate, cotton cloth, rayon cloth, acrylic cloth, silk cloth, polyester cloth, wood-free paper, wood-containing paper, art paper, bond paper, recycled paper, baryta paper, cast-coated paper, corrugated paper and resin-coated paper. In an embodiment according to the present invention, magnetic microparticle, organic polymer compound (plastic substrate) and glass substrate are used as a favorable carrier.
In cases where a magnetic microparticle is used as a carrier in this invention, magnetic microparticle can be selected accordingly, e.g. metal or oxide, ferrite microparticle as a chemically stable ferric oxide, magnetite microparticle and maghemite microparticle. Also, the magnetic microparticle contains such elements as Ag, Li, Mg, Fe, Mn, Co, Ni, Cu, Y, Sm and Zn, and it may be selected according to a purpose of use. In an embodiment according to the present invention, Ag-coated magnetic microparticle as a ferrite microparticle is used as a favorable magnetic microparticle. Magnetic microparticle is not particularly limited in diameter, but it is preferably 10 nm to 500 nm.
A biomolecule-immobilized magnetic microparticle according to the present invention comprises a biomolecule having a membrane-binding peptide and/or a membrane-binding molecule having said membrane-binding peptide and the biomolecule, and a magnetic microparticle coated with an organic membrane containing a phospholipid. Specifically, the biomolecule and/or the membrane-binding molecule are immobilized to the organic membrane covering the magnetic microparticle, with adhesiveness of the membrane-binding peptide and biomolecule functions maintained. It is also possible to control orientation of the biomolecule and/or the membrane-binding molecule. Since biomolecule activity can be maintained and magnetically be collected, the biomolecule-immobilized magnetic microparticle can be used for various purposes. In an embodiment according to the present invention, it comprises a microarray and a separating agent for a biomolecule.
A probe-immobilized carrier using a biomolecule having a membrane-binding peptide and/or a membrane-binding molecule as a probe according to the present invention comprises a probe as a biomolecule and/or a membrane-binding molecule and a carrier coated with an organic membrane containing a phospholipid. Specifically, the probe-immobilized carrier is immobilized to an organic membrane with a biomolecule and/or a membrane-binding molecule covering the carrier, and the biomolecule and/or the membrane-binding molecule function as a probe.
A carrier of a probe-immobilized carrier using a biomolecule having a membrane-binding peptide and/or a membrane-binding molecule as a probe in this invention can be selected accordingly as long as the probe is immobilized to a carrier coated with an organic membrane containing a phospholipid and target substances can be successfully detected or separated using the probe-immobilized carrier, e.g. the aforementioned carrier, preferably solid carrier, and in case of microarray, glass substrate and plastic substrate are preferable in view of target substance detection and versatility, and alkali-free glass substrate and quartz substrate are more preferable. In an embodiment according to the present invention, 96-hole plate and glass slide-embedded MTP-Slide-AdaptorII (Bruker) are used.
An enzyme which is used as a biomolecule in this invention is not particularly limited, but e.g. oxidoreductase, transferase, hydrolase, lyase, isomerase and ligase. In this invention, it is preferably transferase or hydrolase, and more preferably glycosyl transferase or glycosidase. In this invention, any glycosyl transferase can be used, e.g. fucosyltransferase, galactose transferase, sialidase, N-acetylgalactosamine transferase, N-acetylglucosaminyl transferase, mannosyltransferase, β-glucuronyl transferase, β-xylosyltransferase and glucosyltransferase. In an embodiment according to the present invention, fucosyltransferase, maltose-linked protein-fused galactose transferase (MBP-GalT), N-acetylgalactosamine transferase2 (ppGalNAcT2) and sialidase are used.
A sugar chain in this invention is not particularly limited, but it may be a sugar chain composed of O-linked sugar chain or N-linked sugar chain, or a combination of O-linked and N-linked sugar chains. Herein, O-linked sugar chain is generically called as various types of sugar chain structures elongating from N-acetylgalactosamine linked to serine residues existing in a primary structure of a protein or threonine residues, e.g. a sugar chain in which GalNAc is bound to OH group of Ser or Thr through C—O binding. N-linked sugar chain is generically called as various types of sugar chain structures elongating from N-acetylgalactosamine linked to asparagine residues for a primary structure of a protein, e.g. a sugar chain containing GlcNAc linked to NH2 group of Asn of Asn-X-(Ser/Thr) through C—N binding as a reducing terminal.
A method for immobilizing a biomolecule to a carrier according to the present invention is a method for directly or indirectly immobilizing a biomolecule fused or bound using a membrane-binding peptide as a linker, to an organic membrane containing a phospholipid, with bioactivity thereof maintained, specifically a method for immobilizing a biomolecule to an organic membrane by making contacts between a hydrophilic amino-acid side chain of a membrane-binding peptide and a hydrophilic group of the organic membrane, via the membrane-binding peptide (a biomolecule might have the peptide) and the organic membrane (biomimetic membrane). The inventors, etc. successfully completed a method for immobilizing a biomolecule to a carrier according to the present invention after long-term careful researches, by finding that said membrane-binding peptide is provided with an α-helix structure by which a hydrophilic amino-acid side chain is oriented in a prescribed direction, a hydrophilic group of said organic membrane coating the carrier is oriented in the direction of surface, and biomolecule orientation control can immobilize a biomolecule to an organic membrane (biomembrane, biomimetic membrane) by making the hydrophilic amino-acid side chain of the membrane-binding peptide and the hydrophilic group of the organic membrane bind.
By immobilizing a biomolecule to an organic membrane via a membrane-binding peptide (a biomolecule might have the peptide) and an organic membrane (biomimetic membrane), using a method for immobilizing a biomolecule to a carrier according to the present invention, it is possible to control biomolecule orientation and to immobilize the biomolecule to the organic membrane with bioactivity maintained, thereby providing an effect of maintaining biomolecule bioactivity even with a repeated use of resultant substances from immobilization. Since the organic membrane containing a phospholipid can be formed by being readily coated on any carrier, a membrane-binding molecule-immobilized carrier, containing a mixture composed of any biomolecule and any carrier, can be produced by a method for immobilizing a biomolecule to a carrier according to the present invention.
Meanwhile, a method for producing a membrane-binding molecule in vivo according to the present invention comprises the following processes (i) to (v):
(i) A process for amplifying DNA fragments by PCR method by designing a primer encoding an amino acid sequence represented by SEQ ID NO: 1;
(ii) A process for constructing a biomolecule-expressing vector which constructs a vector expressing a biomolecule;
(iii) A process for constructing a membrane-binding molecule-expressing vector which constructs a vector expressing a membrane-binding molecule from DNA fragments and a biomolecule-expressing vector;
(iv) A process for preparing an expressing cell line which prepares an expressing cell line by introducing a membrane-binding molecule-expressing vector; and
(v) A process for collecting a membrane-binding molecule which collects a membrane-binding molecule by culturing an expressing cell line.
A process for amplifying DNA fragments in process (i) is a process for designing a primer encoding an amino acid sequence represented by SEQ ID NO: 1 according to a conventional method and amplifying DNA fragments by PCR method. A typical method is to identify an annealed portion of the primer, perform Blast search using a sequence of a target area, design a primer by homology analysis of a downloaded sequence and perform PCR by confirming a primer. In view of PCR efficiency and other factors, DNA fragments whose amplifying size ranges approximately 500 to 1000 bases are desirable.
A base sequence of target DNA fragments can be confirmed by known methods such as Dideoxynucleotide Chain Termination Method (Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989), and automatic base sequence-determining apparatus (for example, DNA Sequencer PRISM 377 or DNA Sequencer PRISM 310; both from Perkin-Elmer) can be used. Also, DNA fragment modification can be performed using commercially available kits and known methods, such as digestion by restriction enzyme, insertion of synthetic oligonucleotide and appropriate DNA fragments, addition of a linker and insertion of start codon (ATG) and/or termination codon (TAATGA or TAG).
A process for constructing a biomolecule-expressing vector in process (ii) is a process for constructing a biomolecule-expressing vector according to a conventional method, such as a method described in Berg, P. et al., Proc. Nat. Acad. Sci. USA, 69, 2904-2909, 1972. Herein, a vector used in this invention can be selected by those skilled in the art accordingly, and it is not specifically limited, but it can be, e.g. pCOS1, pME18S, pEF-BOS, pCDM8, pRSVneo, pSV2-neo, pcDNAI/Amp, pcDNAI, pAMoERC3Sc, pAGE107, pREP4, pAGE103, pAMoA, pAS3-3, pCAGGS, pBK-CMV, pcDNA3.1 and pZeoSV.
In addition, a biomolecule-expressing vector can be constructed using a commonly used promoter, such as human polypeptide chain elongation factor 1α (HEF-1α). An expression vector containing HEF-1α promoter is, e.g. pEF-BOS (Mizushima, S. et al., Nuc. Acid. Res. 18, 5322, 1990). A gene promoter can be virus promoter such as cytomegalovirus, retrovirus, polyomavirus, adenovirus, simian virus 40 (SV40), and promoter derived from mammal cell.
Also, a biomolecule-expressing vector can contain a selectable marker gene, such as phosphotransferase APH (3′) II or I (neo) gene, thymidine kinase gene, E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene and dihydrofolate reductase (DHFR) gene.
A process for constructing a membrane-binding molecule-expressing vector in process (iii) is a method for constructing a membrane-binding molecule-expressing vector by conventional methods, using DNA fragments amplified in process (i) and a biomolecule-expressing vector constructed in process (ii). For example, a membrane-binding molecule-expressing vector can be constructed as in process (ii).
A process for preparing an expressing cell line in process (iv) is a process for preparing an expressing cell line by introducing a membrane-binding molecule-expressing vector constructed in process (iii) to a cell by conventional methods. A cell to which a membrane-binding molecule-expressing vector is introduced can be selected by those skilled in the art accordingly, such as animal cell, E. coli and yeast, and it is not specifically limited, but preferably E. coli. A typical cell is JM109 or DH5α. In an embodiment according to the present invention, E. coli JM109 strain is used as a favorable cell.
A membrane-binding molecule-expressing vector is introduced by a known method, such as calcium phosphate method (Chen, C. et al., Mol. Cell. Biol., 7, 2745-2272, 1987), lipofection method (Feigner, P L. et al., Proc. Natl. Acad. Sci. USA, 84, 7413-7417, 1987) and electroporation method (Potter, H. Anal. Biochem., 174, 361-373, 1988). In the electroporation method, gene introduction apparatus (Gene Pulser; Bio-Rad) can be used.
A process for collecting a membrane-binding molecule in process (v) is a process for culturing an expressing cell line prepared in process (iv) by conventional methods and solubilizing and collecting a membrane-binding molecule expressed in an expressing cell line under alkaline conditions. The expressing cell line is crushed by a known method, but in an embodiment according to the present invention, it is preferably a method for crushing an expressing cell line, using an ultrasonic crusher.
Next, a method for producing a biomolecule-immobilized carrier according to the present invention comprises the following processes (i) to (iii):
(i) A process for forming a biomembrane-mimicking carrier which forms a biomembrane-mimicking carrier by coating a carrier with an organic membrane containing a phospholipid;
(ii) A process for preparing an immobilized biomolecule which prepares a biomolecule having a membrane-binding peptide and/or a membrane-binding molecule having said membrane-binding peptide and the biomolecule; and
(iii) A process for immobilizing a membrane-binding molecule to a biomembrane-mimicking carrier in neutrality and in the presence of a phospholipid, using a membrane-binding peptide as a linker.
A process for forming a biomembrane-mimicking carrier in process (i) is a process for forming a biomembrane-mimicking carrier by coating a magnetic microparticle and a carrier such as solid carrier with an organic membrane containing a phospholipid. Specifically, a hydrophobic group of a phospholipid is arrayed in the direction of a carrier by binding the hydrophobic group of a phospholipid to a surface of the carrier, resulting in arraying of the hydrophilic group of a phospholipid outwardly to coat a surface of the carrier on a biomembrane-like organic membrane. In this invention, a carrier whose surface is coated with a biomembrane-like organic membrane is defined as a biomembrane-mimicking carrier. A fixed ligand to be immobilized to a carrier may be modified to a phospholipid as required.
A process for preparing an immobilized biomolecule in process (ii) is a process for preparing a biomolecule having a membrane-binding peptide and/or a membrane-binding molecule having said membrane-binding peptide and the biomolecule in vitro or in vivo. As mentioned above, the process includes a case in which not only a fused molecule in which two individual substances of the membrane-binding peptide and the biomolecule are fused or bound is prepared, but also a biomolecule having the membrane-binding peptide as a substance is prepared.
A process for immobilizing a membrane-binding molecule to a biomembrane-mimicking carrier in process (iii) is a process for immobilizing a biomembrane-mimicking carrier formed by a process for forming a biomembrane-mimicking carrier in process (i) and a biomolecule and/or a membrane-binding molecule prepared by a process for preparing an immobilized biomolecule in process (ii) in neutrality via a membrane-binding peptide in the biomolecule and/or the membrane-binding molecule. Specifically, in a process for immobilizing a membrane-binding molecule to a biomembrane-mimicking carrier in process (iii), a method for immobilizing a biomolecule to a carrier for immobilizing a biomolecule is used with bioactivity maintained in an organic membrane containing a phospholipid according to the present invention.
A membrane-binding molecule-immobilized carrier and a method for immobilizing a biomolecule to a carrier according to the present invention will be described with reference to Examples. A technological scope in this invention is not limited to characteristics as shown in the Examples.
α-1,3 fucosyltransferase derived from Helicobacter pylori includes said membrane-binding peptideon on a C terminal side. Then, the fucosyltransferase is presented to a biomembrane mimetic (organic membrane-coating) magnetic microparticle to measure enzyme activity of fucosyltransferase-immobilized magnetic microparticle obtained.
(1) Expression and Purification of Fucosyltransferase
α-1,3 fucosyltransferase derived from Helicobacter pylori J99B strain was expressed by recombinant E. coli to be purified. Specifically, after a recombinant E. coli expressing α-1,3 fucosyltransferase derived from Helicobacter pylori J99B strain (Taylor, D. E. et al., J. Biol. Chem., 278, 21893-21900, 2003) was cultured in 2 L of a YT medium obtained by diluting twice at 37° C. for 8 hours, isopropylthiogalactoside was added thereto so that a final concentration thereof reached 1 mM and cultured at 20° C. for 20 hours. A culture solution was subjected to centrifugal separation at 10,000×g for 20 minutes to collect bacterial cells. The collected bacterial cells were suspended in 5 mL of 25 mM Tris buffer solution (pH8.0) containing 1 mM mercaptoethanol and 10% (w/v) glycerol and were subjected to ultrasonic crushing by ultrasonic crusher under cooling five times for 2 minutes each. After another centrifugal separation at 10,000×g for 20 minutes, supernatants were collected to perform affinity purification by nickel column (His Trap FF; Amersham Pharmacia Biotech). After fractions containing fucosyltransferase were substituted by dialysis using 25 mM glycine-sodium hydroxide buffer solution (pH10) and subjected to an anion-exchange column (HiTrap Q HP; Amersham Pharmacia Biotech) under alkaline conditions of glycine-sodium hydroxide buffer solution (pH10), they were purified by gel filtration column chromatography (Superdex 200 pg; Amersham Pharmacia Biotech). A purified product obtained was condensed by ultrafiltration to obtain purified fucosyltransferase.
(2) Preparation of Fucosyltransferase-Immobilized Magnetic Microparticle
Fucosyltransferase obtained by purification in this Example 1(1) was presented to a biomembrane-mimicking magnetic microparticle to prepare a fucosyltransferase-immobilized magnetic microparticle. Specifically, after 1 mg of Ag-coated magnetic microparticle (ferrite microparticle; Hitachi Maxell, Ltd.) 200 nm in diameter was washed with 100 μL of ethanol 3 times, the product was reacted in 40 μL of 80 mM phosphatidyl choline linker having thiol on an alkyl chain terminus at room temperature for 16 hours to prepare a biomembrane-mimicking magnetic microparticle, as shown in
Next, 100 μL (734 μg) of fucosyltransferase solution prepared by diluting purified fucosyltransferase obtained in this Example 1(1) with 100 mM Tris buffer solution (pH7.5) was added to a biomembrane-mimicking magnetic microparticle prepared in this Example 1(2) and agitated at 4° C. for 2 hours. Afterward, the biomembrane-mimicking magnetic microparticle was washed with 100 mL of 100 mM Tris buffer solution (pH7.5) 5 times to obtain a fucosyltransferase-immobilized magnetic microparticle in which fucosyltransferase was immobilized to a magnetic microparticle via a membrane-binding peptide and an organic membrane (biomimetic membrane), as shown in
(3) Measurement of Fucosyltransferase Activity
[3-1: Fucosyltransferase Activity Measurement Using Lacto-N-neotetraose]
Subsequently, fucosyltransferase activity for Lacto-N-neotetraose (LNnT) as human milk oligosaccharide of a fucosyltransferase-immobilized magnetic microparticle obtained in this Example 1(2) was measured. Specifically, 1 mg of fucosyltransferase-immobilized magnetic microparticle prepared in this Example 1(2) was reacted in a mixture containing 0.5 μL of 1M manganese chloride, 2.5 μL of 200 mM guanosine 5′-2phosphoric acid-6-L-fucose (GDP-fucose), 25 μL of 40 mM LNnT, 5 μL of 1M Tris buffer solution (pH7.5) and 17 μL of ultrapure water, at room temperature for 60 minutes. The product was analyzed by saccharides analyzer (HPAEC-PAD; Dionex) every 5, 15, 30 and 60 minutes to measure fucosyltransferase activity by quantifying fucosylated pentasaccharide. A chromatograph showing measurement results, corresponding to a reaction time of 5, 15, 30 and 60 minutes, are shown in
As shown in
[3-2: Fucosyltransferase Activity Measurement Using O-Linked Type Sugar Amino Acid]
Fucosyltransferase activity for O-linked type sugar amino acid of a fucosyltransferase-immobilized magnetic microparticle obtained in this Example 1(2) was measured. Specifically, 1 mg of 576 mU/mL fucosyltransferase-immobilized magnetic microparticle was reacted in a mixture containing 1 mg of O-linked type sugar amino acid (R in
MALDI-TOF-MS was measured using 2,5-dihydroxybenzoic acid (DHB; Naruchi, K. et al., J. Org. Chem., 71, 9609-9621, 2006) as a matrix agent. A spectral chart thereof is shown in
In MALDI-TOF-MS analysis as shown in
[3-3: Fucosyltransferase Activity Measurement Using N-Linked Type Sugar Amino Acid]
Similarly, fucosyltransferase activity for N-linked type sugar amino acid of a fucosyltransferase-immobilized magnetic microparticle prepared in this Example 1(2) was measured. Specifically, 1 mg of 576 mU/mL fucosyltransferase-immobilized magnetic microparticle was reacted in a solution containing 5 μL of 10 mM N-linked type sugar amino acid, 0.5 μL of 1M manganese chloride, 5 μL of 200 mM GDP-fucose, 5 μL of 1M Tris buffer solution (pH7.5) and 34.5 μL of ultrapure water at room temperature for 16 hours. A reaction of N-linked type sugar amino acid to be fucosylated is shown in
As shown in
From this Example 1(3) [3-1] to [3-3] described above, fucosyltransferase can be immobilized to a biomembrane-mimicking magnetic microparticle, with activity maintained, and can be used as a fucosyltransferase-immobilized magnetic microparticle.
(4) Examination of a Repeated Use and Conservation Using LNnT
Next, whether a fucosyltransferase-immobilized magnetic microparticle prepared in this Example 1(2) can be repeatedly used or not was discussed. Specifically, as in a method in this Example 1(3) [3-1], 1 mg of fucosyltransferase-immobilized magnetic microparticle having 576 mU/mL activity prepare in this Example 1(2) was reacted in 100 mM Tris buffer solution (0-17.5) containing 10 mM manganese chloride, 10 mM GDP-fucose and 20 mM LNnT at room temperature for 15 minutes. This was repeated 5 times, and in each step fucosyltransferase activity was measured by quantifying fucosylated pentasaccharide using saccharides analyzer (HPAEC-PAD; Dionex). In the 1st to 3rd steps, fucosyltransferase after reaction was washed with 100 μL of 100 mM Tris buffer solution (pH7.5) 5 times and another activity measurement was performed. In the 4th step, fucosyltransferase was washed with 100 μL of 100 mM Tris buffer solution (pH7.5) 5 times and washed with 100 μL of 1M sodium chloride 5 times to perform activity measurement. In the 5th step, fucosyltransferase was washed with 100 μL of 100 mM Tris buffer solution (pH7.5) 5 times and preserved at 4° C., and activity measurement was performed the following day. A chromatograph showing measurement results with numbers of cycles of 1 to 5 are shown in
As shown in
(5) Release Test of Fucosyltransferase of Fucosyltransferase-Immobilized Magnetic Microparticle
A release test of fucosyltransferase of fucosyltransferase-immobilized magnetic microparticle was performed. Specifically, as shown in
As shown in
Herein, fucosyltransferase, as shown in this Example 1(1), under alkaline conditions of glycinesodium hydroxide buffer solution (pH10), was condensed by ultrafiltration to obtain purified fucosyltransferase without precipitation. Also, as shown in this Example 1(2), under neutrality conditions of Tris buffer solution (pH7.5), the purified fucosyltransferase solution was added to a biomembrane-mimicking magnetic microparticle to be reacted, and an α-helix structure of a membrane-binding peptide portion required for binding was induced and fucosyltransferase was successfully immobilized to a magnetic microparticle via a membrane-binding peptide.
The above observations found that fucosyltransferase was immobilized to a magnetic microparticle via a membrane-binding peptide and an organic membrane (biomimetic membrane), with activity maintained under neutrality conditions, and it was released to be solubilized under alkaline conditions, enabling a reversible use of the immobilization and release.
A biomembrane-mimicking well was prepared by coating each well of a 96-hole plate with phosphatidyl choline linker having thiol in an alkyl chain terminus, and enzyme fucosyltransferase activity-immobilized microarray obtained by immobilizing fucosyltransferase to a well via a membrane-binding peptide and an organic membrane (biomimetic membrane) was measured.
(1) Preparation of Fucosyltransferase-Immobilized Microarray
100 μL of 80 mM phosphatidyl choline linker having thiol in alkyl chain terminus was added to each well of a 96-hole plate to be reacted at room temperature for 16 hours to prepare a biomembrane-mimicking well. Subsequently, after the biomembrane-mimicking well was washed with 100 μL of 100 mM Tris buffer solution (pH7.5) 5 times, 100 μL (734 μg) of fucosyltransferase solution prepared by diluting purified fucosyltransferase obtained as in Example 1(1) with 100 mM Tris buffer solution (pH7.5) was added thereto and agitated at 4° C. for 2 hours. Afterward, the biomembrane-mimicking well was washed with 100 μL of 100 mM Tris buffer solution (pH7.5) 5 times, and a fucosyltransferase-immobilized microarray was prepared via a membrane-binding peptide in fucosyltransferase and an organic membrane (biomimetic membrane).
(2) Fucosyltransferase Activity Measurement
Subsequently, fucosyltransferase activity for O-linked 4 sugar amino acid of a fucosyltransferase-immobilized microarray obtained in this Example 2(1) was measured. Specifically, a fucosyltransferase-immobilized microarray prepared in Example 1(2) was reacted in a mixture containing 1 μL of 1M manganese chloride, 5 μL of 200 mM GDP-fucose, 10 μL of 1 mM O-linked 4 sugar amino acid, 10 μL of 1M Tris buffer solution (pH7.5) and 74 μL of ultrapure water at room temperature for one hour. 1 μL of the reaction solution was subjected to MALDI-TOF-MS (Bruker), and as in Example 1(3) [3-2], 1 μL of 10 mg/mL DHB was used as a matrix agent to detect fucosylated O-linked 5 sugar amino acid. In addition, as a control, said solution was added to a fucosyltransferase nonspecific adsorption well which is not coated with phosphatidyl choline linker having thiol in alkyl chain terminus and the product was subjected to MALDI-TOF-MS. The results using a fucosyltransferase nonspecific adsorption well are shown in
As shown in
The above observation found that only by dispensing a sugar chain to a probe-immobilized microarray using a probe in which a biomolecule having a membrane-binding peptide and a membrane-binding molecule having a membrane-binding peptide and biomolecule are immobilized, sugar chain trimming can be performed.
Using a non biomembrane-mimetic (not coating with an organic membrane) magnetic microparticle in the absence of a membrane-binding peptide, a biomolecule nonspecific adsorption magnetic microparticle was prepared and an activity thereof was measured.
(1) Preparation of Sialidase Nonspecific Adsorption Magnetic Microparticle
Specifically, 1 mg of Ag-coated magnetic microparticle 200 nm in diameter (Hitachi Maxell, Ltd.; ferrite microparticle) was firstly washed with 100 μL of ethanol 3 times, and it was not coated with phosphatidyl choline linker having thiol in an alkyl chain terminus, and 100 μL of solution (74 μg), in which sialidase (SIGMA) derived from Vivrio cholerae was diluted with 100 mM Tris buffer solution (pH7.5), was added thereto and agitated at 4° C. for 2 hours. Next, the product was washed with 100 mL of 100 mM Tris buffer solution (pH7.5) 5 times to obtain sialidase nonspecific adsorption magnetic microparticle to which sialidase was nonspecifically adsorbed.
(2) Sialidase Activity Measurement
Subsequently, sialidase activity for 4-methylumbelliferyl sialic acid of a sialidase nonspecific adsorption magnetic microparticle obtained in this Comparative Example 1(1) was measured. Specifically, after 1 mg of sialidase nonspecific adsorption magnetic microparticle prepared in this Comparative Example 1(1) was reacted in 100 mM Tris buffer solution (pH7.5) containing 2 mM calcium chloride and 20 mM 4-methylumbelliferyl sialic acid at room temperature for 10 minutes, the reaction solution was analyzed by saccharides analyzer (HPAEC-PAD; Dionex) and free sialic acid was quantified to measure sialidase activity. The activity was measured by washing the product with 100 μL of 100 mM Tris buffer solution (pH7.5) 5 times after each activity measurement was completed, and another activity measurement was repeatedly performed 3 times. The results are shown in
As shown in
Conformational analysis of membrane-binding peptide was performed in an aqueous solution or a micell solution to examine membrane adhesiveness.
Specifically, firstly, a polypeptide of 42 residues containing a membrane-binding peptide present on a C terminal side of α-1,3 fucosyltransferase derived from Helicobacter pylori J99B strain was prepared by automated peptide synthesizer (APEX3962; Advanced Chem. Tech.) and measured by RP-HPLC and purified. RP-HPLC was measured as in Example 1(3) [3-2]. In conformational analysis of a membrane-binding peptide obtained by purification in an aqueous solution, a solution containing 600 μL of water (pH7.0) was used as a sample so that a membrane-binding peptide reached 62.5 μM in concentration. Meanwhile, in conformational analysis in a micell solution of membrane-binding peptide, a mixture of 240 μL of 156 μM a peptide of said 42 residues and 360 μL of 100 μM dodecylphosphocholine was used as a sample.
Next, CD analysis was performed using the samples. In CD analysis, using circular dichroism spectroscope J-820 (JASCO Corporation), CD was measured at a wavelength ranging from 190 to 250 nm by 10-time integrating. The results are shown in
As shown in
Since membrane adhesiveness of a membrane-binding peptide was confirmed in Example 3, a maltose-linked protein-fused galactose transferase (MBP-GalT) was used as a biomolecule, and by presenting MBP-GalT to which a membrane-binding peptide was fused to a biomembrane-mimicking magnetic microparticle, an MBP-GalT-immobilized magnetic microparticle was prepared in vitro to measure an activity thereof.
(1) Preparation of MBP-GalT and Membrane-Binding Peptide
As shown in
Accordingly, MBP-GalT, and a polypeptide of 42 residues containing a membrane-binding peptide containing triglycine on an N terminal were prepared. Specifically, MBP-GalT was prepared by a method of S. Nishiguchi et al. (Susumu Nishiguchi, Shin-ichiro Nishimura., Chem. Commun., 1944-1945, 2001). A polypeptide of said 42 residues containing triglycine on an N terminal was prepared in a method as in Example 3 to be measured and purified by RP-HPLC. RP-HPLC measurement was performed at a flow rate of 10 mL/min using Inertsil ODS-3 (200 mM×250 mM; GL Sciences Inc.) as a reversed-phase column and 0.1% TFA-containing ultrapure water and 0.1% TFA-containing methyl cyanide as eluate.
(2) Preparation of MBP-GalT-Immobilized Magnetic Microparticle
MBP-GalT obtained in this Example 4(1) and a polypeptide of 42 residues containing a membrane-binding peptide containing triglycine on an N terminal were bound by SrtA, and by presenting an obtained membrane-binding peptide-fused MBP-GalT to a biomembrane mimetic magnetic microparticle, an MBP-GalT-immobilized magnetic microparticle was prepared. Specifically, 2 μL of 1.8 mM SrtA was firstly reacted in a mixture containing 1004 of 4 μM MBP-GalT obtained in this Example 4(1), 44 of polypeptide of said 42 residues containing 1 mM triglycine obtained in this Example 4(1) on an N terminal, 104 of 1M Tris-HCL buffer solution (pH7.5), 14, of 1M calcium chloride, 154 of 2M sodium chloride and 684 of ultrapure water at 37° C. for 20 hours. By transferring the reaction liquid to Microcon YM-50 (Millipore Corporation) and performing centrifugal separation at 10,000×g for 1 minute, an enzyme reaction liquid containing 504 of a membrane-binding peptide-fused MBP-GalT left in a filter was obtained, except for a polypeptide of said 42 residues containing unreacted triglycine on an N terminal.
After 1 mg of biomembrane mimetic magnetic microparticle prepared as in Example 1(2) was added to the enzyme reaction liquid and the product was agitated at 4° C. for hours, all of the solutions were removed and the biomembrane mimetic magnetic microparticle washed with 1004 of 50 mM Tris-HCL buffer solution (pH7.5) 7 times. This step can present MBP-GalT having a membrane-binding peptide on a C terminal to a biomembrane mimetic magnetic microparticle, and an MBP-GalT-immobilized magnetic microparticle, in which MBP-GalT was immobilized to a magnetic microparticle via a membrane-binding peptide and an organic membrane (biomimetic membrane), was obtained.
(3) Preparation of MBP-GalT Nonspecific Adsorption Magnetic Microparticle
As a control, a mixture of 100 μL of 4 μM MBP-GalT and 100 μL of water was added to Ag-coated magnetic microparticle which was not coated with phosphocholine linker (non biomembrane-mimetic), and by the step mentioned above, an MBP-GalT nonspecific adsorption magnetic microparticle, to which MBP-GalT adsorbed nonspecifically, was prepared.
(4) Measurement of MBP-GalT Activity of MBP-GalT-Immobilized Magnetic Microparticle and MBP-GalT Nonspecific Adsorption Magnetic Microparticle
Subsequently, MBP-GalT activity for 4-methylumbelliferyl-N-acetylglucosamine (4MU-GlcNAc) of an MBP-GalT-immobilized magnetic microparticle obtained in this Example 4(2) and an MBP-GalT nonspecific adsorption magnetic microparticle obtained in this Example 4(3) was measured. Specifically, 10 μL of 1M manganese chloride, 25 μL of 25 mM uridine 5′-2phosphoric acid-α-D-galactose (UDP-Gal), 200 μL of 2.5 mM 4MU-GlcNAc, 50 μL of 1M Tris-HCL buffer solution (pH7.5) and 1 μL of 10% (w/v) Triton X-100 were mixed with 714 μL of ultrapure water to prepare a substrate solution. The substrate solution was added to 1 mg of MBP-GalT-immobilized magnetic microparticle and 1 mg of MBP-GalT nonspecific adsorptions magnetic microparticle prepared in the Examples 4(2) and (3), respectively, each by 50 μL to be reacted at room temperature for 30 minutes. 50 μL of each reaction liquid was diluted in 450 mL of ultrapure water and analyzed by RP-HPLC. Measurement results of an MBP-GalT nonspecific adsorption magnetic microparticle as a control are shown in
As shown in
From the above observation, according to Examples 1 and 4, fucosyltransferase having a membrane-binding peptide and MBP-GalT to which a membrane-binding peptide was fused can be immobilized to a biomembrane mimetic magnetic microparticle and each activity was confirmed, resulting in a possibility of using not only a biomolecule having a membrane-binding peptide, but also a membrane-binding molecule to which a membrane-binding peptide was fused. Then, it was found that as long as a biomolecule is immobilized to a magnetic microparticle via a membrane-binding peptide and an organic membrane (biomimetic membrane), a biomolecule-immobilized magnetic microparticle, which is immobilized with biomolecule activity maintained, can be obtained.
(1) Preparation of MBP-GalT-Immobilized Sepharose Resin In Vitro
By immobilizing MBP-GalT used in Example 4 to cyanobromid (BrCN) activated sepharose 4B, MBP-GalT-immobilized sepharose resin was prepared in vitro (FIG. 20). Specifically, 500 μL of 2 μM MBP-GalT obtained by a method as in Example 4(1) (Nishiguchi, S. et al., Chem. Commun, 1944-1945, 2001) was reacted in 100 mM Tris buffer solution (pH8) containing 0.3 mL of BrCN activated sepharose 4B (GE Healthcare Japan Corporation) at 4° C. for 16 hours. The reaction liquid was subjected to centrifugal separation at 1000×g in ULTRAFREE-MC 0.45 μmb Filter-Unit (Millipore Corporation) for 3 minutes to obtain MBP-GalT-immobilized sepharose resin.
(2) MBP-GalT Activity Measurement
Subsequently, MBP-GalT activity for UDP-Gal as glycosyl donor of an MBP-GalT-immobilized sepharose resin obtained in this Comparative Example 2(1) was measured. Specifically, after 0.3 mL of MBP-GalT-immobilized sepharose resin having 0.2 U activity prepared in this Comparative Example 2(1) was reacted in 50 mM Hepes (HEPES) buffer solution (pH7.5) containing 13 mM UDP-Gal and 10 mM receptor in which 2-aminopyridyl-N-acetylglucosamine was bound to polyacrylamide polymer at 37° C. for 24 hours, the product was fractionated by Sephadex G-25 (GE Healthcare Japan Corporation) to measure enzyme activity. The enzyme activity was repeatedly measured 8 times. The activity measurement results are shown in
As shown in
O-linked glycopeptide (mucin-type peptide) as a membrane-binding peptide (glycopeptide) having sugar chain was used as a biomolecule to prepare a membrane-binding peptide-fused O-linked glycopeptide incorporating a polypeptide of 42 residues containing a membrane-binding peptide on a C terminal of the O-linked glycopeptide in vitro, and by presenting the membrane-binding peptide-fused O-linked glycopeptide to a biomembrane-mimicking magnetic microparticle, O-linked glycopeptide-immobilized magnetic microparticle was prepared and an activity thereof was measured.
(1) Preparation of O-Linked Glycopeptide and Membrane-Binding Peptide
O-linked glycopeptide (mucin-type peptide) was prepared according to a method described in Naruchi, K. et al., J. Org. Chem., 71, 9609-9621, 2006, and a polypeptide of 42 residues containing a membrane-binding peptide containing triglycine on an N terminal was prepared by a method as in Example 3 and measured and purified by RP-HPLC by a method as in Example 4(1).
(2) Preparation of O-Linked Glycopeptide-Immobilized Magnetic Microparticle
O-linked glycopeptide (containing SrtA recognition sequence on a C terminal) obtained in this Example 5(1) and a polypeptide of 42 residues containing a membrane-binding peptide containing triglycine on N terminal were bound by SrtA, and by presenting a membrane-binding peptide-fused O-linked glycopeptide obtained to a biomembrane mimetic magnetic microparticle, O-linked glycopeptide-immobilized magnetic microparticle was prepared. Specifically, 1 μL of 1.8 mM SrtA was reacted in a mixture containing 2.54, of 10 mM O-linked glycopeptide obtained in this Example 5(1), 2.5 μL of polypeptide of said 42 residues containing 10 mM triglycine on an N terminal obtained in this Example 5(1), 2.5 μL of 1M Tris buffer solution (pH7.5), 2.54, of 100 mM calcium chloride, 2.5 μL of 3M sodium chloride and 35.54, of ultrapure water at 37° C. for 20 hours. The reaction liquid was transferred to Microcon YM-10 (Millipore Corporation) and subjected to centrifugal separation at 14,000×g for 20 minutes to remove SrtA and obtain a peptide mixture by filtration. The mixture was measured by RP-HPLC by a method as in Example 4(1) and purified to obtain a membrane-binding peptide-fused O-linked glycopeptide. The process for preparing a membrane-binding peptide-fused O-linked glycopeptide is shown in
After 1 mg of biomembrane mimetic magnetic microparticle prepared by a method as in Example 1(2) was added to membrane-binding peptide-fused O-linked glycopeptide obtained and agitated at 4° C. for 2 hours, all of the solutions were removed and biomembrane mimetic magnetic microparticle was washed with 100 μL of 50 mM Tris buffer solution (pH7.5) 5 times. The step can present O-linked glycopeptide having a membrane-binding peptide on a C terminal to a biomembrane mimetic magnetic microparticle to obtain O-linked glycopeptide-immobilized magnetic microparticle in which O-linked glycopeptide was immobilized to magnetic microparticle via a membrane-binding peptide and an organic membrane (biomimetic membrane).
(3) Preparation of O-Linked Glycopeptide-Immobilized Ag-Coated Magnetic Microparticle
As a control, a membrane-binding peptide-fused O-linked glycopeptide was presented to Ag-coated magnetic microparticle which was not coated with phosphocholine linker (non biomembrane-mimetic) to prepare O-linked glycopeptide-immobilized Ag-coated magnetic microparticle. Specifically, after 1 mg of Ag-coated magnetic microparticle 200 nm in diameter washed with 100 μL of ethanol 3 times (Hitachi Maxell, Ltd.; ferrite microparticle) was added to membrane-binding peptide-fused O-linked glycopeptide obtained in this Example 5(2) at 4° C. for 2 hours, all of the solutions were removed and Ag-coated magnetic microparticle was washed with 100 μL of 50 mM Tris buffer solution (pH7.5) 5 times.
(4) Confirmation of Immobilization of Membrane-Binding Peptide-Fused O-Linked Glycopeptide in O-Linked Glycopeptide-Immobilized Magnetic Microparticle and O-Linked Glycopeptide-Immobilized Ag-Coated Magnetic Microparticle
Whether a membrane-binding peptide-fused O-linked glycopeptide is immobilized in O-linked glycopeptide-immobilized magnetic microparticle and O-linked glycopeptide-immobilized Ag-coated magnetic microparticle or not was confirmed. Specifically, O-linked glycopeptide-immobilized magnetic microparticle and O-linked glycopeptide-immobilized Ag-coated magnetic microparticle prepared in the Examples 5(2) and (3) were taken in a small volume by tip end and spotted on a target for MALDI-TOF-MS. 1 μL of 10 mg/mL DHB was mixed therewith and dried, and the product was measured by MALDI-TOF-MS. The results are shown in
As shown in
Using an antibody which specifically recognizes O-linked glycopeptide immobilized to a biomembrane-mimicking magnetic microparticle via a membrane-binding peptide and an organic membrane (biomimetic membrane), a possible use as antibody separating agent was discussed. Specifically, 1004 of solution obtained by diluting primary antibody VU-3C6 (Exalpha Biologicals) which recognizes mucin-type glycopeptide MUC1 containing 50 mM Tris buffer solution (pH7.5) 1000 times was added to a mixture containing O-linked glycopeptide-immobilized magnetic microparticle prepared in a method as in Example 5(2) and Fetal Bovine Serum (FBS) whose final concentration was 1% and reacted at room temperature for one hour. Thereafter, O-linked glycopeptide-immobilized magnetic microparticle was washed with 1004 of 100 mM Tris buffer solution (pH7.5) containing 0.2M NaCl and 0.05% Tween 5 times to be collected, and 100 μL of 1000-time diluted HRP conjugate-mouse monoclonal antibody (Kirkegaard & Perry Laboratories) was mixed therewith and reacted at room temperature for one hour.
After reaction, O-linked glycopeptide-immobilized magnetic microparticle was again washed with 1004 of 100 mM Tris buffer solution (pH7.5) containing 0.2M NaCl and 0.05% Tween 5 times to be collected, and 100 μL of TMB-Microwell-Peroxidase-Substrate-System reagent (Kirkegaard & Perry Laboratories) was added thereto. Thereafter, an absorbance at 450 nm was measured and color development was confirmed. As a control, using a step as described above, color development was confirmed in a biomembrane mimetic magnetic microparticle coated with phosphocholine linker prepared by a method as in Example 1(2) and O-linked glycopeptide-immobilized Ag-coated magnetic microparticle prepared by a method as in Example 5(3). The results are shown in
As shown in
Since it was found that O-linked glycopeptide-immobilized carrier can be used as a practical antibody separating agent in Example 6,0-linked glycopeptide-immobilized microarray was prepared.
(1) Preparation of Substrate of O-Linked Glycopeptide-Immobilized Microarray
In order to prepare substrate of O-linked glycopeptide-immobilized microarray, after gold-coated chip was coated with phosphocholine linker by a method as in Example 2(1), the chip was fixed to a glass slide to be engaged into MTP-Slide-Adaptor II (Bruker). A portion spotted was washed with a mixture containing 5 μL of ethanol, 5 μL of ultrapure water and 5 μL of 50 mM Tris buffer solution (pH7.5) 5 times to prepare a substrate of O-linked glycopeptide-immobilized microarray.
(2) Preparation of O-Linked Glycopeptide-Immobilized Microarray and Confirmation of Immobilization
5 μL of 500 μM membrane-binding peptide-fused O-linked glycopeptide prepared by a method as in Example 5(1) was spotted on a substrate of O-linked glycopeptide-immobilized microarray prepared in this Example 7(1). 5 minutes after the product was allowed to stand, it was washed with 54 of 50 mM Tris buffer solution (pH7.5) 5 times. As a result of the measurement using MALDI-TOF-MS by a method as in Example 1(3) [3-2], immobilization of a membrane-binding peptide-fused O-linked glycopeptide was confirmed. O-linked glycopeptide-immobilized microarray is shown in
As shown in
A biomolecule to which membrane-binding peptide was fused, which can be immobilized to a biomembrane-mimicking magnetic microparticle, was prepared in vivo. Specifically, using sialidase NEU2 derived from as an enzyme which hydrolyzes sialic acid present on sugar chain terminal as a biomolecule, a membrane-binding peptide-fused sialidase incorporating a polypeptide of 42 residues containing a membrane-binding peptide on C terminal of the sialidase NEU2 derived from human was prepared in vivo.
A primer was designed as follows, so that the polypeptide of the above 42 residues is cut from a vector used in preparing α-1,3 fucosyltransferase derived from Helicobacter pylori J99B strain having the polypeptide of the said 42 residues in Example 1(1), and a sequence thereof was amplified under the following conditions by PCR method.
30 cycles, each composed of a step at 95° C. for 30 seconds, a step at 55° C. for 30 seconds and a step at 68° C. for 1 minute and 30 seconds
Sialidase NEU2 was cloned from cDNA derived from small intestines (Invitrogen) to obtain a vector in which sialidase NEU2 was constructed. (Monti E. et al., J.B.C., 279, 3169-3179, 2004).
Fragments encoding the polypeptide of the above 42 residues and the vector, in which the above sialidase NEU2 was constructed, were treated with restriction enzymes of NdeI and BamHI to construct an expression vector of sialidase NEU2, in which a membrane-binding peptide was fused to a C terminal. The vector was transformed to E. coli JM109 strain to prepare E. coli variant. After the product was cultured on LB agar medium at 37° C. overnight, a colony was inoculated on 2.5 mL of LB medium to be cultured at 37° C. overnight. After the culture solution was subcultured on 50 mL of LB medium and was cultured at 37° C. for 2 hours so that an absorbance (OD600) at 600 nm reached 0.7, isopropylthiogalactoside (IPTG) was added thereto so that a final concentration thereof reached 0.25 mM and cultured at 20° C. for 20 hours.
The culture solution was subjected to centrifugal separation at 10,000×g for 20 minutes to collect bacterial cells, and suspended in 5 mL of 25 mM Tris buffer solution (pH8.0) containing 1 mM mercaptoethanol and 10% (w/v) glycerol, and ultrasonic crushing of bacterial cells was performed 5 times for 2 minutes each under cooling by ultrasonic crusher. After the product was again subjected to centrifugal separation at 10,000×g for 10 minutes, supernatants were collected and subjected to SDS-PAGE, the activity was measured to confirm membrane-binding peptide-fused sialidase. Subsequently, after supernatants collected were subjected to Ni column (His Trap FF; Amersham Pharmacia Biotech) under alkaline conditions (pH 8 to 10), they were condensed by ultrafiltration under alkaline conditions (pH 8 to 10) to obtain purified membrane-binding peptide-fused sialidase.
Since preparation of a biomolecule in which membrane-binding peptide was fused in vivo in Example 8 was confirmed, using MBP-GalT as a biomolecule, a membrane-binding peptide-fused MBP-GalT incorporating a polypeptide of 42 residues containing membrane-binding peptide on C terminal of the MBP-GalT was prepared in vivo and by presenting the membrane-binding peptide-fused MBP-GalT to a biomembrane-mimicking magnetic microparticle, MBP-GalT-immobilized magnetic microparticle was prepared and an activity thereof was measured.
(1) Preparation of Membrane-Binding Peptide-Fused MBP-GalT In Vivo
Fragments encoding a polypeptide of 42 residues containing membrane-binding peptide prepared in Example 8 and a vector in which MBP-GalT was constructed (Susumu Nishiguchi, Shin-ichiro Nishimura. Chem. Commun. 1944-1945, 2001) were treated with restriction enzymes of NdeI and BamHI to construct an expression vector of MBP-GalT in which a membrane-binding peptide was fused to a C terminal. The vector was transformed to E. coli JM109 strain to prepare E. coli variant. After the product was cultured on 4 mL of 2×YT medium at 37° C. overnight, the culture solution was subcultured on 200 mL of 2×YT medium and cultured at 37° C. for 2 hours. IPTG was added to the culture solution so that a final concentration thereof reached 1 mM and cultured at 20° C. for 20 hours.
The culture solution was subjected to centrifugal separation at 10,000×g for 20 minutes to collect bacterial cells and suspended in 5 mL of 25 mM Tris buffer solution (pH8.0) containing 1 mM mercaptoethanol and 10% (w/v) glycerol, and ultrasonic crushing of bacterial cells was performed 5 times for 2 minutes each under cooling by ultrasonic crusher. After another centrifugal separation at 10,000×g for 20 minutes, supernatants were collected to be purified by anion-exchange column (DEAE FF; Amersham Pharmacia Biotech). Subsequently, after adsorbed fractions were subjected to Ni column (His Trap FF; Amersham Pharmacia Biotech) under alkaline conditions (pH 8 to 10), they were condensed by ultrafiltration under alkaline conditions (pH 8 to 10) to obtain purified membrane-binding peptide-fused MBP-GalT (1.6 mg/mL).
(2) Preparation of MBP-GalT-Immobilized Magnetic Microparticle
By presenting a membrane-binding peptide-fused MBP-Gal obtained in this Example 9(1) to a biomembrane mimetic magnetic microparticle, MBP-GalT-immobilized magnetic microparticle was prepared. Specifically, 1004 of membrane-binding peptide-fused MBP-Gal solution (160 μg) obtained in this Example 9(1) was added to biomembrane-mimicking magnetic microparticle prepared by a method as in Example 1(2) and agitated at 4° C. for 2 hours. Thereafter, all of the solutions were removed and the biomembrane mimetic magnetic microparticle was washed with 1004, of 100 mM Tris buffer solution (pH7.5) 5 times. The step can present an MBP-GalT having membrane-binding peptide on a C terminal to a biomembrane mimetic magnetic microparticle, and MBP-GalT-immobilized magnetic microparticle in which MBP-GalT was immobilized to magnetic microparticle via a membrane-binding peptide and an organic membrane (biomimetic membrane) was obtained.
(3) Measurement of Activity of MBP-GalT-Immobilized Magnetic Microparticle
Subsequently, MBP-GalT activity for 4MU-GlcNAc of MBP-GalT-immobilized magnetic microparticle obtained in this Example 9(2) was measured and whether MBP-GalT which was immobilized to a magnetic microparticle via a membrane-binding peptide and an organic membrane (biomimetic membrane) has an activity or not was confirmed. Specifically, 50 μL of substrate solution prepared by a method as in Example 4(4) was added to MBP-GalT-immobilized magnetic microparticle prepared in this Example 9(2) and reacted at room temperature for 2 hours. Thereafter, 1 μL of 10 mg/mL DHB was mixed with 1 μL of the reaction liquid to measure an activity thereof by MALDI-TOF-MS by a method as in Example 1(3) [3-2]. As a control, an activity of free membrane-binding peptide-fused MBP-GalT which was not immobilized to a magnetic microparticle was also measured. Measurement results of free membrane-binding peptide-fused MBP-GalT are shown in
As shown in
The above observation found that a membrane-binding peptide-fused MBP-GalT can be immobilized to a biomembrane mimetic magnetic microparticle, with activity maintained, and the product can be used as MBP-GalT-immobilized magnetic microparticle.
Using N-acetylgalactosamine transferase 2 (ppGalNAcT2) which is an enzyme transferring N-acetylgalactosamine to threonine or serine side-chain hydroxyl group as a biomolecule, a membrane-binding peptide-fused ppGalNAcT2 incorporating a polypeptide of 42 residues containing membrane-binding peptide in a C terminal of the ppGalNAcT2 was prepared in vivo, and by presenting the membrane-binding peptide-fused ppGalNAcT2 to biomembrane-mimicking magnetic microparticle, ppGalNAcT2-immobilized magnetic microparticle was prepared and an activity thereof was measured.
(1) Preparation of Membrane-Binding Peptide-Fused ppGalNacT2 In Vivo
ppGalNAcT2 was cloned from cDNA derived from small intestines (Invitrogen) to obtain a vector in which ppGalNAcT2 was constructed (Tabak, L A et al. JBC. 2006, 281, 8613-8619). The vector and fragments encoding a polypeptide of 42 residues containing a membrane-binding peptide prepared in Example 8 were treated with restriction enzymes of NdeI and BamHI to construct an expression vector of ppGalNAcT2 in which membrane-binding peptide was fused to a C terminal. The vector was transformed to methanol utilizing yeast to prepare yeast variant. After the product was cultured in 50 mL of YPAD medium at 37° C. overnight, the culture solution was subcultured in 500 mL of YPAD medium, and by adding 2.5 mL of methanol thereto every 24 hours, the product was further cultured at 20° C. for total 72 hours.
The culture solution was subjected to centrifugal separation at 10,000×g for 20 minutes to remove bacterial cells, and supernatants containing membrane-binding peptide-fused ppGalNAcT2 were collected and subjected to Ni column (His Trap FF; Amersham Pharmacia Biotech) under alkaline conditions (pH 8 to 10). Thereafter, the product was condensed by ultrafiltration under alkaline conditions (pH 8 to 10) to obtain purified membrane-binding peptide-fused ppGalNAcT2v (1.5 mg/mL).
(2) Preparation of ppGalNAcT2-Immobilized Magnetic Microparticle
By presenting a membrane-binding peptide-fused ppGalNAcT2 obtained in this Example 10(1) to a biomembrane mimetic magnetic microparticle, ppGalNAcT2-immobilized magnetic microparticle was prepared. Specifically, 40 μL of membrane-binding peptide-fused ppGalNAcT2 solution (60 μg) obtained in this Example 10(1) was added to a biomembrane-mimicking magnetic microparticle prepared in a method as in Example 1(2) and agitated at 4° C. for 2 hours. Thereafter, all of the solutions were removed and the biomembrane mimetic magnetic microparticle was washed with 100 μL of 50 mM Tris-HCL buffer solution (pH7.5) 7 times. This step can present ppGalNAcT2 having membrane-binding peptide on a C terminal to magnetic microparticle, and ppGalNAcT2-immobilized magnetic microparticle in which ppGalNAcT2 was immobilized to a magnetic microparticle via a membrane-binding peptide and an organic membrane (biomimetic membrane) was obtained.
(3) Preparation of ppGalNAcT2 Nonspecific Adsorption Magnetic Microparticle
As a control, by mixing 40 μL (60 μg) of ppGalNAcT2 with Ag-coated magnetic microparticle using the above-mentioned step, ppGalNAcT2 nonspecific adsorption magnetic microparticle, in which ppGalNAcT2 was nonspecifically adsorbed, was prepared.
(4) Measurement of Activity of ppGalNAcT2-Immobilized Magnetic Microparticle and ppGalNAcT2 Nonspecific Adsorption Magnetic Microparticle
Subsequently, ppGalNAcT2 activity for MUC5AC mucin of ppGalNAcT2-immobilized magnetic microparticle obtained in this Example 10(2) and ppGalNAcT2 nonspecific adsorption magnetic microparticle obtained in this Example 10(3) was measured. Specifically, 5 μL of 100 mM manganese chloride, 10 μL of 10 mM uridine 5′-2phosphoric acid-α-D-N-acetylgalactosamine, 1.25 μL of 10 mM MUC5AC mucin peptide (GTTPSPVPTTSTTSA-NF12) and 2.5 μL of 1M Tris-HCL buffer solution (pH7.5) were mixed with 36.25 μL of ultrapure water to prepare a substrate solution. The substrate solution was added to 1 mg of ppGalNAcT2-immobilized magnetic microparticle and 1 mg of ppGalNAcT2 nonspecific adsorption magnetic microparticle prepared in this Examples 10(2) and (3), respectively, each by 50 μL, and reacted at 37° C. for one hour. 1 μL of 10 mg/mL DHB was mixed with 14 of reaction liquid to measure an activity thereof by MALDI-TOF-MS by a method as in Example 1(3) [3-2]. Measurement results of ppGalNAcT2-immobilized magnetic microparticle are shown in
As shown in
The above observation found that a membrane-binding peptide-fused ppGalNAcT2 can be immobilized to a biomembrane mimetic magnetic microparticle, with activity maintained, and the product can be used as ppGalNAcT2-immobilized magnetic microparticle.
The above-mentioned Examples can provide a biomolecule-immobilized carrier capable of controlling molecular orientation with biomolecule activity maintained due to excellent durability and being readily handled due to solubilization of a membrane-binding molecule, particularly a biomolecule-immobilized magnetic microparticle and a probe-immobilized carrier using a biomolecule having said membrane-binding peptide and/or said membrane-binding molecule as a probe. For example, in case of a biomolecule-immobilized microarray using sugar chain as a biomolecule, a protein (marker) which recognizes and binds sugar chain as a probe can be fished. When sugar chain which is specifically expressed in cancer, etc is immobilized via a membrane-binding peptide and an organic membrane (biomimetic membrane), this invention can be used as a diagnostic tool for identifying cancer only by flowing patient's serum.
A biomolecule-immobilized carrier and a method for immobilizing a biomolecule to a carrier according to the present invention are not limited to the above Examples, but may be altered accordingly.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008-039890 | Feb 2008 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2009/053038 | 2/20/2009 | WO | 00 | 11/29/2010 |
