JP2005287350A - Cell adhesion polypeptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell adhesion polypeptide that has high cell adhesion, can easily and artificially be synthesized, and comprises at most one hundred amino acids. <P>SOLUTION: This polypeptide is composed of (a) a specific amino acid sequence or substantially equal amino acid sequence to the former peptide or (b) in a specific amino acid sequence, one or several amino acids are deleted, substituted or added to obtain a polypeptide that is substantially isogenic to the polypeptide of (a). There are provided with the DNA encoding the polypeptide and a substrate covered with the cell adhesive factor. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a polypeptide cell adhesion factor consisting of the same or substantially the same amino acid sequence as shown in SEQ ID NO: 1, a substrate modified with the cell adhesion factor, and the like.

  Fibronectin, collagen, laminin, etc. are known as cell adhesion factors or cell adhesion factors and are used in the industry as cell adhesion members, but these factors are macromolecular polypeptides that form the extracellular matrix of animal cells. In addition, because of its complexity, it is not suitable for an artificial and efficient production method using a genetic recombination method, and it cannot be said that the adhesive force is strong.

The following patent gazettes are representative examples of the prior art relating to cell adhesion or cell adhesion, and these include laminin 2 (patent document 1) or laminin 5 (patent document 2) as a protein. ), And an invention in which an LFA-3 derivative protein adheres to cells is described (Patent Document 3). There were no examples of proteins showing strong cell attachment ability such as the PIP-1 polypeptide sequence of the present invention.
JP-T-2002-542823 Japanese translation of PCT publication No. 2002-542824 Japanese Patent Laid-Open No. 09-75090

  Therefore, the inventor conducted intensive screening by applying a phage library prepared from human normal cDNA in order to identify a molecule consisting of 100 or less amino acids with high cell adhesion ability and easy artificial synthesis. As a result, they found an extremely powerful cell adhesion factor (hereinafter abbreviated as “PIP-1”) that promotes cell attachment and migration, and completed the present invention.

  That is, the present invention provides, as a first aspect, (a) an amino acid sequence represented by SEQ ID NO: 1, or a polypeptide comprising substantially the same amino acid sequence as that; or (b) an amino acid sequence represented by SEQ ID NO: 1. And a DNA encoding a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and having substantially the same biological activity as the polypeptide of (a).

  As a second aspect of the present invention, the following DNA (a) or (b): (a) DNA consisting of the base sequence represented by SEQ ID NO: 2; (b) complementary to the base sequence of (a) The present invention relates to a DNA that hybridizes with a DNA comprising a base sequence under stringent conditions and encodes a polypeptide having substantially the same biological activity as the polypeptide encoded by the base sequence of (a).

  Furthermore, this invention relates to the expression vector containing said DNA as a 3rd aspect.

  As a fourth aspect, the present invention provides a polypeptide comprising the following polypeptide (a) or (b): (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence substantially identical thereto Or (b) an amino acid sequence represented by SEQ ID NO: 1, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and biology substantially the same as the polypeptide of (a) It relates to a polypeptide having an activity.

  Furthermore, the present invention relates to, as a fifth aspect, a cell adhesion factor comprising the above polypeptide, a substrate modified with the polypeptide, and a fusion protein comprising the polypeptide.

    Furthermore, the present invention relates to, as a sixth aspect, a method for treating cells, which comprises adhering a cell typified by an animal cell such as a mammalian cell to a substrate via the cell adhesion factor.

  When the fusion protein GST-PIP-1 containing the polypeptide (PIP-1) of the present invention is prepared by a recombinant method and beads coated thereon are added to the culture medium of adherent animal cells, the beads are added after 30 minutes. Cell adhesion and migration (Migration) due to cell migration and adhesion on the top, cell adhesion over the entire bead after 24 hours, and adherence of the cells even after washing with a buffer solution. It was shown that the polypeptide of the present invention has a strong cell adhesion ability.

  The DNA of the present invention comprises (a) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence substantially identical thereto; or (b) one or a number in the amino acid sequence represented by SEQ ID NO: 1. DNA encoding a polypeptide consisting of an amino acid sequence in which one amino acid has been deleted, substituted or added, and having substantially the same biological activity as the polypeptide of (a), more specifically, for example, A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 which hybridizes under stringent conditions with the DNA comprising the base sequence represented by SEQ ID NO: 2 or the DNA comprising the base sequence complementary to the DNA. DNA encoding a polypeptide (protein) having the same biological activity as

Examples of DNA that can hybridize with the DNA consisting of the base sequence represented by SEQ ID NO: 2 under such stringent conditions include, for example, a degree of homology with the entire base sequence of the DNA of about 90 on average as a whole. % Or more, preferably about 95% or more, more preferably about 98% or more. Here, “stringent conditions” refers to, for example, hybridization buffer (50% formamide, 5 × SSC,
Oligos under conditions that wash membranes in 5X SSC, 0.5% SDS solution (1xSSC is 0.15M NaCl, 0.015M sodium citrate) that has been hybridized overnight with 0.5% SDS. The conditions are such that they are hybridized to the human DNA probe of the present invention by array analysis.

In addition, hybridization itself is molecular cloning (Molecular Cloning) 3rd edition (J.
Sambrook et al., Cold Spring Harbor Lab. Press, 2001). Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual.

  The DNA of the present invention can be prepared according to the following examples. Further, based on the specific base sequence of SEQ ID NO: 2 described in the present specification, those skilled in the art can easily synthesize the nucleic acid molecule of the present invention by chemical synthesis methods well known in the art. I can do it.

  On the other hand, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 has a degree of homology with the entire amino acid sequence represented by SEQ ID NO: 1 on average of about 90% or more, preferably Means an amino acid sequence that is about 95% or more, more preferably about 98% or more. Therefore, as a polypeptide consisting of an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 of the present invention, for example, the above homology with the amino acid sequence represented by SEQ ID NO: 1 is used. And a polypeptide having substantially the same biological activity as the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1. Here, “substantially the same quality” means that their activities are homogeneous in nature, and specifically means that they have the ability to attach cells as shown in the Examples.

  The polypeptide of the present invention comprises, for example, an amino acid sequence obtained by deleting, substituting or adding one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence combining them. A polypeptide having substantially the same biological activity as the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is also included.

  The polypeptide of the present invention can be easily prepared by any means known to those skilled in the art using an expression vector containing the DNA of the present invention. Such an expression vector can be prepared according to a method known in the art. For example, it can be produced by (1) cutting out the DNA of the present invention or a DNA fragment containing the DNA of the present invention and (2) ligating the DNA fragment downstream of a promoter in an appropriate expression vector. Expression vectors include plasmids derived from E. coli (eg, pBR322, pBR325, pUC18, pUC118), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), λ phage, etc. Or an expression vector using a sequence derived from an animal virus such as SV40, CMV virus, retrovirus, vaccinia virus, baculovirus, bovine papilloma virus, or the like. The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when the host is Escherichia coli, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, T7 promoter, or a promoter in which these promoters are combined or combined, and when the host is Bacillus subtilis, When the host is yeast such as SPO1 promoter, SPO2 promoter, penP promoter, etc., PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When animal cells are used as a host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter, HSP promoter, metallothionein promoter and the like can be mentioned.

  In addition to the above, an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori), etc., are added to the expression vector, if desired. be able to. If necessary, the polypeptide encoded by the DNA of the present invention can be expressed as a fusion protein with other proteins (for example, glutathione S transferase, histidine tag, calmodulin binding protein, protein A, etc.). It is. Such a fusion protein can be cleaved using an appropriate protease and separated into each.

Examples of host cells that can be used include Escherichia, Bacillus, yeast, insect cells, insects, animal cells, and the like. Specific examples of the genus Escherichia include DH1 derived from Escherichia coli K12 (Proc. Natl. Acad. Sci. USA, 60, 160 (1968)), JM103 (Nucleic Acids Research, 9, 309). (1981)), JA221 (Journal of Molecular Biology, 120, 517 (1978)), and HB101 (Journal of Molecular Biology, 41, 459 (1969)), or Escherichia coli (Escherichia
coli) B strain and the like are used. Examples of the Bacillus bacterium include Bacillus subtilis MI114 (Gene, 24, 255 (1983)), 207-21 [Journal of Biochemistry, 95, 87 (1984)]. Examples of yeast include Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris (Pichia pastoris)
pastoris) and the like are used. Examples of animal cells include monkey kidney cell-derived COS-1, COS-7, Vero cells, Chinese hamster CHO cells (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter referred to as CHO (dhfr-)). Abbreviated cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3 cells, human FL cells, human Hela cells, or human myeloma cells.

Transformation of these host cells can be performed according to methods known in the art. For example, the following documents can be referred to. Proc. Natl. Acad. Sci. USA, 69, 2110 (1972); Gene, 17, 107 (1982);
& General Genetics, 168, 111 (1979); Methods in Enzymology, 194, 182-187 (1991); Proc.
Natl. Acad. Sci. USA), 75, 1929 (1978); Cell Engineering Supplement 8 New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha); and Virology, 52, 456 (1973).

  The thus obtained transformant transformed with the expression vector containing the DNA of the present invention can be cultured according to a method known in the art. For example, when the host is an Escherichia bacterium, the culture is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration or agitation can be added. When the host is a genus Bacillus, the culture is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration or agitation can be added. When cultivating a transformant whose host is yeast, the culture is usually performed at a temperature of about 20 to 35 ° C. for about 24 to 72 hours using a medium adjusted to a pH of about 5 to 8, with aeration and agitation as necessary. Can also be added. When culturing a transformant whose host is an animal cell, the culture is usually performed at about 30 to 40 ° C. for about 15 to 60 hours using a medium whose pH is adjusted to about 6 to 8, and if necessary, aeration or agitation. Can also be added.

In order to separate and purify the polypeptide of the present invention from the above culture, for example, after culturing, cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and / or freezing. After disrupting cells or cells by thawing or the like, a crude protein extract is obtained by centrifugation or filtration. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the protein is secreted into the culture solution, the cells or cells are separated from the supernatant by a known method after completion of the culture, and the supernatant is collected. Purification of the protein contained in the thus obtained culture supernatant or extract can be performed by appropriately combining known separation / purification methods. In particular, when the polypeptide of the present invention is expressed as a fusion protein, it can be easily purified by affinity column chromatography using an appropriate ligand known to those skilled in the art.

  The polypeptide of the present invention thus obtained can be converted into a salt by a known method or a method analogous thereto, and conversely, when it is obtained as a salt, it can be isolated as a free form or others by a known method or a method analogous thereto. Can be converted to the salt. Further, the protein produced by the recombinant is purified before or after purification using methionine aminopeptidase to remove the N-terminal methionine, using myristoyltransferase to perform myristoylation of the N-terminal amino acid, Amino acids can be arbitrarily modified by acetylating the N-terminal amino acid using transferase, or by using other modifying enzymes. In addition, the C-terminal amino acid can be modified by the action of a processing carboxyl peptidase that modifies the C-terminus, a C-terminal amidating enzyme, or the like. Furthermore, the polypeptide can also be partially removed by acting an appropriate protein-degrading enzyme such as trypsin, chymotrypsin, FactorXa, thrombin, KEX2 protease, or PreScission protease. When it is produced as a fusion protein, an unnecessary polypeptide portion can be removed using a suitable protein-degrading enzyme.

In the polypeptide of the present invention, the C-terminus is usually a carboxyl group (—COOH) or carboxylate (—COO—), but the C-terminus may be an amide (—CONH ₂ ) or an ester (—COOR). Good. Here, R in the ester is, for example, a C1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, for example, phenyl, α -C6-12 aryl groups such as naphthyl, for example, C7-14 aralkyl groups such as phenyl-C1-2 alkyl groups such as benzyl and phenethyl or α-naphthyl-C1-2 alkyl groups such as α-naphthylmethyl, Pivaloyloxymethyl ester, which is widely used as an oral ester, is used.

When the polypeptide of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the present invention. As the ester in this case, for example, the C-terminal ester described above is used. Furthermore, the protein of the present invention includes those in which the amino group of the N-terminal methionine residue is protected with a protecting group (for example, a C1-6 acyl group such as formyl group, acetyl group, etc.) or cleaved in vivo. The resulting N-terminal glutamic acid residue is pyroglutamated, and is present on the side chain of the amino acid in the molecule, for example, OH, COOH, NH ₂ , SH, etc. are suitable protecting groups (for example, formyl group, Examples thereof include those protected by a C1-6 acyl group such as an acetyl group) or complex proteins such as so-called glycoproteins to which sugar chains are bound.

  The salt of the polypeptide of the present invention is particularly preferably a physiologically acceptable acid addition salt. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

Alternatively, the polypeptide of the present invention or a salt thereof or an amide thereof can also be prepared by using a chemical synthesis method known in the art based on the amino acid sequence information shown in SEQ ID NO: 1. As a peptide synthesis method, either a solid phase synthesis method or a liquid phase synthesis method may be used. Examples of known condensation methods and protecting group elimination include the methods described in the following (1) to (3).
(1) Nobuo Izumiya et al., Peptide synthesis basics and experiments,
Maruzen Co., Ltd. (1975)
(2) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV,
205, (1977) (3) Supervised by Haruaki Yajima, Development of follow-up medicines Volume 14: Peptide synthesis Hirokawa Shoten.

  For example, a commercially available resin for protein synthesis is used, and an amino acid with an α-amino group and a side chain functional group appropriately protected is subjected to various condensation methods known in the art according to the sequence of the target protein. Condensation on the resin. At the end of the reaction, the protein is cut out from the resin, and at the same time, various protecting groups are removed, and an intramolecular disulfide bond forming reaction is performed in a highly diluted solution to obtain the target protein, its partial peptide, or their amides. Regarding the condensation of the above protected amino acids, for example, for protein synthesis represented by carbodiimides such as DCC, N, N′-diisopropylcarbodiimide, and N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide. Various activating reagents that can be used can be used. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (for example, HOBt, HOOBt), or the protected amino acid is activated in advance as a control anhydride, HOBt ester or HOOBt ester. Once done, it can be added to the resin.

  Solvents used for the activation of protected amino acids and condensation with resins include acid amides, halogenated hydrocarbons, alcohols, sulfoxides, ethers, and the like that can be used in protein condensation reactions in the industry. It may be appropriately selected from known solvents. The reaction temperature is appropriately selected from a range that is known to be usable for protein bond forming reaction, and is usually selected appropriately from a range of about -20 to 50 ° C. The activated amino acid derivative is usually used in an excess of 1.5 to 4 times. As a result of a test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation is not obtained even after repeating the reaction, the unreacted amino acid can be acetylated with acetic anhydride or acetylimidazole so as not to affect the subsequent reaction. As a protective group such as each amino group, carboxyl group, and serine hydroxyl group of the raw material, groups usually used in this technical field can be used. The protection of the functional group that should not be involved in the reaction of the raw material, the protection group, the removal of the protective group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.

  For purification after the reaction, the peptide of the present invention can be purified and isolated by a combination of methods known per se, such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. When the peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method. Conversely, when it is obtained as a salt, it can be converted into a free form by a known method. it can.

  The polypeptide of the present invention thus obtained has a remarkable cell adhesion ability and can function as a cell adhesion factor, as shown in the Examples of the present specification. Therefore, by modifying various substrates with the polypeptide of the present invention, cell adhesion of the substrate can be promoted, and various cells can be attached to the substrate.

For example, by modifying the pharmaceutical composition with the polypeptide of the present invention, the affinity of each pharmaceutical composition to various cells is increased, or the polypeptide of the present invention is applied to the surface of a medical transplant device such as an artificial blood vessel or an artificial bone. By coating them, their biocompatibility is improved, or when transplanting specific cells in regenerative treatment, they are attached to a substrate such as biodegradable beads coated with the polypeptide of the present invention. This makes it possible to increase the engraftment ability of the cells in vivo.

  In addition, trapping test cells on a substrate such as a substrate coated with the polypeptide of the present invention, or proliferating the trapped cells, thereby producing a cytodiagnostic chip that enables cell diagnosis and cell profiling. Is also possible.

  There are no particular restrictions on the material, shape, size, etc. of the “substrate”. Various substrates used in the fields of application to enzyme engineering / genetic engineering or artificial organ engineering / biomaterial engineering can be used. As a material of the substrate, for example, a material having high cell affinity such as glass, plastic and metal can be used when used in vitro. In addition, a substance exhibiting magnetism may be included in the substrate for the purpose of separation using magnetic force. When the substrate is used in vivo, a biocompatible substance that can maintain cell growth is preferable. Examples of such biocompatible substances include biodegradable polymer compounds such as polylactic acid, collagen, regenerated collagen, polyglycolic acid, polygalactose, and polyethylene glycol or derivatives thereof, polystyrene, dextran, DEAE, high molecular polymer made of cellulose, collagen, etc., liposome made of phospholipid, hydroxyapatite particles made of calcium phosphate, etc., biocompatible metals such as titanium and stainless steel, ceramic substances including artificial materials such as hydroxyapatite, And medical devices made from or coated with polymers and biomolecules including polyester and nylon, polystyrene, polyacrylate, and polytetrafluoroethylene, or coated with those materials. I can do it.

  The modification of the substrate with the polypeptide of the present invention can be performed by any method known to those skilled in the art. As used herein, “modification of a substrate by a polypeptide” means a state in which the substrate is subjected to some action by the polypeptide. For example, any substrate known to those skilled in the art, such as binding, coating, and complex formation. Includes form.

For example, a polypeptide of the invention can be coated on the substrate by binding to such substrate directly or through a suitable linker. In the case of chemical bonding, use is made of a substrate on which a spacer having an amino group or a carboxyl group contained in the polypeptide of the present invention, in particular, a reactive functional group that can be chemically bonded to an amino group, is used. Good. For example, a spacer having a tosyl group, a carboxyl group, an amino group, a thiol group or other reactive reactive group used as a protein cross-linking agent may be introduced on the surface of the substrate.

When the polypeptide of the present invention is bound to another protein as a fusion protein, a ligand that specifically binds to the other protein (for example, glutathione in the case of a fusion protein with glutathione S transferase) Can be used as a linker.

The binding between the polypeptide of the present invention and the substrate can be easily carried out by any method / means known to those skilled in the art depending on the material, shape, size, etc. of the substrate. For example, the polypeptide of the present invention can be easily coated on the surface of the substrate by holding the substrate in a suitable buffer containing the polypeptide of the present invention for a suitable time. The substrate does not need to be modified with the polypeptide of the present invention in its entirety, and a substrate whose arbitrary part is modified (coated) with the polypeptide of the present invention can be used according to the purpose. It is a range.

  Furthermore, in order to promote cell growth / differentiation, in addition to the polypeptide of the present invention, desired cell growth / differentiation factors such as various growth factors, various cytokines, colony stimulating factors, collagen, laminin, fibronectin, vitronectin, cadherin, The substrate may be coated with entactin / nidogen, α-dystroglycan, other glycoproteins, proteoglycan, heparin sulfate, proteoglycan, glycosaminoglycan, or a polymer compound derived therefrom, or a mixed composition. In this way, for example, it can be expected to obtain more effects in regenerative treatment.

  The polypeptide of the present invention can be a fusion protein with other proteins. Here, there is no restriction | limiting in the objective and use of this other protein. For example, as described above, a fusion protein can be prepared for the convenience of separation and purification of the polypeptide of the present invention. Further, by creating a fusion protein with another protein (polypeptide) for the purpose of treatment and functional analysis, introduction of the other protein into the cell can be promoted. Such a fusion protein can be prepared by any method known to those skilled in the art. For example, DNAs encoding these can be ligated and introduced into an expression vector, and can be easily prepared by the above method.

The polypeptide of the present invention and other proteins can be bound via an appropriate linker or spacer (sequence). For example, if a fusion protein in which the polypeptide of the present invention and another protein are fused via an intracellular peptidase recognition sequence, only the target protein can be released in the cell.

  The protein as a component of the fusion protein of the present invention can be any known to those skilled in the art depending on the purpose of the fusion protein. For example, for therapeutic purposes, the above-mentioned various cell growth / differentiation factors can be mentioned.

  The present invention makes use of the above-mentioned substrate to collect cells via the cell adhesion factor of the present invention, for example, to collect / collect or remove leukocytes from blood collected for use in diagnosis. It also relates to various methods of treating cells, including attaching to a substrate. By using the polypeptide of the present invention, cells can be grown on the substrate after the cells are attached to the substrate. Furthermore, the polypeptide of the present invention can be solidified on a substrate together with the protein or gene to be introduced into the cell, and the introduction of the target molecule into the cell can be promoted by culturing the cell on the surface. Here, there is no restriction | limiting in particular in the specific aspect of the processing method of a cell, Various operations currently implemented in this industry are included.

The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1] This shows the amino acid sequence (number of amino acids: 37) of the polypeptide of the present invention.
[SEQ ID NO: 2] This shows the base sequence (111 base pairs) of DNA encoding the polypeptide of the present invention having the amino acid sequence shown by SEQ ID NO: 1.

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited thereto. Various gene manipulations in the examples are described in Molecular cloning, 3rd edition (J.
Sambrook et al., Cold Spring Harbor Lab. Press, 2001).

First collagen 6 cm diameter were plated cultured human cells HEK293 cells 6 X10 ⁵ cells to confluency in culture dishes coated. Next, after the phage library was increased to 1.6 × 10 ¹¹ pfu / ml, 500 μl was added to the culture dish and incubated at 37 ° C. for 30 minutes. The phage library used is a phage library (human normal T7 Selected cDNA library; Novagen # 70637) in which a brain-derived cDNA library is incorporated and the amino acids encoded thereby are displayed on the virus surface. RIPA after washing 5 times with care not to peel off cells with D-MEM broth (GIBCO BRL; # 11885-084)
buffer (50 mM
Cells were disrupted with Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton x-100, 1% Sodium deoxycholate, 0.1% SDS). After centrifugation at 15.000 rpm for 5 min, the supernatant was cultured overnight in LB medium. E. coli BLT5615
(Novagen #
69905-3) and cultured for about 2 hours until further lysis occurred. In this way, only the phage displaying the amino acid attached to the cell surface of the living cell was selectively proliferated. By repeating this operation multiple times, it is possible to further increase the selectivity. In this screening, the titer of the cell lysate is checked each time, and the force once decreases to about 10 ² pfu / ml. The above procedure was repeated until the value began to rise again and reached the maximum. Eventually, infection and panning were repeated 5 times. As a result, it was possible to select multiple clones of recombinant phage having strong cell adhesion ability.

  The obtained phage was plated on a plate and analyzed for 14 plaques. It was found that 11 were derived from the same recombinant phage. The amino acid sequences of the human cDNA-derived polypeptides presented by these recombinant phages and the base sequences encoding them are described as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

SEQ ID NO: 1:
SSQPGRQERNSVSKKKNKTNKKKTRNIPFWLQYTCLF

SEQ ID NO: 2:
TCAAGCCAGCCTGGGCGACAAGAGCGAAACTCCGTCTCAAAAAAAAAAAACAAAACAAACAAAAAAAAAACAAGAAACATTCCCTTTTGGTTACAGTATACATGTTTGTTC

Next, in order to confirm whether the polypeptide having this amino acid sequence (PIP-1) really recognizes the molecule on the surface of the animal cell and adheres to the animal cell, this sequence is expressed as a GST fusion protein in E. coli as the host. Adhesion to the cells was observed. That is, the gene sequence derived from the human cDNA library incorporated in the phage was excised by restriction enzyme digestion with EcoRI / XhoI, and then pGEX-4T-3 plasmid (Amersham; # 27-4538-01: desired peptide sequence Was incorporated into the EcoRI / XhoI site of a commercially available expression vector that is expressed as a fusion protein with the Glutatione S-transferase sequence. The resulting recombinant plasmid is introduced into Escherichia coli K12-derived DH5α strain to produce recombinant Escherichia coli, and the recombinant Escherichia coli is cultured and grown using LB liquid medium.
Induced expression with IPTG (isopropyl &acirc; -D-thiogalactoside), then glutathione-sepharose
beads
(Glutathione Sepharose 4B, Amersham; # 17-0756-01) was used to purify the target GST-PIP-1 fusion protein (Coligan, JE, Dunn, BM, Speicher , DW, and Wingfield, PJ,
1997, Current Protocols in Protein, John Wiley & Sons, Inc. New Jersey, pp. 6.6.1-6.6.21.). Next, 100 μg of purified GST-PIP-1 fusion protein (hereinafter referred to as GST-PIP-1 protein) or 100 μg of GST protein (used as a negative control: control) was added to 50 μl of glutathione-sepharose beads. Combined.

Specifically, 100 ml of each E. coli culture solution in which GST protein or GST-PIP1 fusion protein was expressed was centrifuged at 8,000 rpm for 5 minutes at 4 ° C. to precipitate each E. coli to prepare E. coli pellets. The prepared pellets are suspended in 10 ml of TBST buffer (50 mM Tris-HCl (pH 8.0), 0.1 M NaCl, 1 mM EDTA, Tween 20 or 0.1% TX-100), and an ultrasonic crusher (Sonifier 250) is used. Each was crushed by ultrasonic waves. The suspension containing the crushed cells was centrifuged at 11,000 rpm for 30 minutes at 4 ° C., and the supernatant was collected. Each collected supernatant contains soluble GST or GST-PIP1 fusion protein. Next, glutathione-sepharose beads were added to each protein-containing solution and gently shaken at 4 ° C. for 1 hour to bind the target soluble protein to the beads. The supernatant was removed by centrifugation at 4.700 rpm for 5 minutes at 4 ° C, and the precipitated protein-bound beads were washed twice with TBST buffer to remove excess protein adsorbed non-specifically on the beads. . For the amount of each protein adsorbed specifically to the beads, GST protein or GST-PIP1 fusion protein was used using glutathione buffer (10 mM glutathione, 0.1 M Tris-HCl (pH 8.0), 0.2 M NaCl). The solution containing the released protein was released from the beads, dialyzed overnight at 4 ° C in PBS, and after removing glutathione contained in the solution, the protein concentration was measured by the Bradford method. Can be detected (about 1 mg of protein for about 100 ml of solution), and it can be confirmed that the GST protein or GST-PIP1 fusion protein is sufficiently bound to the glutathione-sepharose beads used. It was.

The Bradford method is a standard method for determining the amount of protein contained in a purified enzyme preparation. In this method, bovine serum albumin (BSA) standard protein solution (0.5 mg / ml) was dispensed in a test tube so that the amount of protein was 0, 5, 10, 15, 20, 25, 30 μg. Two samples with the same concentration were prepared to avoid errors. In order to keep the volume constant, water was added and the total volume was adjusted to 0.1 ml. Next, 0.5 ml of a staining reagent was added and mixed. After 5 minutes, the sample was placed in a plastic cuvette and the absorbance at 595 nm was measured to prepare a standard curve using BSA. Each solution containing the protein in which the GST protein or GST-PIP1 fusion protein was released from the beads was placed in a 10 μl test tube, water was added to adjust the total volume to 0.1 ml, and the protein concentration was determined. As a result, glutathione-sepharose to which the GST protein was bound under the conditions indicated above.
Glutathione-sepharose bound to beads or GST-PIP1 fusion protein
Since it was found that beads could be produced, these two kinds of beads were produced by the above method and subjected to the cell adhesion experiment shown below.

When these two types of beads are added to the culture medium of various cultured cells each attached to the culture dish and the culture is continued, no particular change is observed in the GST protein, but in the GST-PIP-1 protein, the beads are brought to the cell surface in a few minutes. When the cells were attached and further cultured, the cells were observed to swell so as to surround the beads (FIG. 1).

That is, in the experiment shown in FIG. 1, when GST protein (control) or GST-PIP-1 protein-bound beads were added to the culture medium of HEK293 cells and compared after 0.5 hour, GST-PIP The -1 beads quickly attached to the cells, but the control GST beads did not attach at all, and were present alone in a portion away from the cells. 24 hours after the addition of GST-PIP-1 beads, beads with a diameter of 100 μm or less appear to be phagocytosed, and beads with a diameter of 100 μm or more are 24 hours later or 48 Many cells swelled on the beads after a while and were attached and proliferated. Further, after 24 hours of culture, the culture solution was aspirated and washed with PBS, and then fixed with 70% methanol and stained with hematoxylin (Wako, # 131-09665). From the observation results of cells and beads using a microscope, it was found that beads bound with GST protein were completely washed away, but beads bound with GST-PIP-1 protein remained together with the cells without being washed away. . Since the cell nuclei were deeply stained blue-violet by hematoxylin staining, multiple cell-derived nuclei were observed on the beads in the GST-PIP-1 beads.

Since the cells used here are adherent cells, it can be interpreted that the PIP-1 protein of the present invention induces the activity of migration or migration of adherent cells. In addition, the same findings were observed in all the cells examined (MDCK; dog, HCT116; human, HEK293; human, COS7; monkey). It is thought to occur beyond.

  As is clear from the above examples, animal cells can be attached to various substrates having a diameter of about 100 μm or more modified with the polypeptide of the present invention. Therefore, when liposomes or particles are used as a substrate, it is possible to introduce various drugs, polymer compounds, protein factors and the like contained in the cells into the cells. Furthermore, it can be used as a nanomachine by incorporating magnetic polymer compounds, magnetic particles, various metal fine particles, various light emitters, etc. into liposomes or particles, and future applications in these fields will be possible. Be expected.

  In addition, it is possible to provide a novel medical device, a pharmaceutical composition, a cell carrier for regenerative treatment, a diagnostic composition, etc., to which cell adhesion ability is imparted or promoted by coating with the polypeptide of the present invention. Since it can also be used as a DDS (drug delivery system), it is extremely useful industrially.

Beads bound with GST protein (control) or GST-PIP-1 protein were respectively added to the culture medium of HEK293 cells, and the time course was observed with a transmission microscope. 1) When GST beads or GST-PIP-1 beads are added and compared 0.5 hours later, GST-PIP-1 beads (right, second from the top) quickly attach to cells, but control GST The beads (right, first from the top) do not adhere at all, and are present alone in a part away from the cells. The first and second photomicrographs from the top on the left show the appearance of cells and beads at 0 hours after addition of beads as a control. As shown in the third micrograph from the top left, 24 hours after the addition of GST-PIP-1 beads, beads with a diameter of 100 μm or less (arrows) appear to be phagocytosed by cells. The fourth micrograph from the top left is a photograph further magnified 4 times (showing a bar 50 μm). The third and fourth photomicrographs from the top on the right show images of many cells rising and adhering and growing on beads with a diameter of 100 μm or more after 24 hours and 48 hours, respectively (bar 200 μm). . GST protein (control) or GST-PIP-1 protein-bound beads were added to the HEK293 cell culture medium. After 24 hours, the culture medium was aspirated, washed with PBS, and then fixed with 70% methanol and stained with hematoxylin (Wako). (# 131-09665), the beads bound with GST protein are completely washed away (top), but the beads bound with GST-PIP-1 protein remain with the cells (picture) Bottom: arrow). Since cell nuclei are stained in blue-violet with hematoxylin, in the case of GST-PIP-1 beads (bottom photo), a deep staining of a plurality of cell-derived nuclei is observed on the beads.

Claims (10)

DNA encoding the following polypeptide: (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence substantially identical thereto; or (b) an amino acid sequence represented by SEQ ID NO: 1. A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added, and having substantially the same biological activity as the polypeptide of (a). DNA of the following (a) or (b): (a) DNA consisting of the base sequence represented by SEQ ID NO: 2; (b) DNA consisting of a base sequence complementary to the base sequence of (a) and stringent A DNA encoding a polypeptide that hybridizes under conditions and has substantially the same biological activity as the polypeptide encoded by the base sequence of (a). An expression vector comprising the DNA according to claim 1 or 2. A polypeptide comprising the following polypeptide (a) or (b): (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 or a substantially identical amino acid sequence thereto; or (b) SEQ ID NO: 1 A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by the formula (a) and having biological activity substantially the same as the polypeptide of (a). A cell adhesion factor comprising the polypeptide according to claim 4. A substrate modified with the polypeptide of claim 4. A fusion protein comprising the polypeptide according to claim 4. A method for treating a cell, comprising attaching a cell to a substrate via the cell adhesion factor according to claim 5. 9. The method of claim 8, wherein the cell is an animal cell. The method of claim 8, wherein the cell is a mammalian cell.

Images