JP2010178640A - Polypeptide capable of controlling adsorption of cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a polypeptide which can control the non-specific adsorption of cells to a solid surface, when used as a solid surface-modifying molecule. <P>SOLUTION: The polypeptide comprises polar amino acids and non-charged amino acids. The polypeptide comprises at least 50 amino acid residues. The polypeptide in which one unit of amino acid sequence is repeatedly arranged, wherein an amino acid sequence comprising several amino acid residues is one unit. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a polypeptide capable of controlling nonspecific adsorption of cells to the surface of a solid phase when used as a solid phase surface modifying molecule, and more specifically composed of a polar amino acid and an uncharged amino acid. Regarding a polypeptide, specifically, a polypeptide comprising at least 50 amino acid residues, wherein an amino acid sequence comprising a plurality of amino acid residues is defined as one unit, and the amino acid sequence of the one unit is repeated. About.

  In developing medical devices such as artificial blood vessels, biomolecule detection and analysis devices such as biochips, and materials for cell-related applications such as magnetic nanoparticles, materials and device surfaces are common issues. Nonspecific adsorption of proteins and cells to In addition, attempts have been made to suppress nonspecific adsorption of proteins and cells to the surface of materials and devices by surface modification using various molecules.

  Polyethylene glycol (PEG) is a polymer most utilized as a surface modifying molecule. PEG is a polar molecule that is non-charged and has strong hydrophilicity, and has excellent biocompatibility. By modifying higher molecular weight PEG on the particle surface, the thickness of the immobilized water layer (FALT) formed on the particle surface increases, and steric hindrance increases the interaction of particles with cells and proteins. It can be reduced (see Non-Patent Documents 1 and 2). In addition, when using particles in vivo such as DDS, the modification of PEG on the particle surface reduces adsorption to serum proteins and capture by macrophages, and the particles are in the reticuloendothelial system (RES). It helps to escape from the foreign body phagocytosis. Thereby, the PEG-modified particles can circulate in the blood for a long time (see Non-Patent Documents 3, 4 and 5).

  In addition to PEG, as a surface-modifying molecule that has the effect of preventing nonspecific adsorption of particles to cells and proteins, it is commonly used as a dextran or blocking agent that is a non-charged and hydrophilic polysaccharide like PEG. Has been reported (see Non-Patent Documents 6 and 7). In particular, phosphatidylcholine (PC) is used as a modifying molecule for the device surface. PC is a major phospholipid constituting the outer cell membrane of animals. Cells distribute and transmit information selectively by avoiding non-specific interactions with extracellular proteins, sugars, and other biomolecules by placing neutral phospholipid PC on the surface. ing. Non-specific adsorption of serum proteins and blood components on the surface of artificial biological membranes made by self-assembly on a hydrophobic substrate using 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer with PC groups in the side chain It was possible to almost completely suppress the adhesion and activation of (see Non-Patent Document 8).

  In addition, as magnetic fine particles that can be applied to medical devices such as biosensors, magnetic fine particles with little non-specific adsorption and magnetic fine particles combined with a functional protein for specifically binding to a target substance are disclosed (patents) References 1 and 2).

JP 2006-314314 A JP-A-8-228782

Sadzuka, Y., A. Nakade, R. Hirama, A. Miyagishima, Y. Nozawa, S. Hirota and T. Sonobe (2002) .Effects of mixed polyethyleneglycol modification on fixed aqueous layer thickness and antitumor activity of doxorubicin containing lipid. Int J Pharm 238 (1-2): 171-80. Fang, C., B. Shi, YY Pei, MH Hong, J. Wu and HZ Chen (2006) .In vivo tumor targeting of tumor necrosis factor-alpha-loaded stealth nanoparticles: effect of MePEG molecular weight and particle size.Eur J Pharm Sci 27 (1): 27-36. Jeong, YI, SJ Seo, IK Park, HC Lee, IC Kang, T. Akaike and CS Cho (2005). Cellular recognition of paclitaxel-loaded polymeric nanoparticles composed of poly (gamma-benzyl L-glutamate) and poly (ethylene glycol) ) diblock copolymer endcapped with galactose moiety. Int J Pharm 296 (1-2): 151-61. Martina, MS, JP Fortin, C. Menager, O. Clement, G. Barratt, C. Grabielle-Madelmont, F. Gazeau, V. Cabuil and S. Lesieur (2005). Generation of superparamagnetic pores revealed as highly efficient MRI contrast agents for in vivo imaging. J Am Chem Soc 127 (30): 10676-85. Dixit, V., J. Van den Bossche, DM Sherman, DH Thompson and RP Andres (2006) .Synthesis and grafting of thioctic acid-PEG-folate conjugates onto Au nanoparticles for selective targeting of folate receptor-positive tumor cells.Bioconjug Chem 17 (3): 603-9. Lewin, M., N. Carlesso, CH Tung, XW Tang, D. Cory, DT Scadden and R. Weissleder (2000). Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat Biotechnol 18 (4 ): 410-4. Wilhelm, C., C. Billotey, J. Roger, J. N. Pons, J. C. Bacri and F. Gazeau (2003). Intracellular uptake of anionic superparamagnetic nanoparticles as a function of their surface coating. Biomaterials 24 (6): 1001-11. Futamura, K., R. Matsuno, T. Konno, M. Takai and K. Ishihara (2008). Rapid development of hydrophilicity and protein adsorption resistance by polymer surfaces bearing phosphorylcholine and naphthalene groups. Langmuir 24 (18): 10340-4 .

  The following analysis is given by the present invention. Each of the above-mentioned patent documents and non-patent documents is incorporated in the present specification by reference.

  The following two methods are applied to the surface of the device / material used for the cell-related applications as described above. One is that the antibody is modified to add binding / specificity to the target cell, and the second is polyethylene glycol (PEG), phosphatidylcholine ( It is that resistance from non-specific adsorption of cells is added by modifying phosphatidylcholine (PC), dextran, etc. In order to realize a highly accurate application, it is necessary to simultaneously add the above two elements to the surface of the device / material, and further, it is necessary to modify two kinds of molecules. When PEG or the like is modified by a chemical crosslinking method on the surface on which the antibody is immobilized, it is necessary to consider a modification mechanism and a size (length) of the modified molecule that do not inhibit the activity of the antibody. However, in many cases, the effect of suppressing nonspecific adsorption of cells cannot be sufficiently exhibited by considering them. Therefore, a form in which an antibody is immobilized on the surface of a device / material using a molecule such as PEG as a linker has been studied. However, this method requires a stepwise high-accuracy reaction of each molecule, and there are problems of complicated operation and long reaction time.

  Therefore, the present invention has been made in view of the above, and an object of the present invention is to provide a polypeptide capable of controlling the adsorption of cells. More specifically, the present invention is used as a solid phase surface modifying molecule. An object of the present invention is to provide a polypeptide capable of controlling nonspecific adsorption of cells to the solid surface.

  As a result of intensive studies to solve the above problems, the inventors of the present invention are able to control nonspecific adsorption of cells on a solid phase surface by a polypeptide composed of polar amino acids and uncharged amino acids. I found out.

  That is, in one aspect of the present invention, the polypeptide is a polypeptide composed of a polar amino acid and an uncharged amino acid. As long as this polypeptide has an amino acid sequence composed of at least 50 amino acid residues, the amino acid sequence may be any sequence, and as long as it does not affect the expression system of the cell in which the polypeptide is used, The length of is not particularly limited. The polypeptide is characterized in that an amino acid sequence composed of a plurality of amino acid residues is defined as one unit, and the amino acid sequence of the one unit is repeatedly arranged.

  According to the present invention, when separating or recovering or detecting a protein or cell as a target substance using a solid phase, the target substance is provided with hydrophilicity and steric hindrance by arranging the polypeptide on the surface of the solid phase. Nonspecific adsorption to the solid phase surface can be suppressed.

  The polypeptide may have a structure in which an amino acid sequence of one unit composed of a plurality of amino acid residues is repeatedly arranged. Thus, when designing such a polypeptide, the type and residue length of the amino acid to be used can be optimized in units, and the amino acid sequence can be efficiently selected. The amino acid sequence constituting this 1 unit can be arbitrarily selected by appropriately selecting the amino acid.

  Examples of polar amino acids and uncharged amino acids constituting one unit include asparagine (N), glutamine (Q), threonine (T), tyrosine (Y) and serine (S). Among them, hydrophilicity and steric hindrance From the viewpoint of imparting properties, asparagine having high hydrophilicity and a small side chain size is preferable. Moreover, serine may be contained in a part of the amino acid sequence of the whole unit or the entire polypeptide. By adding serine to the amino acid sequence, flexibility can be imparted to the polypeptide, and it can be expected to contribute to the improvement of the ability to suppress nonspecific adsorption of the target substance.

Such an amino acid sequence of one unit is a combination of the first type amino acid × a and the second type amino acid × b, where a and b are arbitrary integers, for example, the selected amino acid symbol and the values of a and b. When displayed (NQTYS × a, NQTYS × b), various combinations are possible. For example, glutamine (Q) is selected as the first type of amino acid, serine (S) is selected as the second type of amino acid, and a combination of the values of a and b (for example, a, b = 2,1; 3,1; 3 , 2; 4,1; 4,2; 5,1; 5,2 ,,), (QaSb) is (Q ₂ S), (Q ₃ S), (Q ₃ S ₂ ) , (Q ₄ S), (Q ₄ S ₂ ), (Q ₅ S), (Q ₅ S ₂ ), etc. are all possible. In the present invention, specifically, one unit is a combination of four asparagine and one serine (N ₄ S), and this one unit is a repetitive sequence ((N ₄ S) _n ). Peptides can control nonspecific adsorption of cells to the solid surface when used as a solid surface modifying molecule. In this case, in the arrangement | sequence in 1 unit, the arrangement position of serine does not have limitation in particular, What is necessary is just to design in arbitrary positions in 1 unit. In addition, the number of repetitions of the same arrangement of one unit is not particularly limited as long as steric hindrance can be secured, but generally, as the number of repetitions increases, it becomes difficult to obtain due to cost and ease of synthesis. Repeating about 20 times is preferable. In the present invention, the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 is particularly preferable, and may be artificially synthesized using an amino acid synthesizer or the like.

  In addition, the polypeptide of the present invention can be incorporated as a fusion protein by adding amino acids of functional proteins such as antibodies applied to the surface of devices and materials used for cell-related applications. In addition, in order to use the target substance captured by this functional protein for further research and the like, it is necessary to recover from the polypeptide, for example, by providing a recognition site for a specific restriction enzyme within the polypeptide sequence, The captured target substance can be easily recovered.

  Moreover, according to another viewpoint of this invention, this invention can provide the plasmid containing the base sequence which codes the said polypeptide. A plasmid containing the base sequence shown in SEQ ID NO: 2 is preferable. This allows the cells to be transformed so that the polypeptide can be expressed. In addition, the present invention can provide a cell transformed with the plasmid, and the cell is preferably a magnetic bacterium.

  According to still another aspect of the present invention, in the magnetic bacterium, the plasmid is introduced into the magnetic bacterium, the magnetic bacterium is cultured, and the synthesized magnetic bacterium particles are extracted, whereby non-specificity to the cell is obtained. It is possible to provide a method for producing magnetic bacterial particles obtained by synthesizing a polypeptide capable of controlling adsorption. In addition, any of the above-described polypeptides can be expressed by the above-described method. Specifically, magnetic bacterial particles expressed on the magnetic bacterial particle film of magnetic bacteria can be obtained.

  Furthermore, according to another aspect of the present invention, by using the magnetic bacterial particles, a method for suppressing nonspecific adsorption of the particles to cells, a method for improving the dispersibility of the particles, and a method for separating cells I will provide a. As a result, nonspecific adsorption of the cells to the particles can be controlled by the hydrophilicity and steric hindrance caused by the polypeptide in the particle having the polypeptide arranged on the particle surface. In addition, the above-mentioned polypeptide on the particle surface contributes to suppression of aggregation between particles and can improve the dispersibility of the particles. In addition, as a result of the nonspecific adsorption of the cells being suppressed by the polypeptide on the particle surface, for example, the target cells can be separated from peripheral blood with high purity and high efficiency.

According to the present invention, nonspecific adsorption to cellular particles can be controlled by the polypeptide composed of the polar amino acid and the uncharged amino acid of the present invention. A polypeptide composed of at least 50 amino acid residues and an amino acid sequence composed of a plurality of amino acid residues as one unit, wherein the amino acid sequence of the one unit is repeated, specifically, at least two types A polypeptide composed of a combination of the above amino acids, for example, (N ₄ S) _n polypeptide is arranged on the particle surface, and due to the hydrophilicity and steric hindrance caused by the polypeptide, non-specificity to cell particles Adsorption can be controlled. In addition, the (N ₄ S) _n polypeptide on the particle surface contributes to the suppression of aggregation between particles and can improve the dispersibility of the particles. Furthermore, by extending the polypeptide chain length, it is possible to increase the effect on the interaction between particles and cells or between particles. In addition, when the polypeptide of the present invention is modified on a material (magnetic particle) used for magnetic cell separation, the antibody-binding protein and the polypeptide of the present invention are displayed as a fusion protein so that they are oriented on the magnetic nanoparticles. It is possible to immobilize antibodies that retain their properties. By using the antibody-immobilized magnetic nanoparticles, only target cells can be magnetically labeled with high accuracy and can be magnetically separated with high purity, in accordance with avoidance of nonspecific adsorption of cells. Furthermore, since the dispersibility of the particles is improved by modifying the polypeptide of the present invention, the reaction efficiency of the antibody-immobilized magnetic nanoparticles with the target cells is improved, and magnetic separation at a high recovery rate is possible. is there.

FIG. 2 shows a schematic diagram for preparing a plasmid for protein G-NS polypeptide linker (100AA) -Mms13 fusion protein expression on BacMPs in Example 1. FIG. FIG. 2 shows a schematic diagram for preparing a plasmid for protein G-NS polypeptide linker (50AA) -Mms13 fusion protein expression on BacMPs in Example 1. FIG. The conceptual diagram of Protein G-NS polypeptide linker (100AA) -Mms13 fusion protein expression BacMP (protein G-linker (100) -BacMP) in this invention is shown. The figure of the expression confirmation by SDS-PAGE of the protein G-NS polypeptide linker (100AA) -Mms13 fusion protein on BacMPs in Example 1 is shown. It is a figure which shows the number of rabbit IgG antibody fix | immobilized on BacMPs depending on Protein G in Example 2. FIG. (A) And (B) is a figure which shows the particle size distribution of BacMPs in Example 3. FIG. It is a figure which shows observation of BacMPs by the transmission electron microscope (Transmission electron microscopic) (TEM) in Example 4. FIG. It is a figure which shows the correlation of the expression of NS polypeptide linker on BacMPs in Example 5, and nonspecific adsorption | suction to the cell of BacMPs. It is a figure which shows the magnetic recovery rate of the Raji cell using the anti-CD19 antibody fixed BacMPs in Example 6. FIG. 6 shows direct magnetic separation of CD19 + cells from peripheral blood using anti-CD19 antibody-immobilized BacMPs in Example 7.

  The polypeptide of the present invention is a polypeptide capable of controlling non-specific adsorption of cells to a solid phase surface as a solid phase surface modification molecule in a cell-related application material such as magnetic nanoparticles. And

  More specifically, the polypeptide of the present invention is a polypeptide of polar amino acids and uncharged amino acids. As long as this polypeptide has an amino acid sequence composed of at least 50 amino acid residues, the amino acid sequence may be any sequence, and unless this affects the expression system of the cell in which the polypeptide is used, The length of the polypeptide is not particularly limited. This polypeptide is characterized by a polypeptide in which an amino acid sequence composed of a plurality of amino acid residues is taken as one unit, and the amino acid sequence of the one unit is repeated.

  The amino acid constituting the polypeptide of the present invention is not particularly limited as long as it is a polar amino acid and an uncharged amino acid. Asparagine (N), glutamine (Q), threonine (T), tyrosine corresponding to these amino acids Although it is arbitrarily selected from (Y) and serine (S), it is preferably used by selecting two or more from the group consisting of asparagine, glutamine and serine. Moreover, polar amino acids and uncharged amino acids are also hydrophilic amino acids, and the amino acids constituting the polypeptide of the present invention are also hydrophilic. The peptide sequence within one unit is preferably composed of a sequence of five amino acids, but is not limited thereto.

More preferably, the polypeptide of the present invention is specifically arranged by arranging four first type amino acids (eg, asparagine) and then one second type amino acid (eg, serine). A polypeptide composed of a peptide having (N ₄ S) as one unit is preferable, but here, the sequence of five amino acids in one unit is not limited. That is, in one unit, the sequence of only one second-type amino acid (serine) and four first-type amino acids (asparagine) may be arbitrarily arranged. For example, when a and b are arbitrary integers, one unit is composed of a combination of the first type of amino acid × a and the second type of amino acid × b, and the sequence of one unit is asparagine (N), Amino acid sequences consisting of various combinations arbitrarily selected from glutamine (Q), threonine (T), tyrosine (Y) and serine (S) are possible.

The polypeptide of the present invention is a polypeptide in which one unit composed of the amino acid sequences of the above four asparagines and one serine (hereinafter referred to as “(N ₄ S) _n polypeptide”). ) Is preferred. In particular, asparagine is preferable from the viewpoint of imparting hydrophilicity and steric hindrance to the polypeptide, and serine can impart structural flexibility to the polypeptide and improve the nonspecific adsorption inhibition ability of the target substance. Can contribute. Here, the repeating number (n) of the one unit is not particularly limited and can take any value. In the polypeptide of the present invention, it is at least several times, preferably about 10 times or more, particularly about 20 times. Repeat is preferred.

  In the polynucleotide of the present invention, the polypeptide chain length can be adjusted by adjusting the number of repetitions of the one unit. Depending on the polypeptide chain length, the control of nonspecific adsorption can be adjusted. Is possible.

Thus, the polypeptide of the present invention can be appropriately prepared according to the above-mentioned conditions depending on the degree of nonspecific adsorption control. In the present invention, the above (N ₄ S) _n polypeptide is preferable. In particular, (N ₄ S) ₂₀ polypeptide in which one unit of (N ₄ S) repeats 20 times is preferred. In the present invention, the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 is preferred.

  Furthermore, the polypeptide of the present invention can be used as a linker for displaying a functional protein when developing a material for cell-related applications, for example, when modifying the surface of a magnetic nanoparticle. In this case, the functional protein can be integrated with the polypeptide of the present invention. That is, when preparing a functional protein, it can be used as a fusion protein with the polypeptide of the present invention, and the amino acid sequence of the functional protein can be incorporated into the polypeptide of the present invention. Examples of functional proteins include immune-related proteins such as antigens, antibodies, protein A, and protein G, binding proteins having binding ability such as lectin, avidin, and biotin, coenzymes, hydrolases, and oxidoreductases. , Enzymes such as isomerase, transferase, desorption enzyme, restriction enzyme, various receptors, marker proteins such as GFP, and the like, peptides composed of a part of the above functional proteins, HA ( Hemagglutinin), FLAG, Myc and other epitope tags, GST, maltose binding protein, biotinylated peptide, oligohistidine and other affinity tags can be exemplified, but are not limited to these, and one or more The functional protein or functional peptide may be fused functional protein.

  In this case, the target substance captured by the functional protein can be easily recovered by providing a recognition site for a specific restriction enzyme in the sequence of the polypeptide of the present invention. It can be used for further research and experiments.

  In addition, the polypeptide of the present invention can be artificially synthesized with an amino acid synthesizer. In the present invention, the base sequence encoding the polypeptide, that is, the polypeptide of the present invention which is a specific amino acid sequence. A base sequence for synthesizing in a cell such as bacteria, the base sequence is introduced into a vector such as a plasmid by a known gene technique, then, by a known introduction technique such as microinjection or electroporation, The plasmid can be synthesized by introducing it into a host cell comprising an expression system that can express the polypeptide.

  Examples of host cells include bacterial prokaryotic cells such as magnetic bacteria and Escherichia coli, eukaryotic cells such as yeast, insect cells, animal cells, and plant cells. The polypeptide of the present invention is used for cell-related applications. In particular, it is preferable to use a magnetic bacterium since it can be formed on the magnetic nanoparticles.

  The magnetic bacterium is a bacterium having the ability to accumulate magnetic bacterium particles (also referred to as magnetic fine particles) in the body, and examples of the magnetic bacterium (Magnetospirillum sp.) Usable in the present invention include AMB-1 (FERM BP). -5458), MS-1 (IFO15272, ATCC31632, DSM3856), MSR-1 (IFO15272, DSM6361), RS-1 (FERM BP-13283), MGT-1 (FERM P-16617), etc. However, the present invention is not limited to these examples.

  The polypeptide of the present invention can be produced using the host magnetic bacterium as magnetic fine particles that contain the functional protein and are expressed on the magnetic fine particle film. As such a production method, for example, DNA encoding all or a part (at least a membrane-binding portion) of a membrane protein Mms13 that is inherently bound to an organic membrane, or a functional protein and a polypeptide are encoded in the DNA. By culturing a magnetic bacterium transformed with a recombinant plasmid containing a DNA sequence fused with the DNA to be treated and a promoter sequence having a high transcription activity such as the mms16 promoter, the polypeptide of the present invention (function Examples thereof include a method of generating magnetic fine particles that are expressed on the magnetic fine particle film in bacteria, including a sex protein. The magnetic fine particles in which the polypeptide of the present invention (including functional protein) is expressed on the magnetic fine particle film are obtained by crushing or dissolving the magnetic bacteria grown by culture by a known method, and using a magnet. Easy to collect.

The magnetic bacterial particles obtained in this way are non-specific to cellular particles due to the hydrophilicity and steric hindrance that the polypeptides provide by placing (N ₄ S) _n polypeptide chains on the particle surface. Can control the adsorption. In addition, the (N ₄ S) _n polypeptide on the particle surface contributes to the suppression of aggregation between particles and can improve the dispersibility of the particles. Furthermore, by extending the polypeptide chain length, it is possible to increase the effect on the interaction between particles and cells or between particles.

  When the polypeptide of the present invention is used as a linker for displaying functional proteins when modifying the surface of particles such as magnetic nanoparticles, the polypeptide of the present invention has a constant orientation of the proteins. It is useful to display on the surface of the device material while keeping For example, by displaying protein G, which is an antibody-binding protein, and the polypeptide of the present invention as a fusion protein, it is possible to immobilize an antibody with orientation maintained on magnetic nanoparticles. By using the antibody-immobilized magnetic nanoparticles, only target cells can be magnetically labeled with high accuracy and can be magnetically separated with high purity, in accordance with avoidance of nonspecific adsorption of cells. Moreover, since the dispersibility of the particles is improved by modifying the polypeptide of the present invention, the reaction efficiency of the antibody-immobilized magnetic nanoparticles with the target cells is improved, and magnetic separation with high recovery rate is possible .

  Hereinafter, the present invention will be described more specifically with specific examples. However, the scope of the present invention is not limited to these examples.

In this example, (N ₄ S) _n polypeptide (also referred to as NS polypeptide) was synthesized as a novel particle surface modifying molecule for suppressing nonspecific adsorption of nanoparticles to cells in cell-related applications. In order to verify the effect of NS polypeptide, in each Example, magnetic bacterial particles (hereinafter referred to as BacMPs) in which NS polypeptide is introduced as a linker when protein G is expressed (magnetic particles) , Also called magnetic nanoparticles), and analyzed the interaction between protein G-NS polypeptide-expressing BacMPs and cells.

Production of protein G-NS polypeptide linker expressing BacMPs (protein G-linker-BacMPs)
A plasmid was constructed for the purpose of displaying protein G-NS polypeptide linker fusion protein on BacMPs (FIGS. 1 and 2). Plasmid (pUC18) containing gene (315 bp) (SEQ ID NO: 2) encoding NS polypeptide linker ((N ₄ S) ₁₈ + LVPRGS + (N ₄ S) (SEQ ID NO: 1)) The linker gene was excised from the order) using the restriction enzyme SspI and introduced into the SspI site of pUMP16M13. This was designated as an NS polypeptide linker (100AA) expression plasmid (pUM13L (100)) (FIG. 1). Furthermore, an Hpa I site and a stop codon were introduced into the center of the NS polypeptide linker gene on pUM13L (100) by site-direct mutagenesis. This was designated as an NS polypeptide linker (50AA) expression plasmid (pUM13L (50)) (FIG. 2). The restriction enzyme Ssp from a plasmid (pPCR-Script) (ordered gene synthesis from OPERON) containing the gene (174 bp) (SEQ ID NO: 4) encoding the IgG binding domain (B1-domain) (SEQ ID NO: 3) of Protein G The B1-domain gene was excised using I and introduced into the Afe I site of pUM13L (100) and the Hpa I site of pUM13L (50). These were used as protein G-NS polypeptide linker (100 AA or 50AA) expression plasmids (pUM13L (100) B1, pUM13L (50) B1). Note that SEQ ID NOs: 6 and 7 were used in the preparation of pUM13L (50) B1. pUM13L (100) B1 contains a fusion gene of Mms13 gene + ((N ₄ S) ₁₈ + LVPRGS + (N ₄ S)) polypeptide gene + protein G gene, and ampicillin resistance gene. pUM13L (50) B1 contains a fusion gene of Mms13 gene + (N ₄ S) ₁₀ polypeptide gene + protein G gene, and ampicillin resistance gene. The amino acid sequence of (N ₄ S) ₁₀ polypeptide is represented by SEQ ID NO: 5. For both plasmids, mms16 promoter was used as the promoter. The amino acid (LVPRGS) in the NS polypeptide linker ((N ₄ S) ₁₈ + LVPRGS + (N ₄ S)) is a digestion site of thrombin enzyme.

In this example, a restriction enzyme recognition site for recovery of a target substance after capture was provided in a polypeptide composed of asparagine and serine ((N ₄ S) _n polypeptide). A polypeptide ((N ₄ S) ₂₀ polypeptide) in which one unit of four asparagines and one serine is repeated 20 times can be obtained. In addition, in FIG. 3, the conceptual diagram of Protein G-NS polypeptide linker (100AA) -Mms13 fusion protein expression BacMP (protein G-linker (100) -BacMP) in this invention is shown.

  After introducing pUM13L (100) B1 and pUM13L (50) B1 into magnetic bacterium AMB-1 by electroporation, BacMPs extracted from the transformant is boiled in 1% SDS for 30 minutes to recover BacMPs membrane protein Then, the expression of the target protein: protein G-NS polypeptide linker in the BacMPs membrane was confirmed by SDS-PAGE (acrylamide: 12.5%). As a result, a band corresponding to the fusion protein of anchor protein: Mms13 and protein G-NS polypeptide linker was confirmed in SDS-PAGE of the membrane protein of BacMPs extracted from the transformant (FIG. 4). By introducing plasmid pUM13L (100) B1 or pUM13L (50) B1 into magnetic bacteria, protein G-NS polypeptide linker (100AA) -expressing BacMPs (protein G-linker (100) -BacMPs) or protein G-NS It was shown that BacMPs expressing protein linker (50AA) (protein G-linker (50) -BacMPs) can be produced.

Evaluation of the number of immobilized antibodies on Protein G-linker-BacMPs Protein G-linker (100) -BacMPs Protein G-linker (50) -BacMPs and protein G-BacMPs were extracted. After washing with HEPES, each BacMPs: 20 μg was reacted with ALP-labeled rabbit-derived anti-goat IgG or ALP-labeled chicken-derived anti-rat IgG antibody solution: 20 μl (20 μg / ml) (RT, 30 minutes). After washing by magnetic separation, Lumifos 530: 80 μl was added to BacMPs suspension: 20 μl, and the luminescence intensity after 5 minutes was measured. Based on a calibration curve prepared using an ALP-labeled antibody, the amount immobilized on BacMPs was calculated from the measured luminescence intensity. Further, the number of antibodies immobilized on BacMP 1 particles was calculated from the amount of antibody immobilized on BacMPs using the following conditions (antibody molecular weight: 150 kDa, BacMP diameter: 75 nm, BacMP density: 5.2 g / cm ³ ).

  As a result, in the three types of BacMP evaluated, about 20 molecules of antibody were immobilized on BacMP 1 particles in a protein G-dependent manner (FIG. 5). Protein G-linker (100) -BacMP, protein G-linker (50) -BacMP, protein G-BacMP Number of rabbit-derived IgG antibodies bound nonspecifically from the number of rabbit-derived IgG antibodies immobilized on one particle The subtracted value was defined as the number of antibodies bound on protein G dependently expressed on BacMP 1 particles. From this, protein G-linker (100) -BacMP, protein G-linker (50) -BacMP, protein G-BacMP 1 particle has approximately the same number (20 molecules / a single BacMP) of functional protein G. It was thought to be expressed. In the expression system of protein G on BacMPs, introduction of NS polypeptide linker was considered not to affect the expression and function of the fusion protein. From the above, it was shown that the prepared BacMPs are useful for immobilizing antibodies.

Correlation analysis of BacMPs dispersibility and expressed NS polypeptide linker chain length
Protein G-linker (100) -BacMPs, protein G-linker (50) -BacMPs, protein G-BacMPs are suspended in super pure water or PBS (5μg BacMPs / ml) and immediately after ultrasonic stirring. The particle size distribution was measured using a dynamic light scattering type particle size distribution measuring apparatus, and the dispersibility was evaluated.

  As a result, the average particle size of each BacMPs in super pure water is (protein G-linker (100) -BacMPs: 52.9 ± 16.8 nm, protein G-linker (50) -BacMPs: 50.7 ± 13.5 nm and protein G-BacMPs: 61.9 ± 50.5 nm) (FIG. 6A). On the other hand, the particle size indicated by the main peak of each BacMPs in PBS is (protein G-linker (100) -BacMPs: 66.8 ± 10.6 nm, protein G-linker (50) -BacMPs: 105.2 ± 29.3 nm, protein G -BacMPs: 211.8 ± 42.8 nm) (FIG. 6B), and the dispersibility of BacMPs was improved in proportion to the NS polypeptide linker chain length. In super pure water, electrical repulsion between BacMPs occurs due to the negative charge derived from phosphatidylethanolamine, the main phospholipid that covers BacMPs, and BacMPs are monodispersed. I thought it was easy to keep. On the other hand, in PBS, which is a buffer solution, it was considered that the surface charge of BacMPs was canceled by a large amount of ions, and the formation of aggregates due to the magnetic force of each BacMP was promoted. However, it was thought that aggregation of BacMPs was suppressed by steric hindrance by arranging NS polypeptide linker on BacMPs. Furthermore, since NS polypeptide is hydrophilic, it was suggested that an immobilized water layer (see Non-Patent Document 1) may be formed on the surface of BacMPs as well as the effect of PEG. From the above, it was shown that the NS polypeptide linker display can maintain the dispersibility of BacMPs even in a buffer solution.

Observation of protein G-linker-BacMPs by transmission electron microscope (TEM)
Protein G-linker (100) -BacMPs and protein G-BacMPs were observed with a transmission electron microscope STEM.

  As a result, a thicker layer was observed on the surface of protein G-linker (100) -BacMPs compared to Protein G-BacMPs (FIG. 7). In TEM, it was considered that NS polypeptide expressed on protein G-linker (100) -BacMPs was accumulated on the surface of BacMPs to observe a sample in vacuum, and it was observed as a thick layer. This was considered to be a result supporting the expression of NS polypeptide on BacMPs and the steric hindrance of NS polypeptide.

Evaluation of NS polypeptide display effect on non-specific adsorption of BacMPs to cells Cultured cell suspension: 70μl (2 × 10 ⁵ cells), protein G-linker (100) -BacMPs, protein G-linker ( 50) -BacMPs, protein G-BacMPs: 30 μl (1-30 μg) was reacted (4 ° C., 10 minutes). After centrifugal washing and magnetic separation, the number of cells recovered by nonspecific adsorption of BacMPs to cells was counted, and the nonspecific cell recovery rate was calculated. As cultured cells, Raji cells (B cell line), JM cells (T cell line), THP-1 cells (monocyte line), and RAW 264.7 cells (macrophage line) were used.

  The result is shown in FIG. When Protein G-BacMPs was used, the nonspecific recovery rate of Raji cells and RAW264.7 cells increased with nonspecific adsorption of BacMPs in proportion to the reaction amount. In addition, when protein G-linker (50) -BacMPs was used, Raji cells were hardly recovered nonspecifically, but in RAW264.7 cells, it was proportional to the amount of BacMPs reaction and was not specific. The number of recovered cells increased. Compared with Raji cells, which are B cell line cultured cell lines, the high adhesion of RAW264.7 cells, which are macrophage line cultured cell lines, was thought to promote nonspecific adsorption of BacMPs to the cell surface. On the other hand, when protein G-linker (100) -BacMPs was used, both Raji cells and RAW264.7 cells were hardly recovered non-specifically. When 30 μg of protein G-linker (100) -BacMPs was reacted with RAW264.7 cells, the non-specific recovery rate was 0.9% (1/5 when protein G-BacMPs was used). In addition, in the evaluation using JM cells and THP-1 cells, very few cells were recovered nonspecifically by the action of protein G-linker (100) -BacMPs (nonspecific recovery rate ≤ 0.2%). It was considered that the nonspecific adsorption of BacMPs to cells was suppressed as a result of the decrease in interaction between BacMPs and cells due to steric hindrance due to the presence of NS polypeptide linker on the surface of BacMPs. It was considered that the longer the NS polypeptide linker chain length, the higher the effect of suppressing nonspecific adsorption of BacMPs to cells. Since the same effect is shown also in the nanoparticle surface modification using PEG (refer nonpatent literature 2), it was thought that NS polypeptide linker is acting like PEG on BacMPs. From the above, it was shown that nonspecific adsorption of cells to BacMPs can be reduced by displaying NS polypeptide linker on the surface of BacMPs.

Evaluation of the effect of NS polypeptide display on cell separation ability of antibody-immobilized BacMPs
Anti-CD19 monoclonal antibody immobilized protein G-linker (100) -BacMPs and protein G-BacMPs: 30 μl (1-25 μg) were reacted to Raji cell suspension: 70 μl (3 × 10 ⁴ cells) (4 ° C, 10 minutes). After centrifugal washing and magnetic washing, the number of collected cells was counted, and the cell recovery rate was calculated.

  The result is shown in FIG. As a result of reacting the same amount of particles, 1.2-1.9 times more cells were observed when anti-CD19 monoclonal antibody immobilized protein G-linker (100) -BacMPs was used than when protein G-BacMPs was used. It was recovered. It was considered that the dispersibility of BacMPs in aqueous solution improved by NS polypeptide display contributed to the improvement of antigen (cell) binding ability of antibody-immobilized BacMPs. From the above, it was considered that separation of target cells with higher recovery rate can be achieved by using antibody-immobilized protein G-linker (100) -BacMPs as a magnetic carrier for magnetic cell separation.

Cell isolation from whole blood using Protein G-linker (100) -BacMPs Human peripheral blood: 500 μl, mouse IgG1-derived anti-CD19 immobilized protein G-linker (100) -BacMPs and protein G-BacMPs: 160 μl (40 μg) was reacted (4 ° C., 10 minutes). Furthermore, 60 μl of PE-labeled anti-CD19 mAb was reacted (4 ° C., 10 minutes). After centrifugal washing and magnetic separation washing, the fluorescence of the separated cells was analyzed by flow cytometry. Hemolysis (buffer) (VersaLyse) was used as the suspension only for the second magnetic separation washing. In addition, the following experiment was conducted to evaluate the proportion of CD19 ⁺ cells contained in human peripheral blood. Human peripheral blood: 100 μl was reacted with 10 μl of PE-labeled anti-CD19 mAb (4 ° C., 10 minutes). Furthermore, VersaLyse: 1 ml was added and reacted (RT, 10 minutes). After centrifugal washing, the fluorescence of the collected cells (leukocytes) was analyzed by flow cytometry. In addition, the number of cells was counted using a hemacytometer, and the number of cells contained in the peripheral blood was calculated.

As a result of evaluating the content of CD19 ⁺ cells in leukocytes using antibodies, CD19 ⁺ cells contained 4.1% of leukocytes. Since leukocytes accounted for about 0.1% of peripheral blood-derived blood cells, it was considered that CD19 ⁺ cells were contained in the peripheral blood-derived blood cells by about 0.4 × 10 ⁻² %. FIG. 10 shows the results of magnetic cell separation of CD19 ⁺ cells from human peripheral blood using anti-CD19-immobilized protein G-linker (100) -BacMPs and protein G-BacMPs. CD19 ⁺ cells, which contained approximately 0.4 x 10 ^-2 % in peripheral blood cells, were 95.1% pure and 58.1% when anti-CD19 immobilized protein G-linker (100) -BacMPs was used. Separated in recovery. When anti-CD19-immobilized protein G-BacMPs was used, the purity was 81.9% and the recovery rate was 63.6%. By displaying NS polypeptide linker on the surface of BacMPs, nonspecific adsorption of cells to BacMPs was suppressed. As a result, when protein G-linker (100) -BacMPs was used, target cells were isolated from peripheral blood with high purity. It was separable.

  As can be seen from the above examples, the NS polypeptide linker expressed on the surface of BacMPs exerted an effect on the interaction between BacMPs and between BacMPs and cells in proportion to the chain length.

  NS polypeptide linker also contributed to maintaining the dispersibility of BacMPs in a buffer solution by suppressing aggregation between BacMPs by steric hindrance effect. Thereby, the cell separation efficiency depending on the cell binding efficiency of the antibody-immobilized BacMPs was improved.

  Furthermore, it was effective in suppressing nonspecific adsorption of BacMPs to cells. In particular, the effect of suppressing nonspecific adsorption of BacMPs to macrophage cells (RAW 264.7 cells) having higher adhesion than other blood cells was remarkable.

  In this example, NS polypeptide designed with hydrophilicity and non-charge as indicators is expressed on the surface of BacMPs to obtain the same effect as that obtained by surface modification of nanoparticles using conventional PEG. Was possible. In addition, in this example, NS polypeptide can be arranged on the surface of BacMPs without affecting the functional activity of protein G by incorporating NS polypeptide as a linker when protein G is expressed. there were.

  As described above, NS polypeptide has been developed as a novel surface-modifying molecule capable of improving the dispersibility of particles and suppressing nonspecific binding of cells to the particle surface. In addition, NS polypeptide can function even when used as a linker, so as a linker used when immobilizing functional proteins on solid phases including the particle surface. Is also useful. In addition, by incorporating amino acid sequences that reversibly change the structure and properties due to changes in heat, light, pH, etc. as linkers, non-specific adsorption to cells is reduced, and various functions are added to BacMPs. It can be added.

  In addition, by using antibody-immobilized protein G-linker (100) -BacMPs as a magnetic carrier for magnetic cell separation, target cells can be separated more efficiently. It is useful as a basic tool that supports a wide range of fields, from basic research to applied research, cell diagnosis, and cell therapy.

  By fusing the polypeptide of the present invention and a functional protein as a fusion protein, it becomes possible to immobilize the functional protein on the surface of the material / device while maintaining the orientation of the functional protein. Specific adsorption can be suppressed. In addition, when modified onto particles such as magnetic nanoparticles, it also contributes to improved dispersibility of the particles. As described above, the surface modification technology using the polypeptide of the present invention, which can solve two or three important elements at once, is a medical device such as an artificial blood vessel, and a living body such as a biochip / cell chip. It is expected to be widely used as an important technology when developing materials for cell-related applications such as molecular / cell detection / analysis devices and magnetic nanoparticles.

  In particular, it is most expected to be used as a surface modification molecule for particles used in cell-related applications. So far, in cell-related applications, (nano) particles have been used for the separation of specific cells from a cell population, application as drug carriers for drug delivery systems (DDS), and gene transfer into cells. It is used in various applications such as as a carrier, and for magnetic nanoparticles, it is used as a magnetic hyperthermia that uses the heat generation phenomenon associated with hysteresis loss due to AC magnetic field irradiation, and as a sensitizer for Magnetic Resonance Imaging (MRI). Has also been actively researched. Therefore, it is expected that the range of application of the polypeptide of the present invention is wide-ranging.

  From the above, the present invention is expected to be used in a wide range of fields ranging from in vitro to in vivo, from basic research to applied research or clinical application.

  Special Notes: The nucleotide sequence disclosed by the present invention is allowed to contain one or more of several deletions, additions or substitutions within a range that does not substantially affect the overall properties. The same applies to the amino acid sequence.

Claims (21)

  A polypeptide composed of a polar amino acid and an uncharged amino acid.   The polypeptide according to claim 1, which is composed of at least 50 amino acid residues.   The polypeptide according to claim 1 or 2, wherein an amino acid sequence composed of a plurality of amino acid residues is defined as one unit, and the amino acid sequence of the one unit is repeatedly arranged.   The amino acid constituting the one-unit amino acid sequence is selected from the group consisting of asparagine, glutamine, threonine, tyrosine and serine, and serine is contained in one unit or in a part of the polypeptide. The described polypeptide.   The polypeptide according to claim 4, wherein the unit amino acid sequence is determined by a combination of the two kinds of selected amino acids and the number of each of the two kinds of amino acids.   The polypeptide according to claim 3, wherein the amino acid sequence of one unit is an arbitrary sequence of four asparagines and one serine.   The polypeptide according to any one of claims 3 to 6, which constitutes an amino acid sequence in which the amino acid sequence of the one unit is repeated 20 times.   The polypeptide shown in SEQ ID NO: 1.   The polypeptide according to any one of claims 1 to 8, further comprising an amino acid sequence of a functional protein.   The polypeptide according to claim 9, wherein the functional protein is an antibody-binding protein.   The polypeptide according to any one of claims 1 to 7, further comprising a restriction enzyme recognition site in the sequence of the polypeptide.   A plasmid comprising a base sequence encoding the polypeptide according to any one of claims 1 to 11.   A plasmid comprising the base sequence shown in SEQ ID NO: 2.   A cell transformed with the plasmid according to claim 12 or 13.   The cell according to claim 14, wherein the cell is a magnetic bacterium.   A method for producing a magnetic bacterial particle comprising a polypeptide capable of controlling nonspecific adsorption to cells, wherein the plasmid according to claim 12 or 13 is introduced into the magnetic bacterium, the magnetic bacterium is cultured, and synthesized. A method comprising the steps of extracting magnetic bacterial particles.   The method according to claim 16, wherein the polypeptide is the polypeptide according to any one of claims 1 to 11.   Magnetic bacterial particles in which the polypeptide according to any one of claims 1 to 11 is expressed on a magnetic bacterial particle film.   A method for suppressing nonspecific adsorption of the particles of the magnetic bacteria according to claim 18 to cells.   A method for improving the dispersibility of the particles using the magnetic bacterial particles according to claim 18.   A method for separating cells using the magnetic bacterial particle according to claim 18.