JP2018131426A - Laminin e8 production method and cell culture method using laminin e8 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique that cultures efficiently cells such as stem cells.SOLUTION: Cells such as stem cells are cultured using a cell culture vessel coated with laminin E8, from which a carbohydrate chain is removed, or laminin E8, which is expressed from a microorganism as a host.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing laminin E8 and a cell culture method using laminin E8.

Stem cells, especially pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) are expected to be applied to regenerative medicine. Usually, feeder cells are used for culturing stem cells. However, development of a technique for substituting feeder cells is being promoted due to limitations in clinical application.

For example, it is known to cultivate stem cells using a cell adhesion protein such as laminin or its E8 fragment laminin E8 as a scaffold for cell culture. Laminin E8 has a heterotrimeric structure composed of an α chain E8 fragment (α chain E8), a β chain E8 fragment (β chain E8), and a γ chain E8 fragment (γ chain E8). As laminin E8, for example, human laminin 511E8, which is an E8 fragment of human laminin 511, is useful as a scaffold for cell culture. As human laminin 511E8, for example, those heterologously expressed using CHO-S cells derived from Chinese hamster ovary as a host, and those heterologously expressed using silkworm as a host are commercially available.

  In addition, it has been reported that E8 fragment of each subunit of mouse laminin is expressed using Escherichia coli as a host, and heterotrimeric structures such as laminin 111E8, laminin 121E8, laminin 211E8, and laminin 221E8 are reconstituted ( Non-patent document 1).

  However, it has not been known that stem cells can be efficiently cultured by using laminin E8 from which sugar chains have been removed or laminin E8 expressed using a microorganism as a host as a scaffold for cell culture.

A Utani, et al., Laminin chain assembly. J. Biol. Chem. 1994 269: 19167-19175.

  An object of the present invention is to provide a technique for efficiently culturing cells such as stem cells.

  As a result of intensive studies, the present inventor has found that stem cells can be efficiently cultured by using laminin E8 from which sugar chains have been removed and laminin E8 expressed using a microorganism as a host as a scaffold for cell culture. Completed the invention.

That is, the present invention can be exemplified as follows.
[1]
Laminin E8 having the following properties (A) or (B):
(A) N-type sugar chain addition rate is 20% or less;
(B) It has a microbial sugar chain structure.
[2]
Said laminin E8 except for laminin 111E8, laminin 121E8, laminin 211E8, and laminin 221E8 in mice without sugar chains.
[3]
The laminin E8, which does not have an N-type sugar chain.
[4]
The laminin E8 having a sugar chain structure of yeast type.
[5]
The laminin E8, which is a laminin 111E8, laminin 121E8, laminin 211E8, laminin 221E8, laminin 332E8, laminin 421E8, laminin 411E8, laminin 511E8, or laminin 521E8.
[6]
The laminin E8, which is a laminin 511E8.
[7]
The laminin E8,
The α chain E8 constituting the laminin E8 is the protein described in the following (1a), (1b), or (1c):
(1a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2, 13, 19, 20, 21, or 22;
(1b) comprising an amino acid sequence comprising substitutions, deletions, insertions or additions of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, 13, 19, 20, 21, or 22, and a protein that forms a heterotrimer with β-chain E8 and γ-chain E8 and exhibits a scaffold function for cells;
(1c) an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2, 13, 19, 20, 21, or 22, and heterotrimeric with β chain E8 and γ chain E8 A protein that forms a body and exhibits a scaffold function for cells;
The β chain E8 constituting the laminin E8 is the protein described in the following (2a), (2b), or (2c):
(2a) a protein comprising the amino acid sequence shown in SEQ ID NO: 3, 7, 23, or 24;
(2b) comprising an amino acid sequence comprising a substitution, deletion, insertion or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 3, 7, 23 or 24, and α chain E8 and a protein that forms a heterotrimer with γ-chain E8 and exhibits a scaffold function for cells;
(2c) an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 3, 7, 23, or 24, and forming a heterotrimer with α chain E8 and γ chain E8 A protein that exhibits a scaffold function for cells;
Laminin E8, wherein the γ chain E8 constituting the laminin E8 is a protein described in (3a), (3b), or (3c) below:
(3a) a protein comprising the amino acid sequence shown in SEQ ID NO: 4, 8, 25, or 26;
(3b) in the amino acid sequence shown in SEQ ID NO: 4, 8, 25, or 26, comprising an amino acid sequence comprising substitution, deletion, insertion, or addition of 1 to 10 amino acid residues, and α chain E8 and a protein that forms a heterotrimer with β-chain E8 and exhibits a scaffold function for cells;
(3c) an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 4, 8, 25, or 26, and forming a heterotrimer with α chain E8 and β chain E8 A protein that exhibits a scaffold function for cells.
[8]
A method for producing laminin E8, comprising expressing laminin α chain E8, β chain E8, and γ chain E8 using a microorganism as a host.
[9]
The above-mentioned method, except that Escherichia coli is used as a host to express mouse laminin 111E8, laminin 121E8, laminin 211E8, and laminin 221E8.
[10]
The laminin E8 is laminin 111E8, laminin 121E8, laminin 211E8, laminin 221E8,
Laminin 332E8, Laminin 421E8, Laminin 411E8, Laminin 511E8, or Laminin 521E8
Said method.
[11]
The method, wherein the laminin E8 is laminin 511E8.
[12]
Said method comprising:
The α chain E8 is the protein described in the following (1a), (1b), or (1c): (1a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2, 13, 19, 20, 21, or 22 ;
(1b) comprising an amino acid sequence comprising substitutions, deletions, insertions or additions of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, 13, 19, 20, 21, or 22, and a protein that forms a heterotrimer with β-chain E8 and γ-chain E8 and exhibits a scaffold function for cells;
(1c) an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2, 13, 19, 20, 21, or 22, and heterotrimeric with β chain E8 and γ chain E8 A protein that forms a body and exhibits a scaffold function for cells;
The β chain E8 is a protein according to the following (2a), (2b), or (2c): (2a) a protein comprising the amino acid sequence shown in SEQ ID NO: 3, 7, 23, or 24;
(2b) comprising an amino acid sequence comprising a substitution, deletion, insertion or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 3, 7, 23 or 24, and α chain E8 and a protein that forms a heterotrimer with γ-chain E8 and exhibits a scaffold function for cells;
(2c) an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 3, 7, 23, or 24, and forming a heterotrimer with α chain E8 and γ chain E8 A protein that exhibits a scaffold function for cells;
A method wherein the γ chain E8 is a protein according to the following (3a), (3b), or (3c):
(3a) a protein comprising the amino acid sequence shown in SEQ ID NO: 4, 8, 25, or 26;
(3b) in the amino acid sequence shown in SEQ ID NO: 4, 8, 25, or 26, comprising an amino acid sequence comprising substitution, deletion, insertion, or addition of 1 to 10 amino acid residues, and α chain E8 and a protein that forms a heterotrimer with β-chain E8 and exhibits a scaffold function for cells;
(3c) an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 4, 8, 25, or 26, and forming a heterotrimer with α chain E8 and β chain E8 A protein that exhibits a scaffold function for cells.
[13]
The method, wherein at least the β chain E8 and the γ chain E8 are expressed together by a single microorganism.
[14]
The method, wherein the α chain E8, β chain E8, and γ chain E8 are expressed together by a single microorganism.
[15]
The method, wherein the α chain E8, β chain E8, and γ chain E8 are separately expressed by a plurality of microorganisms.
[16]
The method, wherein the microorganism is a bacterium or a yeast.
[17]
The method, wherein the microorganism is a bacterium belonging to the family Enterobacteriaceae or a coryneform bacterium.
[18]
The method, wherein the microorganism is Escherichia coli or Corynebacterium glutamicum.
[19]
The method, wherein the microorganism is Saccharomyces cerevisiae or Pichia pastoris.
[20]
Constructing laminin E8 from said α chain E8, β chain E8, and γ chain E8. [21]
The method, comprising obtaining the β-chain E8 and the γ-chain E8 as a heterodimer, and constituting laminin E8 from the α-chain E8 and the heterodimer.
[22]
A composition comprising laminin E8.
[23]
A composition comprising laminin E8 produced by the method.
[24]
The composition, which is a cell scaffold material.
[25]
The composition described above, wherein the cell scaffold material is used for culturing stem cells.
[26]
The composition, wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell.
[27]
The said composition which is a coating agent of a cell culture container.
[28]
The composition described above, wherein the cell culture container is for stem cell culture.
[29]
The composition, wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell.
[30]
An instrument comprising the laminin E8.
[31]
An instrument comprising laminin E8 produced by the method.
[32]
Said instrument, which is a medical or research instrument.
[33]
The instrument, which is a cell culture vessel.
[34]
The device, which is a cell scaffold material.
[35]
The instrument, wherein the cell culture surface is coated with the laminin E8.
[36]
The device for culturing stem cells.
[37]
The device, wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell.
[38]
A method for producing cultured cells, comprising culturing cells with the device.
[39]
The method, wherein the cultured cells are stem cells.
[40]
The method, wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell.

  According to the present invention, cells such as stem cells can be efficiently cultured.

It is a figure (photograph) which shows the result of SDS-PAGE before and after sugar chain cleavage of laminin 511E8. It is a figure which shows the result of having coated the sugar chain cutting | disconnection laminin 511E8 on a cell culture plate, and evaluating the iPS cell scaffold function over 3 weeks. The horizontal axis indicates “week”. It is a figure (photograph) which shows the result of SDS-PAGE of the cell disruption liquid of an E. coli human laminin alpha5 chain E8 expression strain. The sample loaded for each lane is as follows. 1: BSA 0.25 μg, 2: BSA 0.5 μg, 3: BSA 1.0 μg, 4: Cell disruption solution (diluted 5 times with respect to the culture solution) 10 μL. The black arrow head indicates the band of human laminin α5 chain E8. YDK010 :: phoS (W302C) / pPK4_CspB6Xa-LE8β strain, YDK010 :: phoS (W302C) / pPK4_CspB6Xa-LE8γ strain, and YDK010 :: phoS (W302C) / pPK4_CspB6Xa-LE8β-CspB6Xa-supernatant It is a figure (photograph) which shows the result used for reduction | restoration SDS-PAGE. It is a figure which shows the ESI-TOF-MS spectrum which carried out the deconvolution process of the protein produced | generated from the culture supernatant of the co-secretion expression strain of human laminin (beta) 1 chain | strand E8 and human laminin (gamma) 1 chain | strand E8. It is a figure (photograph) which shows the result of having used the flow-through, washing | cleaning fraction, and elution fraction in affinity chromatography of denatured laminin 511E8 to SDS-PAGE. It is a figure (photograph) which shows the result of having used the flow-through in the affinity chromatography of non-denaturing laminin 511E8, the wash fraction, and the elution fraction for SDS-PAGE. It is a figure (photograph) which shows the result of having used the flow-through, washing | cleaning fraction, and elution fraction in the affinity chromatography of a diluted sample for SDS-PAGE. It is a figure which shows the result of having coated the cell culture plate with (alpha) (beta) (gamma) trimer reconstituted from the urea modified | denatured laminin 511E8, and evaluating the iPS cell scaffold function. It is a figure (photograph) which shows the result of having used the flow-through, washing | cleaning fraction, and elution fraction in the affinity chromatography of a diluted sample for SDS-PAGE. It is a figure which shows the result of having coated the cell culture plate with (alpha) (beta) (gamma) trimer reconstituted from reductive denatured laminin 511E8, and evaluating the iPS cell scaffold function.

  Hereinafter, the present invention will be described in detail.

<1> Protein of the present invention and production thereof <1-1> Protein of the present invention The protein of the present invention is laminin E8.

  Laminin is a protein having a heterotrimeric structure composed of an α chain, a β chain, and a γ chain. Laminin includes mammalian laminin. Mammals include primates such as humans, monkeys and chimpanzees, rodents such as mice, rats, hamsters and guinea pigs, and various other mammals such as rabbits, horses, cows, sheep, goats, pigs, dogs and cats. It is done. As laminin subunit chains (that is, α chain, β chain, and γ chain), five types of α chains (α1 to α5), three types of β chains (β1 to β3), and three types of γ chains (γ1) ~ Γ3). The amino acid sequences of laminin subunit chains derived from various organisms and the base sequences of genes encoding them can be obtained from public databases such as NCBI. The NCBI accession numbers of human and mouse laminin subunit chains are shown in Table 1.

  Laminin forms various isoforms by combining these subunit chains. Specific examples of laminin include those having a heterotrimeric structure shown in Table 2.

“Laminin E8” refers to an E8 fragment of laminin. Specifically, laminin E8 has a heterotrimeric structure composed of an α chain E8 fragment (α chain E8), a β chain E8 fragment (β chain E8), and a γ chain E8 fragment (γ chain E8). It has a protein. The subunit chains of laminin E8 (that is, α chain E8, β chain E8, and γ chain E8) are collectively referred to as “E8 subunit chain”. Examples of the E8 subunit chain include the E8 fragment of the above-exemplified laminin subunit chain. Laminin E8 forms various isoforms by combining these E8 subunit chains. Specific examples of laminin E8 include laminin E8 fragments having the heterotrimeric structure shown in Table 2. Laminin E8, especially laminin 111E8, laminin 121E8, laminin 211E8
Laminin 221E8, laminin 332E8, laminin 421E8, laminin 411E8, laminin 511E8, laminin 521E8. Laminin E8 is also named similarly to laminin depending on its heterotrimeric structure. That is, for example, “laminin 511E8” refers to an E8 fragment of laminin 511, specifically, an α8 chain E8 fragment (α5 chain E8), a β1 chain E8 fragment (β1 chain E8), and a γ1 chain A protein having a heterotrimeric structure composed of E8 fragments (γ1 chain E8).

  The “α chain E8” may be a region near the C-terminus of the α chain, specifically, may be a C-terminal fragment excluding the spherical domains 4 and 5 of the α chain, more specifically. Or a fragment of C-terminal 780-830 (for example, 790-800) amino acid residues excluding the globular domains 4 and 5 of the α chain. The “β chain E8” may be a C chain C-terminal fragment of the β chain, specifically, a fragment of 220 to 230 amino acid residues at the C terminal of the β chain. The “γ chain E8” may be a C-terminal fragment of the γ chain, specifically, a fragment of 240 to 250 amino acid residues at the C-terminal of the γ chain.

Table 1 shows the positions of E8 fragments in the human laminin subunit chain and the amino acid sequences of those E8 fragments (N-terminal added with an initial methionine). That is, for example, human α5 chain E8 corresponds to the site of amino acid numbers 2534-3327 of NP_005551. The human β1 chain E8 corresponds to the site of amino acid numbers 1561 to 1786 of NP_002282. The human γ1 chain E8 corresponds to the site of amino acid numbers 1364-1609 of NP_002284.

  That is, the E8 subunit chain is, for example, the amino acid sequence of the site corresponding to the E8 fragment in the amino acid sequence of the laminin subunit chain disclosed in the above database (for example, the amino acids shown in SEQ ID NOs: 3, 4, 13, 19 to 26). A protein having a sequence). Similarly, the gene encoding the E8 subunit chain (also referred to as “E8 subunit chain gene”) encodes a site corresponding to the E8 fragment in the base sequence of the laminin subunit chain gene disclosed in the above database, for example. It may be a gene having the base sequence of the site to be processed. In addition, the expression “having an (amino acid or base) sequence” includes the case of “including the (amino acid or base) sequence” and the case of “consisting of the (amino acid or base) sequence”.

  As long as the original function is maintained, the E8 subunit chain may be a variant of the E8 subunit chain exemplified above. Similarly, the E8 subunit chain gene may be a variant of the E8 subunit chain gene exemplified above as long as the original function is maintained. Such a variant in which the original function is maintained may be referred to as a “conservative variant”. Examples of conservative variants include the E8 subunit chain and the E8 subunit chain gene homologs and artificially modified forms exemplified above.

  “The original function is maintained” means that the variant of the gene or protein has a function (activity or property) corresponding to the function (activity or property) of the original gene or protein. In other words, “the original function is maintained” means that in the E8 subunit chain, the protein variant forms a heterotrimer with other E8 subunit chains and functions as a scaffold for cells such as stem cells. Indicates that `` Other E8 subunit chain '' means β chain E8 and γ chain E8 for α chain E8, α chain E8 and γ chain E8 for β chain E8, α chain E8 and β for γ chain E8 Refers to chain E8. As the other E8 subunit chain, those desired to be used as the subunit chain of the protein of the present invention can be used without particular limitation. As other E8 subunit chains, α5 chain E8, β1 chain E8, and γ1 chain E8 can be used. As other E8 subunit chains, more particularly, human α5 chain E8, β1 chain E8, and γ1 chain E8 can be used. “The original function is maintained” means that in the E8 subunit chain gene, the variant of the gene is a protein that maintains the original function (that is, a heterotrimer with other E8 subunit chain). It encodes a protein that forms a body and exhibits a scaffold function for cells such as stem cells. Whether or not the protein exhibits a scaffold function for cells such as stem cells can be confirmed, for example, by culturing cells such as stem cells using a culture vessel in which the cell culture surface is coated with the protein. That is, when the proliferation of cells such as stem cells is improved by coating the cell culture surface with the protein, it can be determined that the protein exhibits a scaffold function for cells such as stem cells.

  The homologue of the E8 subunit chain is obtained, for example, as an E8 fragment of the homologue of the laminin subunit chain. In addition, the homologue of the E8 subunit chain gene can be obtained, for example, as a fragment encoding a site corresponding to the E8 fragment of the homologue of the laminin subunit chain gene. A homologue of a laminin subunit chain or a laminin subunit chain gene can be obtained, for example, by BLAST search or FASTA search using the amino acid sequence of the laminin subunit chain or the base sequence of the laminin subunit chain gene disclosed in the above database as a query sequence. It can be easily obtained from public databases. In addition, homologs of laminin subunit chain genes can be obtained by PCR using, as primers, oligonucleotides prepared based on the base sequences of laminin subunit chain genes disclosed in the above database using chromosomes of various organisms as templates. Can be acquired.

  Hereinafter, conservative variants of the E8 subunit chain and E8 subunit chain genes will be exemplified.

  As long as the original function is maintained, the E8 subunit chain has one or several positions in the amino acid sequence of the E8 subunit chain (for example, the amino acid sequences shown in SEQ ID NOs: 3, 4, 13, 19 to 26). Or a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added. The above “one or several” varies depending on the position and type of the protein in the three-dimensional structure of the amino acid residue, and specifically, for example, 1 to 50, 1 to 40, 1 to 30, Preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5, particularly preferably 1 to 3.

One or several amino acid substitutions, deletions, insertions, and / or additions described above are conservative mutations in which the function of the protein is maintained normally. A typical conservative mutation is a conservative substitution. Conservative substitution is a polar amino acid between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. In this case, between Gln and Asn, when it is a basic amino acid, between Lys, Arg, and His, when it is an acidic amino acid, between Asp and Glu, when it is an amino acid having a hydroxyl group Is a mutation that substitutes between Ser and Thr. Specifically, substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln
, Lys or Asp substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val Or Phe, Lys to Asn, Glu, Gln, His or Arg, Met to Ile, Leu, Val or Phe, Phe to Trp, Tyr, Met, Ile or Leu, Ser To Thr or Ala, Thr to Ser or Ala, Trp to Phe or Tyr, Tyr to His, Phe or Trp, and Val to Met, Ile or Leu Can be mentioned. In addition, substitution, deletion, insertion, and / or addition of amino acids as described above are caused by naturally occurring mutations (mutants or variants) such as cases based on individual differences or species differences of the organism from which the protein is derived. Also included.

  Further, as long as the original function is maintained, the E8 subunit chain is 80% or more, preferably 90% or more, more preferably 95% or more, and still more preferably, with respect to the entire amino acid sequence of the E8 subunit chain. May be a protein having an amino acid sequence having a homology of 97% or more, particularly preferably 99% or more. In the present specification, “homology” refers to “identity”.

In addition, the E8 subunit chain is stringent with a probe that can be prepared from the base sequence of the E8 subunit chain gene, for example, a complementary sequence to the whole or a part of the base sequence, as long as the original function is maintained. It may be a protein encoded by DNA that hybridizes under conditions. Such a probe can be prepared, for example, by PCR using an oligonucleotide prepared based on the base sequence as a primer and a DNA fragment containing the base sequence as a template. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, highly homologous DNAs, for example, 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, particularly preferably 99% or more. Is hybridized and DNAs with lower homology do not hybridize with each other, or normal Southern hybridization washing conditions of 60 ° C, 1 × SSC, 0.1% SDS, preferably 60 ° C, 0.1 × SSC , 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, a salt concentration and temperature corresponding to 0.1% SDS, and conditions for washing once, preferably 2 to 3 times. For example, when a DNA fragment having a length of about 300 bp is used as the probe, hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.

  In addition, the E8 subunit chain gene may be one in which an arbitrary codon is replaced with an equivalent codon. That is, the E8 subunit chain gene may be a variant of the E8 subunit chain gene exemplified above due to codon degeneracy. For example, the E8 subunit chain gene may be modified to have an optimal codon according to the codon usage of the host to be used.

  The percentage sequence identity between two sequences can be determined, for example, using a mathematical algorithm. Non-limiting examples of such mathematical algorithms include the Myers and Miller (1988) CABIOS 4: 11-17 algorithm, the Smith et al (1981) Adv. Appl. Math. 2: 482 local homology algorithm, Needleman and Hunology alignment algorithm of Wunsch (1970) J. Mol. Biol. 48: 443-453, Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85: 2444-2448 (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877, an improved algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264 Can be mentioned.

  A program based on these mathematical algorithms can be used to perform sequence comparison (alignment) to determine sequence identity. The program can be appropriately executed by a computer. Such programs include, but are not limited to, the PC / Gene program CLUSTAL (available from Intelligents, Mountain View, Calif.), The ALIGN program (Version 2.0), and the Wisconsin Genetics Software Package, Version 8 (Genetics Computer Group (GCG), 575 Science Drive, available from Madison, Wis., USA) GAP, BESTFIT, BLAST, FASTA, and TFASTA. Alignment using these programs can be performed using initial parameters, for example. For the CLUSTAL program, Higgins et al. (1988) Gene 73: 237-244, Higgins et al. (1989) CABIOS 5: 151-153, Corpet et al. (1988) Nucleic Acids Res. 16: 10881-90, Huang et al. (1992) CABIOS 8: 155-65 and Pearson et al. (1994) Meth. Mol. Biol. 24: 307-331.

In order to obtain a nucleotide sequence having homology with the nucleotide sequence encoding the protein of interest, specifically, for example, a BLAST nucleotide search can be performed with the BLASTN program, score = 100, word length = 12. In order to obtain an amino acid sequence having homology with the protein of interest, specifically, for example, BLAST protein search, BLASTX program,
This can be done with score = 50 and word length = 3. Please refer to http://www.ncbi.nlm.nih.gov for BLAST nucleotide search and BLAST protein search. In addition, Gapped BLAST (BLAST 2.0) can be used to obtain an alignment with a gap added for the purpose of comparison. PSI-BLAST (BLAST 2.0) can also be used to perform iterative searches that detect distant relationships between sequences. For Gapped BLAST and PSI-BLAST, see Altschul et al. (1997) Nucleic Acids Res. 25: 3389. When utilizing BLAST, Gapped BLAST, or PSI-BLAST, for example, the initial parameters of each program (eg, BLASTN for nucleotide sequences, BLASTX for amino acid sequences) can be used. The alignment may be performed manually.

  Sequence identity between two sequences is calculated as the percentage of residues that match between the two sequences when the two sequences are aligned for maximum matching.

The E8 subunit chain may contain other amino acid sequences as long as its function is not impaired. Examples of other amino acid sequences include a signal peptide (also referred to as a signal sequence), a peptide tag, a protease recognition sequence, and an amino acid sequence including Gln-Glu-Thr.
Examples of the E8 subunit chain containing these other amino acid sequences include proteins having the amino acid sequences shown in SEQ ID NOs: 2, 7, and 8. That is, the E8 subunit chain may be, for example, a protein having these amino acid sequences or a variant thereof.

The signal peptide is not particularly limited as long as it functions in a host that expresses the E8 subunit chain. Examples of signal peptides that function in bacteria such as coryneform bacteria include signal peptides recognized by the Sec-type secretory pathway and signal peptides recognized by the Tat-type secretory pathway (WO2016 / 171224). Examples of the signal peptide recognized by the Sec-type secretory pathway include a signal sequence of a cell surface protein of coryneform bacteria. Specific examples of cell surface proteins of coryneform bacteria include C1 glutamicum PS1 and PS2 (CspB)
C. stationis SlpA (CspA) (Japanese Patent Laid-Open No. 10-108675). Specific examples of signal peptides recognized in the Tat secretion pathway include TorA signal sequence of E. coli, SufI signal sequence of E. coli, PhoD signal sequence of Bacillus subtilis, LipA signal sequence of Bacillus subtilis, Arthrobacter globiformis An IMD signal sequence may be mentioned. In the E8 subunit chain, the signal peptide may be present at the N-terminus. The signal peptide can be used, for example, for secretory production of the E8 subunit chain. When the E8 subunit chain is secreted and produced using a signal peptide, the signal peptide is typically cleaved during secretion, and the E8 subunit chain not having the signal peptide can be secreted outside the cell. That is, "E8 subunit chain has a signal peptide" means that the E8 subunit chain is expressed in a form having a signal peptide, and it does not matter whether the mature protein of the E8 subunit chain has a signal peptide. Shall.

Specific peptide tags include His tags, FLAG tags, GST tags, Myc tags, MBP (maltose binding protein), CBP (cellulose binding protein), TRX (Thioredoxin), GFP (green fluorescent protein), HRP (horseradish) peroxidase), ALP (Alkaline Phosphatase), and antibody Fc region. An example of a His tag is a 6xHis tag. The peptide tag can be used, for example, for detection and purification of the expressed E8 subunit chain.

Specific examples of protease recognition sequences include Factor Xa protease recognition sequences and proTEV protease recognition sequences. The protease recognition sequence can be used, for example, to cleave the expressed E8 subunit chain.

Examples of the amino acid sequence containing Gln-Glu-Thr include those described in WO2013 / 062029. The amino acid sequence containing Gln-Glu-Thr is a sequence consisting of 3 amino acid residues or more from the N-terminus of the mature protein of the cell surface protein CspB of coryneform bacteria (hereinafter also referred to as “mature CspB”). Good. Examples of coryneform bacteria include those described later. The amino acid sequence containing Gln-Glu-Thr is, for example, 3 to 50 from the amino acid residue at position 1 of mature CspB.
It may be an amino acid sequence up to any amino acid residue. In particular, the amino acid sequence containing Gln-Glu-Thr may be an amino acid sequence from the amino acid residue at position 1 of mature CspB to any one of amino acid residues at positions 3, 8, 17, and 50. The amino acid sequence containing Gln-Glu-Thr may more particularly be an amino acid sequence from the amino acid residue at position 1 to any of amino acid residues at positions 4, 6, 17, and 50 of mature CspB. . As amino acid sequences containing Gln-Glu-Thr, specifically, Gln-Glu-Thr-Asn-Pro-Thr (SEQ ID NO: 14), Gln-Glu-Thr-Gly-Thr-Tyr (SEQ ID NO: 15), Gln-Glu-Thr-Thr-Val-Thr (SEQ ID NO: 16), Gln-Glu-Thr-Pro-Val-Thr (SEQ ID NO: 17), Gln-Glu-Thr-Ala-Val-Thr (SEQ ID NO: 18) Is mentioned. In particular, an amino acid sequence containing Gln-Glu-Thr can be suitably used in combination with a Sec system-dependent signal peptide. The amino acid sequence containing Gln-Glu-Thr can be placed, for example, between the signal sequence and other sites. The amino acid sequence containing Gln-Glu-Thr can be placed in particular immediately after the signal sequence. By using an amino acid sequence containing Gln-Glu-Thr, for example, an increase in secretory production of the E8 subunit chain is expected.

  The E8 subunit chain may or may not be derived from the same organism. The E8 subunit chain may be selected, for example, from all three human subunits and variants thereof, or all three may be selected from mouse subunits and variants thereof, It may be a combination of a variant and a mouse subunit or a variant thereof.

  The protein of the present invention or each subunit chain thereof (each E8 subunit chain) may or may not have a sugar chain. As sugar chains, N-type sugar chains (N-linked sugar chains; those added to asparagine residues) and O-type sugar chains (O-linked sugar chains; added to serine residues or threonine residues). Thing). Examples of sugar chains include N-type sugar chains.

  The protein of the present invention or each subunit chain thereof (each E8 subunit chain) may have, for example, a microbial sugar chain structure. “Microbial-type sugar chain structure” refers to the structure of a sugar chain that can be added when a protein is expressed using a microorganism as a host (pattern of sugar chain modification that can be added when a protein is expressed using a microorganism as a host). Say. The microbial sugar chain structure may be different from a natural mammalian sugar chain structure, that is, a sugar chain structure added by a sugar chain modification system inherent in a mammalian cell. The microbial sugar chain structure may be different from a natural insect sugar chain structure, that is, a sugar chain structure added by a sugar chain modification system inherently possessed by insect cells. A microbial sugar chain (a sugar chain constituting a microbial sugar chain structure) may contain, for example, neither a fucose residue nor a sialic acid residue. Examples of the microorganism include those described later such as bacteria and yeast. In particular, the microorganism includes yeast. As yeast-type sugar chains (sugar chains constituting the yeast-type sugar chain structure), N-type sugar chains include mannan-type sugar chains and galactomannan-type sugar chains, and O-type sugar chains include O-mannose-type sugar chains. Can be mentioned.

  The protein of the present invention or each subunit chain thereof (each E8 subunit chain), for example, may not substantially have a sugar chain (that is, the sugar chain as a whole regardless of the type of sugar chain). Do not have to have). “The protein of the present invention or each subunit chain thereof substantially has no sugar chain” means that the addition rate of the sugar chain in the protein of the present invention or each subunit chain thereof is 20% or less, 10% or less. 5% or less, 2% or less, 1% or less, or 0 (zero). “Addition rate of sugar chain” means that sugar chains are added to the total number of amino acid residues to which sugar chains can be added (specifically, the total number of asparagine residues, serine residues, and threonine residues). It refers to the ratio of the number of amino acid residues (specifically, the total number of asparagine residues, serine residues, and threonine residues to which sugar chains are added).

  In addition, the protein of the present invention or each subunit chain thereof (each E8 subunit chain) substantially has, for example, a specific sugar chain (for example, one or both of an N-type sugar chain and an O-type sugar chain). It does not have to be. Specifically, for example, the protein of the present invention or each subunit chain thereof may be substantially free of at least an N-type sugar chain, and may further contain substantially no O-type sugar chain. Also good. “The protein of the present invention or each subunit chain thereof has substantially no specific sugar chain” means that the addition rate of the specific sugar chain in the protein of the present invention or each subunit chain thereof is 20%. Hereinafter, it means 10% or less, 5% or less, 2% or less, 1% or less, or 0 (zero). “Specific sugar chain addition rate” means the total number of amino acid residues to which the specific sugar chain can be added (specifically, the number of asparagine residues for the N-type sugar chain, The number of amino acid residues to which the specific sugar chain is added relative to the total number of serine residues and threonine residues (specifically, the number of asparagine residues to which sugar chains are added for N-type sugar chains) , For O-type sugar chains, the ratio of the total number of serine residues and threonine residues to which sugar chains are added).

In one embodiment, mouse laminin E8 may be excluded from the protein of the present invention. In one embodiment, mouse laminin 111E8, laminin 121E8, laminin 211E8, and laminin 221E8 may be excluded from the protein of the present invention. In one embodiment, the protein of the present invention may be used to exclude mouse laminin 111E8, laminin 121E8, laminin 211E8, and laminin 221E8 that have substantially no sugar chain (eg, no sugar chain). Good. In one embodiment, the protein of the present invention includes mouse laminin 111E8, laminin 121E8, laminin 211E8, and laminin 221E8 substantially free of a specific sugar chain (for example, having no specific sugar chain). May be removed. In the description of the case where the protein of the present invention or each subunit chain thereof substantially does not have a sugar chain or a specific sugar chain, mouse laminin E8 does not substantially have a sugar chain or a specific sugar chain It can be applied mutatis mutandis.

<1-2> Production of protein of the present invention The means for producing the protein of the present invention is not particularly limited.

  The protein of the present invention can be produced, for example, by expressing the protein of the present invention using a microorganism as a host. That is, the present invention provides a method for producing the protein of the present invention, which comprises expressing the protein of the present invention using a microorganism as a host. This method is also referred to as “production method of the present invention”. “Expression of the protein of the present invention” specifically refers to expression of E8 subunit chains (α chain E8, β chain E8, and γ chain E8). That is, the production method of the present invention specifically includes expressing the E8 subunit chain (α chain E8, β chain E8, and γ chain E8) using a microorganism as a host. It is.

  The E8 subunit chain may be expressed together by a single microorganism, or may be expressed separately by a plurality of microorganisms, for example. That is, for example, α chain E8, β chain E8, and γ chain E8 may be expressed by three types of microorganisms, respectively. In addition, for example, any one of α chain E8, β chain E8, and γ chain E8 may be expressed by a microorganism, and the remaining two may be expressed together by another single microorganism. Specifically, for example, at least β chain E8 and γ chain E8 may be expressed together by a single microorganism. That is, specifically, for example, the α chain E8 may be expressed by a microorganism, and the β chain E8 and the γ chain E8 may be expressed together by another single microorganism. That is, the microorganism used as the host may be a single microorganism or a combination of a plurality of microorganisms. In the context of the present invention, the term “microorganism” in the singular form (for example “a microorganism” or “the microorganism”) depends on the context as at least one microorganism, ie as a single microorganism or a combination of microorganisms, You may replace it.

  The microorganism used as a host has E8 subunit chain genes (α chain E8 gene, β chain E8 gene, and γ chain E8 gene). When the microorganism used as the host is a single microorganism, the single microorganism alone has all three E8 subunit chain genes. When the microorganism used as the host is a combination of a plurality of microorganisms, the plurality of microorganisms has all three E8 subunit chain genes as a whole. The plurality of microorganisms may or may not be the same as each other except for the type of E8 subunit chain gene possessed by the plurality of microorganisms. For example, the plurality of microorganisms may or may not be microorganisms derived from the same genus, species or strain. A microorganism having an E8 subunit chain gene can be obtained by introducing the E8 subunit chain gene into an appropriate microorganism.

  The microorganism used as the host is not particularly limited as long as it can express the E8 subunit chain. Microorganisms include bacteria and yeast.

Examples of bacteria include gram negative bacteria and gram positive bacteria. Gram-negative bacteria include Escherichia bacteria, Enterobacter bacteria,
Examples include bacteria belonging to the family Enterobacteriaceae such as Pantoea bacteria. Examples of Gram-positive bacteria include coryneform bacteria such as Bacillus bacteria and Corynebacterium bacteria. Examples of the genus Escherichia include Escherichia coli. Specific examples of Escherichia coli include, for example, Escherichia coli K-12 strains such as W3110 strain (ATCC 27325) and MG1655 strain (ATCC 47076); Escherichia coli K5 strain (ATCC 23506); BL21 (DE3) strain And Escherichia coli B strains such as the BLR (DE3) strain, which is a recA-strain thereof, and derivatives thereof. Examples of coryneform bacteria include Corynebacterium bacteria such as Corynebacterium glutamicum and Corynebacterium ammoniagenes (Corynebacterium stationis). . As Corynebacterium glutamicum,
Specific examples include Corynebacterium glutamicum AJ12036 strain (FERM BP-734) and Corynebacterium glutamicum YDK010 strain (WO2002 / 081694) which is a deficient strain of cell surface protein PS2 (CspB).

Examples of yeast include Saccharomyces cerevisiae and other Saccharomyces yeasts, Candida utilis and other Candida yeasts, Pichia pastoris and other Pichia yeasts, and Hansenula polymorpha (Hansenula). polymorpha) and the like, and Schizosaccharomyces pombe such as Schizosaccharomyces pombe. Examples of yeast include Saccharomyces yeast and Pichia yeast. More particularly, yeasts include Saccharomyces cerevisiae and Pichia pastoris. Examples of Saccharomyces cerevisiae include Saccharomyces cerevisiae Y006 strain (FERM BP-11299), Saccharomyces cerevisiae BY4743 strain (ATCC 201390), and Saccharomyces cerevisiae S288C strain (ATCC 26108).

  The combination of the microorganism and the expressed E8 subunit chain is not particularly limited. For example, all of the α chain E8 gene, β chain E8 gene, and γ chain E8 gene may be expressed by any one of the microorganisms described above. In addition, for example, α chain E8 is expressed by Enterobacteriaceae bacteria such as Escherichia coli or yeast such as Saccharomyces cerevisiae and Pichia pastoris, and β chain E8 and γ chain E8 are corynebacterium such as Corynebacterium glutamicum. It may be expressed by type bacteria.

The microorganism used as the host may have desired properties as long as it can express the E8 subunit chain. For example, coryneform bacteria may have reduced cell surface protein activity (WO2013 / 065869, WO2013 / 065772, WO2013 / 118544, WO2016 / 171224, WO2013 / 062029).
Coryneform bacteria may be modified so that the activity of penicillin-binding protein is reduced (WO2013 / 065869). The coryneform bacterium may be modified so that expression of a gene encoding a metallopeptidase is increased (WO2013 / 065772). The coryneform bacterium may be modified to have a mutant ribosomal protein S1 gene (mutant rpsA gene) (WO2013 / 118544). Coryneform bacteria may be modified to have a mutant phoS gene (WO2016 / 171224). Examples of the mutant phoS gene include a gene encoding a mutant PhoS protein in which the tryptophan residue (W302) at position 302 of the wild-type PhoS protein is substituted with an amino acid residue other than an aromatic amino acid and histidine. Specific examples of “amino acid residues other than aromatic amino acids and histidine” include K (Lys), R (Arg), A (Ala), V (Val), L (Leu), I (Ile), G (Gly), S (Ser), T (Thr), P (Pro), C (Cys), M (Met), D (Asp), E (Glu), N (Asn), Q (Gln) It is done. More specifically, as “amino acid residues other than aromatic amino acids and histidine”, K (Lys), A (Ala), V (Val), S (Ser), C (Cys), M (Met), And D (Asp) and N (Asn). Examples of the wild-type phoS gene include a phoS gene of coryneform bacteria. “The tryptophan residue (W302) at position 302 of the wild-type PhoS protein” means an amino acid residue corresponding to the tryptophan residue at position 302 of the PhoS protein of the C. glutamicum YDK010 strain in any wild-type PhoS protein. To do. The position of the amino acid residue indicates a relative position, and the position may be changed by amino acid deletion, insertion, addition or the like. Coryneform bacteria may have an enhanced Tat secretion apparatus (WO2016 / 171224). Due to these properties or modifications, for example, an increase in the secretory production amount of the E8 subunit chain is expected. These properties or modifications can be used alone or in appropriate combination.

These strains can be sold, for example, from the American Type Culture Collection (address 12301 Parklawn Drive, Rockville, Maryland 20852 PO Box 1549, Manassas, VA 20108, United States of America). That is, a registration number corresponding to each strain is given, and it is possible to receive a sale using this registration number (see http://www.atcc.org/). The registration number corresponding to each strain is described in the catalog of American Type Culture Collection. The BL21 (DE3) strain is available, for example, from Life Technologies (product number C6000-03). The BLR (DE3) strain is available, for example, from Merck Millipore (product number 69053).

  The presence or absence of a sugar chain in the E8 subunit chain and the sugar chain structure can be determined according to various conditions such as the type of microorganism. For example, by expressing an E8 subunit chain using yeast as a host, typically, an E8 subunit chain having a yeast-type sugar chain structure can be produced. In addition, for example, by expressing an E8 subunit chain using a bacterium as a host, typically, an E8 subunit chain having no sugar chain can be produced.

  In one embodiment, the production method of the present invention may exclude the case where mouse laminin E8 is expressed using Escherichia coli as a host. In one embodiment, the production method of the present invention may exclude the case where mouse laminin 111E8, laminin 121E8, laminin 211E8, and laminin 221E8 are expressed using Escherichia coli as a host.

The E8 subunit chain gene can be obtained, for example, by cloning from an organism having a laminin subunit chain gene such as human or mouse. For cloning, nucleic acids such as genomic DNA and cDNA containing the same gene can be used. The acquired gene can be used as it is or after being appropriately modified. The gene can be modified by a known method. For example, a target mutation can be introduced into a target site of DNA by a site-specific mutation method. That is, the coding region of a gene can be altered, for example, by site-directed mutagenesis such that the amino acid residue at a particular site of the encoded protein includes substitutions, deletions, insertions, and / or additions. . As site-directed mutagenesis, a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. In Enzymol., 154, 382 (1987)) and methods using phage (Kramer, W. and Frits, HJ,
Meth. In Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. In Enzymol., 154, 367 (1987)). The E8 subunit chain gene can also be obtained, for example, by chemical synthesis (Gene, 60 (1), 115-127 (1987)).

The method for introducing the E8 subunit chain gene into the host is not particularly limited. In the host, the E8 subunit chain gene may be retained so that it can be expressed under the control of a promoter that functions in the host. In the host, the E8 subunit chain gene may be present on a vector that autonomously replicates outside the chromosome, such as a plasmid, or may be introduced on the chromosome. The host may have only one copy of each E8 subunit chain gene or may have two or more copies. The host may have only one type of gene for each E8 subunit chain gene, or may have two or more types of genes. Moreover, when introducing two or more genes, each gene should just be hold | maintained in a host so that expression is possible. For example, all the genes may be held on a single expression vector, or all may be held on a chromosome. Moreover, each gene may be separately hold | maintained on several expression vector, and may be separately hold | maintained on the single or several expression vector and chromosome. Further, an operon may be constructed by introducing two or more genes.

The promoter for expressing the E8 subunit chain gene is not particularly limited as long as it functions in the host. The “promoter that functions in the host” refers to a promoter having promoter activity in the host. The promoter may be a host-derived promoter or a heterologous promoter. The promoter may be a promoter specific to the laminin subunit chain gene from which the E8 subunit chain gene is derived, or may be a promoter of another gene. The promoter may be a stronger promoter than the native promoter of the laminin subunit chain gene from which the E8 subunit chain gene is derived. Examples of strong promoters that function in Enterobacteriaceae bacteria such as Escherichia coli include, for example, T7 promoter, trp promoter, trc promoter, lac promoter, tac promoter, tet promoter, araBAD promoter, rpoH promoter, PR promoter, and PL promoter is mentioned. In addition, as a strong promoter that functions in coryneform bacteria, for example, the artificially modified P54-6 promoter (Appl. Microbiol. Biotechnolo., 53, 674-679 (2000)) Pta, aceA, aceB, adh, amyE promoters that can be induced with acetic acid, ethanol, pyruvate, etc., cspB, SOD, tuf (EF-Tu) promoters (Journal of Biotechnology) 104 (2003) 311-323, Appl Environ Microbiol. 2005 Dec; 71 (12): 8587-96.), Lac promoter, tac promoter, trc promoter. Examples of strong promoters that can be used in yeast include promoters of genes such as PGK1, PDC1, TDH3, TEF1, HXT7, and ADH1, which are known high expression promoters. Moreover, as a promoter, you may acquire and utilize the highly active type of a conventional promoter by using various reporter genes. For example, the activity of the promoter can be increased by bringing the −35 and −10 regions in the promoter region closer to the consensus sequence (International Publication No. 00/18935). Examples of highly active promoters include various tac-like promoters (Katashkina JI et al. Russian Federation Patent application 2006134574) and pnlp8 promoter (WO2010 / 027045). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

Moreover, a terminator for termination of transcription can be arranged downstream of the E8 subunit chain gene. The terminator is not particularly limited as long as it functions in the host. The terminator may be a host-derived terminator or a heterologous terminator. The terminator may be a specific terminator of the laminin subunit chain gene from which the E8 subunit chain gene is derived, or may be a terminator of another gene. Specific examples of the terminator include T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.

The E8 subunit chain gene can be introduced into a host using, for example, a vector containing the gene. A vector containing the E8 subunit chain gene is also referred to as an expression vector or a recombinant vector for the E8 subunit chain gene. An expression vector for the E8 subunit chain gene can be constructed, for example, by ligating a DNA fragment containing the E8 subunit chain gene with a vector that functions in the host. By transforming the host with an expression vector for the E8 subunit chain gene, a transformant introduced with the vector can be obtained, that is, the gene can be introduced into the host. As the vector, a vector capable of autonomous replication in a host cell can be used. The vector is preferably a multicopy vector. Further, the vector preferably has a marker such as an antibiotic resistance gene in order to select a transformant. Moreover, the vector may be equipped with a promoter or terminator for expressing the inserted gene. The vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, or a phagemid. As vectors capable of autonomous replication in bacteria of the Enterobacteriaceae family such as Escherichia coli, specifically, for example, pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pSTV29 (all available from Takara Bio Inc.), pACYC184, pMW219 (Nippon Gene), pTrc99A (Pharmacia), pPROK vector (
Clontech), pKK233-2 (Clontech), pET vector (Novagen), pQE vector (Qiagen), pCold TF DNA (TaKaRa), pACYC vector, and wide host range vector RSF1010. Specific examples of vectors capable of autonomous replication in coryneform bacteria include, for example, pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901- 2903 (1984)); plasmids having improved drug resistance genes; pCRY30 (Japanese Patent Laid-Open No. 3-210184); pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX (Japanese Patent Laid-Open No. 2-72876, US Pat. No. 5,185,262) PCRY2 and pCRY3 (Japanese Unexamined Patent Publication No. 1-191686); pAJ655, pAJ611, and pAJ1844 (Japanese Unexamined Patent Publication No. 58-192900); pCG1 (Japanese Unexamined Patent Publication No. 57-134500); pCG2 (Japanese Unexamined Patent Publication No. 58-35197); pCG4 and pCG11 (Japanese Unexamined Patent Publication No. 57-183799); pVK7 (Japanese Unexamined Patent Publication No. 10-215883); pVK9 (US2006-0141588); pVC7 (Japanese Unexamined Patent Publication No. 9-070291); pVS7 (WO2013 / 069634). Examples of vectors that function in yeast cells include plasmids having a CEN4 replication origin and multicopy plasmids having a 2 μm DNA replication origin. Specific examples of vectors that function in yeast cells include pAUR123 (Takara Bio) and pYES2 (Invitrogen). When constructing the expression vector, for example, the coding region of the E8 subunit chain may be linked downstream of the promoter as described above and then incorporated into the vector, or the E8 subunit may be placed downstream of the promoter inherent in the vector. A unit chain coding region may be incorporated.

  Vectors, promoters, and terminators that can be used in various microorganisms are described in detail in, for example, “Basic Course of Microbiology 8 Genetic Engineering, Kyoritsu Shuppan, 1987”, and these can be used.

The E8 subunit chain gene can be introduced, for example, onto the host chromosome. Introduction of a gene into a chromosome can be performed, for example, using homologous recombination (Miller, JH Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). As a gene transfer method using homologous recombination, for example, the Red-driven integration method (Datsenko, KA, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97: 6640-6645 (2000)), etc., a method using a linear DNA, a method using a plasmid containing a temperature-sensitive replication origin, a method using a plasmid capable of conjugation transfer, a method using a suicide vector that does not have a replication origin and functions in a host, A transduction method using a phage can be mentioned. Only one copy of the gene may be introduced, or two copies or more may be introduced. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination with a sequence having multiple copies on the chromosome as a target. Examples of sequences having multiple copies on a chromosome include repetitive DNA sequences and inverted repeats present at both ends of a transposon. In addition, homologous recombination may be performed by targeting an appropriate sequence on a chromosome such as a gene unnecessary for carrying out the present invention. The gene can also be randomly introduced onto the chromosome using transposon or Mini-Mu (Japanese Patent Laid-Open No. 2-109985, US Pat. No. 5,882,888, EP805867B1). When the gene is introduced into the chromosome, for example, the coding region of the E8 subunit chain may be linked downstream of the promoter as described above and then incorporated into the chromosome, or the E8 subunit chain may be E8 downstream of the promoter originally present on the chromosome. The coding region of the subunit chain may be incorporated.

  The introduction of a gene onto a chromosome can be attributed to, for example, Southern hybridization using a probe having a base sequence complementary to all or part of the gene, or a primer prepared based on the base sequence of the gene. Can be confirmed by PCR.

The transformation method is not particularly limited, and a conventionally known method can be used. As a transformation method, for example, a method of increasing the permeability of DNA by treating a recipient cell with calcium chloride as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol.
Biol. 1970, 53, 159-162), as described for Bacillus subtilis, a method of preparing competent cells from proliferating cells and introducing DNA (Duncan, CH, Wilson, GA and Young, FE., 1997. Gene 1: 153-167). In addition, as a transformation method, recombinant DNA is prepared by transforming DNA-receptive cells, such as those known for Bacillus subtilis, actinomycetes, and yeast, into a protoplast or spheroplast state that easily incorporates recombinant DNA. (Chang, S. and Choen, SN, 1979. Mol. Gen. Genet. 168: 111-115; Bibb, MJ, Ward, JM and Hopwood, OA 1978. Nature 274: 398- 400; Hinnen, A., Hicks, JB and Fink, GR 1978. Proc. Natl. Acad. Sci. USA 75: 1929-1933). As a transformation method, an electric pulse method (Japanese Patent Laid-Open No. 2-207791) as reported for coryneform bacteria can also be used.

  By culturing a host having the E8 subunit chain gene, the E8 subunit chain can be expressed. At that time, gene expression is induced as necessary. Host culture conditions and gene expression induction conditions may be appropriately selected according to various conditions such as marker type, promoter type, and host type. The medium used for the culture is not particularly limited as long as the host can proliferate and can express the E8 subunit chain. As the medium, for example, a normal medium containing a carbon source, a nitrogen source, a sulfur source, inorganic ions, and other organic components as required can be used.

  Examples of the carbon source include sugars such as glucose, fructose, sucrose, molasses, and starch hydrolysates, alcohols such as glycerol and ethanol, and organic acids such as fumaric acid, citric acid, and succinic acid.

  Examples of the nitrogen source include inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, and aqueous ammonia.

  Examples of the sulfur source include inorganic sulfur compounds such as sulfate, sulfite, sulfide, hyposulfite, and thiosulfate.

  Examples of inorganic ions include calcium ions, magnesium ions, manganese ions, potassium ions, iron ions, and phosphate ions.

  Other organic components include organic trace nutrient sources. Examples of organic micronutrients include required substances such as vitamin B1, yeast extract containing them, and the like.

The culture temperature may be, for example, 20 ° C to 45 ° C, preferably 24 ° C to 45 ° C, more preferably 27 ° C to 37 ° C. The culture is preferably aeration culture. In this case, the oxygen concentration may be adjusted to, for example, 5 to 50%, preferably 20 to 40% with respect to the saturation concentration. The pH during the culture is preferably 5-9.
In addition, inorganic or organic acidic or alkaline substances such as calcium carbonate, ammonia gas, aqueous ammonia, etc. can be used for pH adjustment. The culture time may be, for example, 10 hours to 120 hours.

In particular, when Escherichia coli is used as a host, organic acid accumulation during induction of E8 subunit chain expression is reduced (WO2015 / 178465) and cell growth after induction of E8 subunit chain expression is reduced ( WO2015 / 178466) is expected to be able to efficiently produce E8 subunit chains.

  By culturing a host having the E8 subunit chain gene as described above, the E8 subunit chain accumulates in the medium and / or in the cells. The E8 subunit chain can accumulate, for example, as inclusion bodies in the microbial cells.

  Recovery and quantification of the E8 subunit chain can be performed, for example, by a known method for recovering and quantifying a heterologously expressed protein (for example, “Neurochemistry Laboratory Lecture Protein VI Synthesis and Expression”, Japanese Biochemical Society, Tokyo Kagaku Dojin (1992) pp 183-184).

Hereinafter, a procedure for recovery and quantification in the case where the E8 subunit chain accumulates as inclusion bodies in the microbial cells will be exemplified. First, cells are collected from the culture solution by centrifugation and then suspended in a buffer solution. The bacterial cell suspension is subjected to treatment such as ultrasonic treatment and French press to crush the bacterial cells. Prior to cell disruption, lysozyme may be added to the cell suspension at a final concentration of 0-200 mg / l and left in ice for 30 minutes to 20 hours. The insoluble fraction is then obtained as a precipitate from the crushed material by low-speed centrifugation (6000-15000 rpm, 5-10 minutes, 4 ° C.). The insoluble fraction is appropriately washed with a buffer as necessary. The number of times of washing is not particularly limited, and may be, for example, once, twice, or three times or more. A suspension of E8 subunit chains is obtained by suspending the insoluble fraction in buffer. As a buffer solution for suspending bacterial cells and E8 subunit chains, those having low solubility of E8 subunit chains can be preferably used. Examples of such a buffer include a Tris-HCl buffer containing NaCl. The pH of the buffer solution may be, for example, usually 4 to 12, preferably 6 to 9.
Further, an E8 subunit chain solution can be obtained by dissolving the insoluble fraction with an SDS solution or a urea solution. The recovered E8 subunit chain may contain components such as bacterial cells, medium components, and bacterial metabolic byproducts in addition to the E8 subunit chain. The E8 subunit chain may be purified to the desired extent. The E8 subunit chain can be purified by, for example, a known method used for protein purification. Examples of such a method include ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, isoelectric precipitation, and dialysis. One of these methods may be used alone, or two or more thereof may be used in appropriate combination. The amount of the E8 subunit chain is determined by, for example, subjecting a sample containing the E8 subunit chain, such as a suspension or solution, to SDS-PAGE and staining it, and the intensity of the band corresponding to the molecular weight of the target E8 subunit chain. Can be determined based on Staining can be performed by CBB staining, fluorescent staining, silver staining, or the like. In quantification, a protein with a known concentration can be used as a standard. Examples of such a protein include albumin and an E8 subunit chain whose concentration is separately determined.

The expressed E8 subunit chain (α chain E8, β chain E8, and γ chain E8) is directly as a heterotrimer consisting of α chain E8, β chain E8, and γ chain E8 (ie, as the protein of the present invention). ) May or may not be obtained. That is, the E8 subunit chain can be obtained as a monomer or multimer depending on various conditions such as its expression mode. The E8 subunit chain is, for example, a heterodimer of two or more E8 subunit chains such as each monomer, each homomultimer such as a homodimer, or heterodimer or heterotrimer. It can be obtained as a multimer. For example, when E8 subunit chains are expressed in a state where they do not coexist with each other, such as when the E8 subunit chains are expressed by separate microorganisms, these E8 subunit chains are used as respective monomers or respectively. As a homodimer. In addition, for example, when two or more E8 subunit chains are expressed together in a state where two or more E8 subunit chains are expressed together by a single microorganism, these E8 subunit chains are: It can be obtained as a heteromultimer. Specifically, for example, when the β chain E8 and the γ chain E8 are expressed together in a state where the β chain E8 and the γ chain E8 are expressed together by a single microorganism, the E8 subunit chain is: It can be obtained as a heterodimer consisting of β chain E8 and γ chain E8. Specifically, for example, all of α chain E8, β chain E8, and γ chain E8, such as when all α chain E8, β chain E8, and γ chain E8 are expressed together by a single microorganism. Are expressed as heterotrimers consisting of α-chain E8, β-chain E8, and γ-chain E8 (ie, as a protein of the present invention).

  From the expressed E8 subunit chain (α chain E8, β chain E8, and γ chain E8), a heterotrimer consisting of α chain E8, β chain E8, and γ chain E8 as appropriate (ie, the protein of the present invention) Can be configured. That is, the production method of the present invention may include constructing a heterotrimer from the E8 subunit chain. The means for forming the heterotrimer is not particularly limited. The heterotrimer can be constructed by, for example, a known method used for the construction of protein multimers. Examples of such a method include the method described in A Utani, et al., Laminin chain assembly. J. Biol. Chem. 1994 269: 19167-19175. The heterotrimer can be constructed, for example, by allowing the E8 subunit chain to coexist (mix) in an appropriate solvent. Examples of the solvent include aqueous solvents such as water and aqueous buffers. All E8 subunit chains may be mixed together or sequentially mixed. For example, a heterotrimer composed of an α chain E8, a β chain E8, and a γ chain E8 can be formed by mixing all E8 subunit chains together. Further, for example, a heterodimer consisting of β chain E8 and γ chain E8 is formed by mixing β chain E8 and γ chain E8, and α chain E8, β chain E8, And a heterotrimer consisting of γ-chain E8. Similarly, for example, when the expressed E8 subunit chain is obtained as a heterodimer consisting of β chain E8 and γ chain E8, α chain E8, β chain E8, and γ can be obtained by further mixing α chain E8. A heterotrimer consisting of chain E8 can be constructed. When the expressed E8 subunit chain is obtained as a homomultimer, for example, it may be appropriately dissociated into monomers and then used in the construction of a heterotrimer. The means for dissociating the homomultimer into monomers is not particularly limited. The dissociation into monomers can be performed by, for example, a known method used for protein denaturation. Examples of such a method include urea modification and reduction modification. “Constructing a heterotrimer from an E8 subunit chain” is not limited to constructing a heterotrimer from an E8 subunit chain obtained (expressed) as a monomer. Construct a heterotrimer from β-chain E8 and γ-chain E8 obtained (expressed) as a heterodimer with chain E8, or heterozygous from an E8 subunit chain obtained (expressed) as a homomultimer. Constructing a trimer is also encompassed. The amount ratio of the E8 subunit chain used for the construction of the heterotrimer is not particularly limited, but usually the E8 subunit chain may be used in equimolar amounts.

  The protein of the present invention can be produced, for example, by expressing the protein of the present invention in a cell-free protein synthesis system. By expressing the E8 subunit chain in a cell-free protein synthesis system, typically an E8 subunit chain having no sugar chain can be produced. Also in this case, a heterotrimer composed of an α chain E8, a β chain E8, and a γ chain E8 (that is, the protein of the present invention) can be appropriately formed according to the form of the obtained E8 subunit chain.

Further, among the proteins of the present invention, laminin E8 substantially having no sugar chain can be produced, for example, by removing the sugar chain of laminin E8 having a sugar chain. The means for removing the sugar chain is not particularly limited. As a means for removing the sugar chain, a method using an enzyme such as glycosidase, anhydrous hydrazine, anhydrous TFMS (Anhydrous trifluoromethanesulfonic acid)
) And other chemical substances.

  The use of the protein of the present invention is not particularly limited. The protein of the present invention may be used, for example, for medical use or research use. The protein of the present invention exhibits a scaffold function for cells such as stem cells. Therefore, the protein of the present invention may be used for culturing cells such as stem cells. Specifically, the protein of the present invention may be used, for example, as a scaffold protein for cell culture. Moreover, you may utilize the protein of this invention as a component of instruments, such as a cell culture instrument. Specifically, the protein of the present invention may be used by coating the surface of a device such as a cell culture container. About an instrument and cell culture, description about the instrument of this invention mentioned later and the method of this invention can apply mutatis mutandis.

<2> Composition of the present invention The composition of the present invention is a composition containing the protein of the present invention. The use of the composition of the present invention is not particularly limited. The composition of the present invention may be used, for example, for medical use or research use. That is, the composition of the present invention may be, for example, a medical or research composition. Specifically, the composition of the present invention may be used, for example, for culturing cells such as stem cells. That is, the composition of the present invention may specifically be a composition for cell culture, for example. More specifically, the composition of the present invention may be, for example, a cell scaffold material. “Cell scaffold material” refers to a structure that functions as a cell scaffold, and specifically refers to a structure that functions as a cell scaffold during cell culture. The cell scaffold material can be used for cell culture, for example. That is, cells can be cultured using a cell scaffold material as a scaffold. Cell culture results (cultured cells, cell sheets, tissues, organs, etc.) obtained using cell scaffold materials can be collected from, for example, cell scaffold materials or desired for treatment, etc. It can be used for Further, the cell scaffold material may be implanted into a living body and used for culturing cells in a living body (tissue formation through cell adhesion and proliferation, etc.). More specifically, the composition of the present invention may be a coating agent for an instrument such as a cell culture container. About an instrument and cell culture, description about the instrument of this invention mentioned later and the method of this invention can apply mutatis mutandis.

  The composition of the present invention may or may not consist of the protein of the present invention. The composition of the present invention may be a combination of the protein of the present invention and other components. That is, the protein of the present invention can be used as the composition of the present invention alone or in combination with other components. The other components are not particularly limited as long as they have acceptable properties according to the use mode of the composition of the present invention. Examples of other components include those used by blending with pharmaceuticals and reagents. Specific examples of such components include additives such as excipients, binders, disintegrants, lubricants, stabilizers, corrigents, flavoring agents, fragrances, diluents, and surfactants. Can be mentioned.

  The concentration (content) of the protein of the present invention in the composition of the present invention is not particularly limited. The concentration of the protein of the present invention in the composition of the present invention can be appropriately set according to various conditions such as the use mode of the composition of the present invention. The concentration of the protein of the present invention in the composition of the present invention is, for example, 0.01% (w / w) or more, 0.1% (w / w) or more, 1% (w / w) or more, 5% ( w / w) or more, or 10% (w / w) or more, 100% (w / w) or less, 99.9% (w / w) or less, 70% (w / w) or less, 50% (w / w) or less, 30% (w / w) or less, 10% (w / w) or less, or 5% (w / w) or less, or 1% (w / w) or less Of course, they may be a consistent combination.

The shape of the composition of the present invention is not particularly limited. The shape of the composition of this invention can be suitably set according to various conditions, such as the utilization aspect of the composition of this invention. The composition of the present invention may be formulated in a desired shape. Specifically, as the shape of the composition of the present invention, for example, liquid (solution or suspension), paste, powder, granule, sphere, tablet, foam, sponge, fiber, film, sheet, membrane, mesh Tube.

  The composition of the present invention may be used for a desired application (for example, coating of an instrument), for example, as it is, or appropriately diluted, dispersed, or dissolved in a medium such as water or an aqueous buffer. Needless to say, such dilution, dispersion, or dissolution is also included in the scope of the composition of the present invention.

<3> Instrument of the Present Invention The instrument of the present invention is an instrument comprising the protein of the present invention. The application of the instrument of the present invention is not particularly limited. The device of the present invention may be used, for example, for medical use or research use. That is, the device of the present invention may be, for example, a medical device or a research device. Specifically, the device of the present invention may be used, for example, for culturing cells such as stem cells. That is, the instrument of the present invention may specifically be a cell culture instrument, for example. Cell culture devices include cell culture containers and cell scaffold materials. The device of the present invention which is a cell culture container is also referred to as “the culture container of the present invention”. The device of the present invention which is a cell scaffold material is also particularly referred to as “cell scaffold material of the present invention”. The instrument of the present invention may be provided with the protein of the present invention in an embodiment in which the function of the protein of the present invention is exhibited. The device of the present invention may have, for example, the protein of the present invention on its surface. That is, the device of the present invention may specifically be, for example, a device whose surface is coated with the protein of the present invention. In addition, when the protein of the present invention or the composition of the present invention is configured in a form that can be used as a device as it is, the protein of the present invention or the composition of the present invention may be used as it is as the device of the present invention. . Specifically, for example, when the composition of the present invention is configured as a cell scaffold material, the composition of the present invention may be used as it is as the cell scaffold material of the present invention. For cell culture, the description of the method of the present invention described later can be applied mutatis mutandis.

  Hereinafter, although the instrument of the present invention will be described mainly with reference to the culture vessel, the description can be applied to other instruments. That is, the following description can be applied to other appliances as they are, or appropriately changed according to various conditions such as the aspect and use of the appliance.

  The culture vessel is not particularly limited as long as it has a material and shape capable of culturing cells. The culture container may have, for example, a material and a shape that can hold cells and a culture medium. Specific examples of the shape of the culture vessel include a dish, a multiwell plate, a flask, a cell insert, and a membrane. Specific examples of the culture vessel material include polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), triacetyl cellulose (TAC), polyimide (PI), nylon (Ny), low density polyethylene ( LDPE), medium density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene, urethane acrylate and other plastics, polylactic acid, polyglycolic acid, polycaprolactan, etc. And biodegradable polymers such as copolymers, glasses such as glass and modified glass, ceramics, metals, cellulose, and other various polymer compounds.

The culture container of the present invention comprises the protein of the present invention at least on the cell culture surface. That is, for example, the cell culture surface of the culture container of the present invention may be coated with the protein of the present invention. The “cell culture surface” refers to a surface where cells are cultured, and specifically refers to a portion that can come into contact with cells during cell culture. The protein of the present invention may or may not coat the entire cell culture surface of the culture container as long as cells such as stem cells can be cultured using the culture container of the present invention. The ratio of the area of the portion coated with the protein of the present invention to the total area of the cell culture surface of the culture vessel is, for example, 50% or more, 70% or more, 80% or more, 90% or more, or 95% or more. It's okay. In general, the protein of the present invention preferably coats the entire cell culture surface of the culture vessel. For example, if the culture vessel is in a dish shape, the entire bottom surface is preferably coated with the protein of the present invention.

  The technique for coating the cell culture surface of the culture vessel with the protein of the present invention is not particularly limited. Coating the cell culture surface of the culture vessel with the protein of the present invention can be carried out, for example, using a known technique for coating the surface of the culture vessel with a functional material. For example, the cell culture surface of the culture container can be coated with the protein of the present invention by applying a solution containing the protein of the present invention to the cell culture surface of the culture container. Specifically, for example, the cell culture surface of the culture container is coated with the protein of the present invention by applying a solution containing the protein of the present invention to the cell culture surface of the culture container and drying (vaporizing the solvent). be able to. More specifically, for example, when a time (hereinafter also referred to as “coating time”) elapses after the solution containing the protein of the present invention is applied to the cell culture surface of the culture vessel, cell culture in the culture vessel is performed. The surface can be coated with the protein of the invention. The coating time is not particularly limited as long as the cell culture surface of the culture vessel can be coated with the protein of the present invention to a desired degree. The coating time may be, for example, 30 minutes or more, 1 hour or more, 6 hours or more, 12 hours or more, or 18 hours or more, 72 hours or less, 48 hours or less, or 30 hours or less, A combination thereof may also be used. After the coating time has elapsed, the remaining solution can be removed as appropriate and can be dried. The culture container can be sterilized as appropriate. The culture container can be sterilized, for example, after the cell culture surface of the culture container is coated with the protein of the present invention. For sterilization, for example, alcohol such as ethanol or 2-propanol can be used. The alcohol may be used as a hydrous alcohol such as 70% ethanol. Sterilization can be performed, for example, by applying alcohol to the cell culture surface or immersing the culture container in alcohol. As described above, sterilization before cell culture can be performed using alcohol. However, in order to ensure completeness, the solution containing the protein of the present invention is sterilized by filter sterilization or the like and then applied to the cell culture surface of the culture vessel. The subsequent operations may be performed under aseptic conditions. For filter sterilization, for example, a filter such as Milex manufactured by Merck Millipore can be used. Aseptic operations can be performed as appropriate. The culture container of the present invention may be used immediately after production (after the coating of the protein of the present invention), or may be used after being properly stored. The culture container of the present invention is effective for culturing cells such as stem cells even when not used immediately after production. The storage temperature may be, for example, 0 ° C to 50 ° C, 0 ° C to 40 ° C, 0 ° C to 25 ° C, or 0 ° C to 10 ° C. The storage period may be, for example, 1 year or less, 6 months or less, or 1 month or less. The culture container of the present invention may be stored in a dry state or in a wet state. For example, the cell culture surface may be stored in a state wetted with a buffer solution such as PBS.

The coating amount of the protein of the present invention on the surface of the culture container is not particularly limited as long as cells such as stem cells can be cultured using the culture container of the present invention. Coating amount of the protein of the present invention to the culture vessel surface is, for example, 0.1 [mu] g / cm ² or more, 0.2 [mu] g / cm ² or more, 0.4 [mu] g / cm ² or more, 1 [mu] g / cm ² or more, or 5 [mu] g / cm ² or more, 500 μg / cm ² or less, 250 μg / cm ² or less, 100 μg / cm ² or less, or 50 μg / cm ² or less, or a combination thereof. Specifically, the coating amount of the protein of the present invention on the surface of the culture container is, for example, 0.1 μg / cm ^{2 to} 500 μg / cm ² , preferably 0.2 μg / cm ^{2 to} 250 μg / cm ² , more preferably 0.4μg ^/ cm 2 may be a ~100μg / cm ^2.

Similarly, the cell scaffold material of the present invention can be appropriately obtained. The cell scaffold material is not particularly limited as long as it has a material and shape capable of culturing cells. The cell scaffold material may have, for example, a shape corresponding to the shape of a target cell culture result (cell sheet, tissue, organ, etc.). The cell scaffold material may be configured, for example, in a form having voids such as porous so that cells are filled in the cell scaffold material. The material of the cell scaffold material is preferably a bioabsorbable polymer from the viewpoint of being suitable for implantation in a living body, for example. Specific examples of bioabsorbable polymers include polylactic acid, polyglycolic acid, copolymers of lactic acid and glycolic acid, polycaprolactone, collagen, gelatin, glycosaminoglycan, chitin, chitosan, hyaluronic acid, poly Peptides are mentioned. The cell scaffold material of the present invention can be obtained, for example, by coating the surface of the cell scaffold material (specifically, the cell culture surface) with the protein of the present invention. It should be noted that the “surface of the cell scaffold material” includes the surface inside the void when the cell scaffold material is configured in a form having voids. For example, as described above, the composition of the present invention configured as a cell scaffold material may be used as it is as the cell scaffold material of the present invention.

<4> Method of the Present Invention Cells such as stem cells can be efficiently cultured using the protein of the present invention. Specifically, cells such as stem cells can be efficiently cultured using a device (the device of the present invention) provided with the protein of the present invention. More specifically, cells such as stem cells can be efficiently cultured using a culture instrument comprising the protein of the present invention, such as the culture container of the present invention or the cell scaffold material of the present invention. That is, this invention provides the method of culture | cultivating a cell including culturing a cell with the instrument of this invention, such as the culture container of this invention, and the cell scaffold material of this invention. This method is also referred to as “the method of the present invention”. The protein of the present invention is particularly effective for the proliferation of stem cells such as iPS cells. That is, the protein of the present invention and a cell culture instrument such as a cell culture container or a cell scaffold material provided with the protein can be used, for example, for growing stem cells such as iPS cells. In addition, the protein of the present invention and cell culture devices such as cell culture containers and cell scaffold materials provided with the protein are not limited to the proliferation of stem cells such as iPS cells. For example, the establishment of stem cells such as iPS cells in the previous stage In addition, it can also be used for differentiation of stem cells such as iPS cells in the subsequent stage. That is, in the present invention, “culture of cells” is not limited to cell proliferation, but includes, for example, establishment of cells having differentiation ability such as stem cells and progenitor cells, and other cells having differentiation ability such as stem cells and precursor cells. Is also included. Specifically, “culture of stem cells” is not limited to the proliferation of stem cells. For example, establishment of stem cells (for example, establishment of universal stem cells such as iPS cells and ES cells or pluripotent stem cells) and differentiation of stem cells (For example, differentiation from stem cells such as iPS cells and ES cells into other stem cells, progenitor cells, or mature cells) is also included. By the method of the present invention, cultured cells (cultured cells) can be obtained. That is, one aspect of the method of the present invention is a method for producing cultured cells, comprising culturing cells with the device of the present invention such as the culture container of the present invention and the cell scaffold material of the present invention. The cultured cells obtained by the method of the present invention may or may not be differentiated. That is, the cultured cells obtained by the method of the present invention may be maintained in an undifferentiated state or may have acquired an undifferentiated state, for example. The cultured cells obtained by the method of the present invention may be, for example, cells having differentiation ability such as stem cells and progenitor cells, or may be differentiated cells such as progenitor cells and mature cells. The cultured cells obtained by the method of the present invention may be, for example, free cells (individual independent cells) or organized cells such as cell sheets, tissues, organs and the like.

The type of cells to be cultured is not particularly limited. The method of the present invention can be suitably used particularly for stem cell culture. Specific examples of stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), pluripotent germ stem cells (mGS cells), neural stem cells, mesenchymal stem cells, and hematopoietic stem cells. It is done. More specifically, examples of the stem cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). The cell may be derived from any living tissue. Examples of biological tissues include skin, cornea, liver, digestive organs, mammary gland, prostate, hair root, trachea, and oral mucosa. The organism from which the cells are derived is not particularly limited and can be appropriately selected according to the purpose of use. Examples of organisms from which cells are derived include mammals. Specific examples of mammals include human, rat, mouse, guinea pig, marmoset, rabbit, dog, cat, sheep, pig and chimpanzee. The mammal may be an immunodeficient animal. When cultured cells are used for human therapy, the organism from which the cells are derived is preferably, for example, a mammal selected from humans, pigs, and chimpanzees. In the method of the present invention, only one type of cell may be cultured, or two or more types of cells may be co-cultured.

The culture conditions are not particularly limited as long as desired culture results (cell proliferation, stem cell establishment, cell differentiation, etc.) can be obtained. The culture conditions are the same as, for example, normal conditions used for culturing cells such as ES cells and iPS cells, except that the device of the present invention such as the culture container of the present invention and the cell scaffold material of the present invention is used. It's okay. The culture conditions can be appropriately set according to various conditions such as the aspect of the device of the present invention, the type of cells, the biological species from which the cells are derived.

The composition of the medium can be appropriately set according to various conditions such as the properties of cells to be cultured. Examples of the medium components include carbon sources such as glucose, amino acids, vitamins, phosphates, buffers, growth factors, serum, and serum albumin. Specific examples of the normal medium used for cell culture include α-MEM, DMEM, and KCM. Further, as a normal medium used for culturing embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), specifically, for example, Essential 8 medium (manufactured by Invitrogen), StemFit (R) medium (Made by Ajinomoto Co., Inc.) and other media used in the examples.

  According to the method of the present invention, cells such as stem cells can be efficiently cultured without using feeder cells or other cell adhesion proteins (cell adhesion proteins other than the protein of the present invention). However, this does not preclude the use of feeder cells or other cell adhesion proteins. That is, in the method of the present invention, feeder cells and other cell adhesion proteins (cell adhesion proteins other than the protein of the present invention) may or may not be used. In the method of the present invention, it is preferable to reduce the amount of feeder cells and other cell adhesion proteins used compared to the usual amount used for cell culture. In the method of the present invention, it is more preferable not to use feeder cells or other cell adhesion proteins.

The number of cells to be seeded at the start of the culture may be, for example, 1 × 10 ³ cells / cm ² or more, 1 × 10 ⁴ cells / cm ² or more, or 2 × 10 ⁴ cells / cm ² or more, and 1 × 10 ^It may be ⁶ cells / cm ² or less, 1 × 10 ⁵ cells / cm ² or less, or 5 × 10 ⁴ cells / cm ² or less, or a combination thereof. The culture temperature may be, for example, 36 ° C to 38 ° C, preferably about 37 ° C. The culture pH may be, for example, pH 6.8 to 7.2. The gas phase at the time of culture may be, for example, a mixed gas of air and carbon dioxide. Specifically, for example, the culture may be performed in an atmosphere of 5% carbon dioxide gas and 95% air. The culture period may be, for example, 5 to 30 days, preferably 7 to 16 days. During the culture, the medium may be changed.

  By performing cell culture in this manner, cultured cells are obtained.

  Hereinafter, the present invention will be described more specifically based on examples.

Example 1: Preparation of sugar chain-cleavage laminin 511E8 and evaluation of cell scaffold function <1> Preparation of sugar chain-cleavage laminin 511E8 For commercially available laminin 511E8 (iMatrix-511, Nippi), N-type sugar chain cleavage enzyme Glycopeptidase F ( Takara Bio Inc.) was reacted at 37 ° C. for 15 hours under denaturing conditions and non-denaturing conditions according to the attached protocol. iMatrix-511 is human laminin 511E8 produced by heterologous expression using CHO-S cells derived from Chinese hamster ovary as a host. Thereafter, SDS-PAGE analysis was performed in order to confirm the band shift due to sugar chain cleavage. The result is shown in FIG. As a result, it was confirmed that the band was moved by an amount corresponding to the predicted molecular weight of the sugar chain. From this result, it was possible to prepare sugar chain-cleaved laminin 511E8 (laminin 511E8 from which the sugar chain was removed).

<2> Evaluation of Cell Scaffold Function of Sugar-Cleaved Laminin 511E8 <2-1> Coating on Cell Culture Plate An aqueous solution of sugar-chain-cleaved laminin 511E8 obtained in Example 1 <1> above was added to phosphate buffered saline (
The solution was diluted with Nacalai Tesque) and charged into a 6-well cell culture plate (Corning Falcon culture plate) so that the protein amount was 4.8 μg / well. After standing at 4 ° C. for a whole day and night, the aqueous solution was removed.

<2-2> Evaluation in iPS cell proliferation system Artificial pluripotent stem cells (iPS cells) on the surface coated with glycosylated laminin 511E8
The adhesion growth effect of was evaluated. The 201B7 strain purchased from iPS Portal was used as the iPS cell. Cell culture using StemFit (R) medium (manufactured by Ajinomoto Co., Inc.), was carried out under the conditions of 5% CO _2/37 ℃. The effect was examined by coating sugar chain-cleaved laminin 511E8 on a cell culture plate and using it immediately in culture. A single cell was seeded with 13,000 live cells per well. Y-27632 was added to the medium used at the time of seeding (final concentration: 10 μM, manufactured by Nacalai Tesque), and the following day, the medium was cultured with no Y-27632 added. The culture medium is changed every 1 to 3 days, and after culturing for 6 to 7 days, the number of cells detached by treatment with cell dissociation enzyme (Life Technologies TrypLE Select) is used with a cell number measuring device (Beckman Coulter ViCell). Measured. As a comparative control, commercially available laminin 511E8 (iMatrix-511, manufactured by Nippi Co., Ltd.) without sugar chain cleavage was coated on a cell culture plate according to the method of the product protocol, and evaluated by the same culture method.

The results are shown in FIG. As shown in FIG. 2, sugar chain-cleaved laminin 511E8 is coated by the above method <2-1>, and cultured for 3 weeks with 2 passages in the middle under conditions where 13,000 live cells are seeded. I was able to. During this time, the rate of increase in viable cells did not vary, indicating that stable culture was possible. After culturing for 3 weeks, staining was performed using an alkaline phosphatase staining kit (Sigma Aldrich: 86-R). As a result, it was confirmed that the undifferentiated state of the cells was maintained. This indicates that laminin 511E8 can proliferate while maintaining the undifferentiated state of iPS cells in long-term culture, even without a sugar chain.

Example 2: Expression of human laminin α5 chain E8 in E. coli <1> Construction of E. coli human laminin α5 chain E8 expression strain Plasmid pET22b-LNa expressing the gene of human laminin α5 chain E8 is a pET22b (+) vector ( Novagen) was cleaved with restriction enzymes NdeI and EcoRI, and the fully synthesized human laminin α5 chain E8 gene was inserted using DNA Ligation Kit (TaKaRa). The base sequence of this gene is shown in SEQ ID NO: 1, and the amino acid sequence of human laminin α5 chain E8 encoded by this gene is shown in SEQ ID NO: 2. The human laminin α5 chain E8 of SEQ ID NO: 2 has a 6xHis tag added to the N-terminus. The pET22b-LNa Escherichia in E. coli BL21 (DE3) ΔrecA strain transformed with _{^{(F - ompT hsdS B (r}} B - - m B) gal dcm (DE3) Δ (srl-recA) 306 :: Tn10 (Tet R)) By converting, human laminin α5
A chain E8-producing bacterium BL21 (DE3) ΔrecA / pET22b-LNa strain was obtained. The BL21 (DE3) ΔrecA strain can be obtained by deleting the recA gene from the BL21 (DE3) strain by the λRed method.

<2> Expression of human laminin α5 chain E8 in E. coli The BL21 (DE3) ΔrecA / pET22b-LNa strain obtained above was inoculated into a 500 mL Sakaguchi flask in which the medium shown in (1) was placed. The seed culture solution A was obtained by shaking culture at 0 ° C. for 8 hours. The seed culture solution A was seeded in a 500 mL Sakaguchi flask in which the medium shown in (2) was placed, and shaken at 37 ° C. until all the glucose was consumed to obtain a seed culture solution B. 294 mL of the medium shown in (3) with 6 mL of seed culture B
The main culture was performed under the conditions of 1 vvm, 37 ° C., DO> 24% (DO before seeding is taken as 100%) and pH 6.9. After the start of culture, the feed solution shown in (4) was added from the time when glucose was depleted. When the OD (measured at a wavelength of 620 nm) in the culture solution reached 40, the culture temperature was lowered to 30 ° C 1 mM isopropyl-β-thiogalactopyranoside was added to induce expression of human laminin α5 chain E8. Culturing was terminated 12 hours after the addition of IPTG, and the cells were collected by centrifugation. When cells were disrupted with ultrasound and the cell disruption solution was subjected to SDS-PAGE, a band of the desired size was observed (FIG. 3), confirming the expression of human laminin α5 chain E8.

(1) Pre-seed medium
Yeast extract 5 g / L, Selecto soytone 10 g / L, NaCl 10 g / L, ampicillin sodium
100 mg / L.

(2) Preseed medium glucose 5 g / L, Yeast extract 5 g / L, Selecto soytone 10 g / L, NaCl 10 g / L, ampicillin sodium 100 mg / L.

(3) Production medium glucose 20 g / L, magnesium sulfate heptahydrate 2.5 g / L, ammonium sulfate 5 g / L, Select soytone 1.0 g / L (nitrogen weight conversion), potassium dihydrogen phosphate 5 g / L, Sodium citrate
3 g / L, iron sulfate heptahydrate 40 mg / L, manganese sulfate heptahydrate 40 mg / L, calcium chloride dihydrate 40 mg / L, GD-113 (antifoam) 0.1 ml / L , Ampicillin sodium 100mg / L

(4) Feed medium glucose 600 g / L

Example 3: Secretory expression of human laminin β1 chain E8 and human laminin γ1 chain E8 and simultaneous secretion expression of human laminin β1 chain E8 and human laminin γ1 chain E8 in C. glutamicum <1> Each of human laminin β1 chain E8 and human laminin γ1 chain E8 Construction of secretory expression plasmid and co-secreted expression plasmid of human laminin β1 chain E8 and human laminin γ1 chain E8 The amino acid sequence of human laminin 511E8 obtained by enzymatic treatment of human laminin 511 has already been determined. The amino acid sequences of human laminin β1 chain E8 and human laminin γ1 chain E8 are shown in <SEQ ID 3> and <SEQ ID 4>. Human laminin β1 based on these amino acid sequences
The nucleotide sequences encoding chain E8 and human laminin γ1 chain E8 were designed in consideration of the codon usage of C. glutamicum. Furthermore, CspB signal peptide 30 amino acid residues from C. glutamicum ATCC13869 strain, N-terminal 6 amino acid residues of CspB mature protein derived from the same strain, recognition sequence IEGR of FactorXa protease, human laminin β1 chain E8 or human laminin γ1 chain E8, And a 6xHis-tagged fusion protein (hereinafter referred to as “CspB6Xa-LE8β” or “CspB6Xa-LE8γ”) and a base sequence encoding the fusion protein were designed. The designed base sequences encoding CspB6Xa-LE8β and CspB6Xa-LE8γ are <SEQ ID NO: 5> and <SEQ ID NO: 6>, and the amino acid sequences of CspB6Xa-LE8β and CspB6Xa-LE8γ are <SEQ ID NO: 7> and <SEQ ID NO: 8>. Shown in

Next, CspB derived from C. glutamicum ATCC13869 strain is located upstream of the base sequence described in <SEQ ID NO: 5>.
An expression cassette for CspB6Xa-LE8β was designed and synthesized in total by linking the gene promoter and adding a KpnI site on the 5'-side and a BamHI site on the 3'-side. A fully-synthesized DNA fragment (CspB6Xa-LE8β expression cassette) is treated with restriction enzymes KpnI and BamHI, and then inserted into the KpnI-BamHI site of pPK4 described in JP-A-9-322774. A certain pPK4_CspB6Xa-LE8β was constructed. Similarly, a CspB6Xa in which the promoter of the CspB gene derived from the C. glutamicum ATCC13869 strain was linked upstream of the base sequence described in <SEQ ID NO: 6>, a KpnI site was added to the 5′-side, and a BamHI site was added to the 3′-side. -The expression cassette of LE8γ was designed and completely synthesized. Totally synthesized DNA fragment (expression cassette for CspB6Xa-LE8γ) is converted into restriction enzymes KpnI and BamHI
After the treatment, pPK4_CspB6Xa-LE8γ, a secretion expression plasmid for CspB6Xa-LE8γ, was constructed by inserting it into the KpnI-BamHI site of pPK4 described in JP-A-9-322774.

Using pPK4_CspB6Xa-LE8β as a template, using the primers <SEQ ID NO: 9> and <SEQ ID NO: 10>, a DNA fragment of about 1.5 kbp containing CspB6Xa-LE8β, and the primers <SEQ ID NO: 11> and <SEQ ID NO: 12> The DNA fragments of about 1.5 kbp containing CspB6Xa-LE8γ were each amplified by the PCR method. For PCR, PrimeSTAR GXL® DNA polymerase (Takara Bio) was used, and the reaction conditions followed the protocol recommended by the manufacturer. Each amplified DNA fragment of about 1.5 kbp was purified using Wizard® SV Gel and PCR Clean-Up System (Promega). Using the purified two DNA fragments as templates, using the primers <SEQ ID NO: 9> and <SEQ ID NO: 12>, an about 3.0 kbp DNA fragment containing the ligated CspB6Xa-LE8β and CspB6Xa-LE8γ was amplified by PCR. did. For PCR, KOD-FX® DNA polymerase (TOYOBO) was used, and the reaction conditions followed the protocol recommended by the manufacturer. The amplified DNA fragment of about 3.0 kbp was subjected to agarose gel electrophoresis, and the target band was cut out and recovered from the gel using Wizard® SV Gel and PCR Clean-Up System (Promega).
The recovered DNA fragment is inserted into the KpnI-SalI site of pPK4 described in JP-A-9-322774 by an infusion reaction, whereby a co-secreted expression plasmid of CspB6Xa-LE8β and CspB6Xa-LE8γ is pPK4_CspB6Xa-LE8β-CspB6Xa-LE8γ Got. In-Fusion® HD Cloning Kit (Takara Bio) was used for the infusion reaction, and the reaction conditions followed the protocol recommended by the manufacturer.

<2> Secretory expression of human laminin β1 chain E8 and human laminin γ1 chain E8 and co-secretion expression of human laminin β1 chain E8 and human laminin γ1 chain E8 in C. glutamicum pPK4_CspB6Xa-LE8β, pPK4_CspB6Xa-LE8a LE8β-CspB6Xa-LE8γ was used to transform the C. glutamicum YDK010 :: phoS (W302C) strain described in WO2016 / 171224, and YDK010 :: phoS (W302C) / pPK4_CspB6Xa-LE8β strain, YDK010 :: phoS (W302C ) / pPK4_CspB6Xa-LE8γ strain and YDK010 :: phoS (W302C) / pPK4_CspB6Xa-LE8β-CspB6Xa-LE8γ strain were obtained. The YDK010 :: phoS (W302C) strain is a strain obtained by replacing the wild-type phoS gene of C. glutamicum YDK010 described in WO2002 / 081694 with a mutant phoS (W302C) gene. The mutant phoS (W302C) gene encodes a PhoS protein (W302C) in which the tryptophan residue at position 302 is replaced with a cysteine residue. YDK010 strain is a cell surface protein PS2 (CspB) deficient strain of C. glutamicum AJ12036 strain (FERM BP-734). On March 26, 1984, the AJ12036 strain was established on March 26, 1984 at the Institute of Microbial Technology (currently the National Institute for Product Evaluation Technology Patent Biological Deposit Center, Postal Code: 292-0818, Address: Kisarazu, Chiba, Japan. Kazusa Kamashika 2-5-8 Room 120) was deposited as an international deposit and was given the deposit number FERM BP-734.

Each obtained transformant was mixed with MMTG liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, sulfuric acid containing 25 mg / l kanamycin in a test tube. Iron heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 0.45 mg, biotin 0.45 mg, DL-methionine 0.15 g, soy hydrochloride hydrolyzate (total nitrogen 0.2 g), calcium carbonate 50 g 1 L with water, adjusted to pH 7.0) and cultured at 30 ° C. for 72 hours. After completion of the culture, 2.5 μl of the culture supernatant obtained by centrifuging each culture solution was subjected to non-reducing SDS-PAGE and then stained with Oriole fluorescent gel stain (Bio-Rad). As a result, a protein band presumed to be a homodimer of human laminin β1 chain E8 in the culture supernatant of YDK010 :: phoS (W302C) / pPK4_CspB6Xa-LE8β strain, YDK010 :: phoS (W302C) / pPK4_CspB6Xa-LE8γ strain Protein band presumed to be a homodimer of human laminin γ1 chain E8 in the culture supernatant, human laminin β1 chain E8 and human laminin γ1 in the culture supernatant of YDK010 :: phoS (W302C) / pPK4_CspB6Xa-LE8β-CspB6Xa-LE8γ strain A protein band presumed to be a homodimer or heterodimer of chain E8 was detected, respectively (FIG. 4). After staining, digitize the band intensity of each sample using the image analysis software ImageQuant TL (GE Healthcare), create a calibration curve from the BSA solution (50, 100, 200 ng / lane) that was simultaneously migrated and stained, From this calibration curve, the concentration of dimer in each sample was calculated. The results are shown in Table 3.

<3> Confirmation of human laminin β1γ1E8 dimer by LC-MS analysis in the culture supernatant of co-secreted expression strains of human laminin β1 chain E8 and human laminin γ1 chain E8 YDK010 :: phoS (W302C) obtained in <2> above Proteins presumed to be homodimers or heterodimers of human laminin β1 chain E8 and human laminin γ1 chain E8 were purified from the culture supernatant of / pPK4_CspB6Xa-LE8β-CspB6Xa-LE8γ strain by affinity purification. Take 1 ml of cOmplate His-Tag Purification Resin (Roche) in a 50 ml plastic tube, add 20 ml of binding buffer (50 mM NaH ₂ PO ₄ (pH 8.0), 300 mM NaCl) and mix well. Resin was equilibrated by removing the supernatant with caution. 12-15 ml of the culture supernatant was added to the equilibrated resin and mixed by inverting for 1 hour 30 minutes, and then centrifuged to remove the supernatant. 1 ml of washing buffer (50 mM NaH ₂ PO ₄ (pH 8.0), 300 mM NaCl, 5 mM imidazole) was added and mixed by inverting for 5 minutes. After repeating this 5 times, the supernatant was removed and the elution buffer (50 mM NaH ₂ PO ₄ (pH 8.0), 300
mM NaCl, 250 mM imidazole) was added and mixed by inverting for 10 minutes, and the centrifugation supernatant was recovered.
The centrifugal supernatant was filtered (PVDF, 0.22 μm).

Next, the obtained filtrate was analyzed using LC-ESI-Q-TOF-MS (Agilent Technology).
It was subjected to purification by affinity chromatography and MS measurement. The analysis conditions are as follows.

Column: TSKgel Protein C4-300 (3 μm, 4.6 mm ID x 15 cm, Tosoh Corporation)
Injection volume: 20 μl
Column temperature: 40 ° C
Flow rate: 1 ml / min
Mobile phase A: 0.1% formic acid Mobile phase B: 0.1% formic acid / acetonitrile gradient: Table 4

  The MS spectrum of the obtained multivalent ions was deconvolved using MassHunter BioConfirm software (Agilent Technology). The result is shown in FIG. The calculated molecular weight of human laminin β1γ1E8 dimer (heterodimer of human laminin β1 chain E8 and human laminin γ1 chain E8) is 57272 Da. From FIG. 5, it was confirmed that the molecular weight of the protein in the filtrate was consistent with the calculated value of the human laminin β1γ1E8 dimer. Therefore, it was confirmed that human laminin β1γ1E8 dimer was produced in the medium by culturing the co-secreted expression strain of human laminin β1 chain E8 and human laminin γ1 chain E8.

Example 4: Reconstitution of αβγ trimer from urea-modified commercial laminin 511E8 and evaluation of cell scaffold function <1> αβγ trimer from urea-modified commercial laminin 511E8 (α chain (fold and βγ dimer)) Reconstitution of the polymer After centrifugal concentration of commercially available laminin 511E8 (iMatrix-511, Nippi) to 5 times (v / v), a solution with a final concentration of 8 M Urea, 20 mM Tris-HCl, pH 7.4 was prepared. And left at 4 ° C. overnight. Laminin 511E8 is dissociated into α chain and βγ dimer by denaturation with the addition of Urea. The denatured laminin 511E8 was diluted 10-fold with 20 mM Tris-HCl, pH 7.4, and allowed to stand at 4 ° C. for 16 hours or more. The diluted sample was then mixed with 1/10 volume of cOmplete His-Tag Purification Resin (Roche) and stirred for 1 hour 30 minutes. Collect the flow-through, wash the resin with 6 volumes of 50 mM NaH ₂ PO ₄ , 5 mM imidazole, and adsorb to the resin with 3 volumes of 50 mM NaH ₂ PO ₄ , 250 mM imidazole. The protein that was present was eluted. Similarly, as a control sample, commercial laminin 511E8 not urea-denatured and commercial laminin 511E8 after urea denaturation and before dilution were subjected to adsorption, washing, and elution on the resin. For diluted and control samples, each fraction was subjected to SDS-PAGE. The results are shown in FIGS. In the commercially available laminin 511E8, since a His tag is added to the α chain, when the laminin 511E8 is denatured and dissociated into an α chain and a βγ dimer, the control sample shown in FIG. As in the previous commercial laminin 511E8), βγ dimers are detected in the flow-through and wash fractions and α chains are detected in the eluted fraction. On the other hand, when laminin 511E8 forms a trimer, as shown in the result of the control sample shown in FIG. 7 (commercially available laminin 511E8 which is not urea-denatured), α-chain and βγ-dimer are contained in the eluted fraction. Is detected. In the diluted sample, α chain and βγ dimer were detected in the eluted fraction (FIG. 8). That is, in the diluted sample, laminin 511E8 formed a trimer, and it was shown that αβγ trimer can be reconstituted by dilution. The elution fraction of the diluted sample was collected and sterilized with a 0.22 μm filter to obtain a reconstituted αβγ trimer aqueous solution. The protein concentration in the aqueous solution was measured by the Bradford method (Bio-Rad protein assay concentrated dye reagent, # 5000006, Bio-Rad) using BSA as a standard.

<2> αβ reconstituted from urea-modified commercial laminin 511E8 (α chain fold and βγ dimer)
Evaluation of Cell Scaffold Function of γ Trimer <2-1> Coating on Cell Culture Plate An aqueous solution of αβγ trimer reconstituted from urea-denatured laminin 511E8 obtained in the above <1> was phosphate buffered saline. The solution was diluted with water (Nacalai Tesque) and charged into a 6-well cell culture plate (Corning Falcon culture plate) so that the protein amount was 4.8 μg / well or 9.6 μg / well. After standing at 4 ° C. for a whole day and night, the aqueous solution was removed.

<2-2> Evaluation in iPS Cell Proliferation System α Reconstituted from urea-denatured laminin 511E8 in the same manner as in Example 1 <2-2>
The adhesion proliferation effect of induced pluripotent stem cells (iPS cells) on the surface coated with βγ trimer was evaluated.

The results are shown in FIG. As shown in FIG. 9, αβγ trimer reconstituted from urea-denatured laminin 511E8 is coated with the above method <2-1>, and is a good iPS cell under conditions where 13,000 live cells are seeded. The scaffold function was demonstrated. This indicates that urea-denatured laminin 511E8 is reconstituted as an αβγ trimer by the method <1> and restores iPS cell scaffold function.

Example 5: Reconstitution of αβγ trimer from reduction-modified commercial laminin 511E8 and evaluation of cell scaffold function <1> Reduction-modified commercial laminin 511E8 (α chain (unfold, β chain, and γ chain) from αβγ
Reconstitution of trimer After concentrating commercially available laminin 511E8 (iMatrix-511, Nippi) 10 times (v / v) using 10K ultrafiltration membrane, final concentration 8 M Urea, 20 mM Tris-HCl , 10 mM DTT, pH 7.4 was prepared, heated at 37 ° C. for 1 hour, and allowed to stand at 4 ° C. overnight. Laminin 511E8 is dissociated into α, β, and γ chains by reducing and denaturing by adding Urea and DTT. Reduced denatured laminin 511E8 was added to 20 mM Tris-HCl to a final concentration of 0.8 M Urea, 20 mM Tris-HCl, 1 mM DTT, 1 mM oxidized glutathione, 1 mM reduced glutathione, pH 7.4, The mixture was diluted 10 times (v / v) with pH 7.4 and a redox agent and allowed to stand at 4 ° C. for 16 hours or more. The diluted sample was then mixed with 1/10 volume of cOmplete His-Tag Purification Resin (Roche) and stirred for 1 hour 30 minutes. Collect the flow-through, wash the resin with 5 volumes of 50 mM NaH ₂ PO ₄ , 5 mM imidazole, and adsorb the resin to the resin with 5 volumes of 50 mM NaH ₂ PO ₄ , 250 mM imidazole Was eluted. When each fraction was subjected to SDS-PAGE, α chain and βγ dimer were detected in the eluted fraction (FIG. 10). That is, in the diluted sample, laminin 511E8 formed a trimer, and it was shown that αβγ trimer can be reconstituted by dilution. The eluted fraction was collected and sterilized with a 0.22 μm filter to obtain a reconstituted αβγ trimer aqueous solution. The protein concentration in the aqueous solution was measured by BCA method (Micro BCA Pritein Asay Reagent kit, Thermo Fisher Scientific) using BSA as a standard.

<2> Evaluation of Cell Scaffold Function of αβγ Trimer Reconstituted from Reduced Modified Commercial Laminin 511E8 (α Chain (unfold, β Chain, and γ Chain) <2-1> Coating on Cell Culture Plate < The aqueous solution of αβγ trimer reconstituted from the reduction-denatured laminin 511E8 obtained in 1> was diluted with phosphate buffered saline (Nacalai Tesque) and added to a 6-well cell culture plate (Corning Falcon culture plate). The amount of protein was added so as to be 4.8 μg / well or 9.6 μg / well. After standing at 4 ° C. for a whole day and night, the aqueous solution was removed.

<2-2> Evaluation in iPS Cell Proliferation System In the same manner as in Example 1 <2-2>, α reconstituted from reduction-denatured laminin 511E8
The adhesion proliferation effect of induced pluripotent stem cells (iPS cells) on the surface coated with βγ trimer was evaluated.

  The results are shown in FIG.

As shown in FIG. 3, αβγ trimer reconstituted from reductive laminin 511E8 is coated with the above method <2-1>, and is a good iPS cell under conditions where 13,000 live cells are seeded. The scaffold function was demonstrated. This indicates that reductively modified laminin 511E8 is reconstituted as an αβγ trimer by the method <1> above and restores iPS cell scaffold function.

<Explanation of Sequence Listing>
SEQ ID NO: 1: Nucleotide sequence of a gene encoding human laminin α5 chain E8 with a 6xHis tag added at the N terminus SEQ ID NO: 2: Amino acid sequence of human laminin α5 chain E8 with a 6xHis tag added at the N terminus SEQ ID NO: 3: Human laminin β1 Amino acid sequence of chain E8 SEQ ID NO: 4: amino acid sequence of human laminin γ1 chain E8 SEQ ID NO: 5: nucleotide sequence encoding CspB6Xa-LE8β SEQ ID NO: 6: nucleotide sequence encoding CspB6Xa-LE8γ SEQ ID NO: 7: amino acid sequence of CspB6Xa-LE8β SEQ ID NO: 8: amino acid sequence of CspB6Xa-LE8γ SEQ ID NO: 9-12: primer SEQ ID NO: 13: amino acid sequence of human laminin α5 chain E8 SEQ ID NO: 14-18: amino acid sequence containing Gln-Glu-Thr SEQ ID NO: 19: human laminin α1 chain Amino acid sequence of E8 SEQ ID NO: 20: Amino acid sequence of human laminin α2 chain E8 SEQ ID NO: 21: Amino acid sequence of human laminin α3 chain E8 SEQ ID NO: 22 Amino acid sequence of human laminin α4 chain E8 SEQ ID NO: 23: Amino acid sequence of human laminin β2 chain E8 SEQ ID NO: 24: Amino acid sequence of human laminin β3 chain E8 SEQ ID NO: 25: Amino acid sequence of human laminin γ2 chain E8 SEQ ID NO: 26: Amino acid of human laminin γ3 chain E8 Array

Claims (40)

Laminin E8 having the following properties (A) or (B):
(A) N-type sugar chain addition rate is 20% or less;
(B) It has a microbial sugar chain structure. The laminin E8 according to claim 1, excluding laminin 111E8, laminin 121E8, laminin 211E8, and laminin 221E8 of a mouse having no sugar chain.   The laminin E8 according to claim 1 or 2, which does not have an N-type sugar chain.   The laminin E8 according to claim 1 or 2, which has a yeast-type sugar chain structure. The laminin E8 according to any one of claims 1 to 4, which is laminin 111E8, laminin 121E8, laminin 211E8, laminin 221E8, laminin 332E8, laminin 421E8, laminin 411E8, laminin 511E8, or laminin 521E8.   The laminin E8 according to any one of claims 1 to 5, which is laminin 511E8. The laminin E8 according to any one of claims 1 to 6,
The α chain E8 constituting the laminin E8 is the protein described in the following (1a), (1b), or (1c):
(1a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2, 13, 19, 20, 21, or 22;
(1b) comprising an amino acid sequence comprising substitutions, deletions, insertions or additions of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, 13, 19, 20, 21, or 22, and a protein that forms a heterotrimer with β-chain E8 and γ-chain E8 and exhibits a scaffold function for cells;
(1c) an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2, 13, 19, 20, 21, or 22, and heterotrimeric with β chain E8 and γ chain E8 A protein that forms a body and exhibits a scaffold function for cells;
The β chain E8 constituting the laminin E8 is the protein described in the following (2a), (2b), or (2c):
(2a) a protein comprising the amino acid sequence shown in SEQ ID NO: 3, 7, 23, or 24;
(2b) comprising an amino acid sequence comprising a substitution, deletion, insertion or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 3, 7, 23 or 24, and α chain E8 and a protein that forms a heterotrimer with γ-chain E8 and exhibits a scaffold function for cells;
(2c) an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 3, 7, 23, or 24, and forming a heterotrimer with α chain E8 and γ chain E8 A protein that exhibits a scaffold function for cells;
Laminin E8, wherein the γ chain E8 constituting the laminin E8 is a protein described in (3a), (3b), or (3c) below:
(3a) a protein comprising the amino acid sequence shown in SEQ ID NO: 4, 8, 25, or 26;
(3b) in the amino acid sequence shown in SEQ ID NO: 4, 8, 25, or 26, comprising an amino acid sequence comprising substitution, deletion, insertion, or addition of 1 to 10 amino acid residues, and α chain E8 and a protein that forms a heterotrimer with β-chain E8 and exhibits a scaffold function for cells;
(3c) an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 4, 8, 25, or 26, and forming a heterotrimer with α chain E8 and β chain E8 A protein that exhibits a scaffold function for cells.   A method for producing laminin E8, comprising expressing laminin α chain E8, β chain E8, and γ chain E8 using a microorganism as a host. The method according to claim 8, except for expressing laminin 111E8, laminin 121E8, laminin 211E8, and laminin 221E8 in mice using Escherichia coli as a host. The laminin E8 is laminin 111E8, laminin 121E8, laminin 211E8, laminin 221E8, laminin 332E8, laminin 421E8, laminin 411E8, laminin 511E8, or laminin 521E8
10. The method according to claim 8 or 9, wherein The method according to any one of claims 8 to 10, wherein the laminin E8 is laminin 511E8. The method according to any one of claims 8 to 11, comprising:
The α chain E8 is the protein described in the following (1a), (1b), or (1c): (1a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2, 13, 19, 20, 21, or 22 ;
(1b) comprising an amino acid sequence comprising substitutions, deletions, insertions or additions of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, 13, 19, 20, 21, or 22, and a protein that forms a heterotrimer with β-chain E8 and γ-chain E8 and exhibits a scaffold function for cells;
(1c) an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2, 13, 19, 20, 21, or 22, and heterotrimeric with β chain E8 and γ chain E8 A protein that forms a body and exhibits a scaffold function for cells;
The β chain E8 is a protein according to the following (2a), (2b), or (2c): (2a) a protein comprising the amino acid sequence shown in SEQ ID NO: 3, 7, 23, or 24;
(2b) comprising an amino acid sequence comprising a substitution, deletion, insertion or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 3, 7, 23 or 24, and α chain E8 and a protein that forms a heterotrimer with γ-chain E8 and exhibits a scaffold function for cells;
(2c) an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 3, 7, 23, or 24, and forming a heterotrimer with α chain E8 and γ chain E8 A protein that exhibits a scaffold function for cells;
A method wherein the γ chain E8 is a protein according to the following (3a), (3b), or (3c):
(3a) a protein comprising the amino acid sequence shown in SEQ ID NO: 4, 8, 25, or 26;
(3b) in the amino acid sequence shown in SEQ ID NO: 4, 8, 25, or 26, comprising an amino acid sequence comprising substitution, deletion, insertion, or addition of 1 to 10 amino acid residues, and α chain E8 and a protein that forms a heterotrimer with β-chain E8 and exhibits a scaffold function for cells;
(3c) an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 4, 8, 25, or 26, and forming a heterotrimer with α chain E8 and β chain E8 A protein that exhibits a scaffold function for cells.   The method according to any one of claims 8 to 12, wherein at least the β chain E8 and the γ chain E8 are expressed together by a single microorganism.   The method according to any one of claims 8 to 13, wherein the α chain E8, β chain E8, and γ chain E8 are expressed together by a single microorganism. The α chain E8, β chain E8, and γ chain E8 are each expressed separately by a plurality of microorganisms.
The method according to any one of claims 8 to 12.   The method according to any one of claims 8 to 15, wherein the microorganism is a bacterium or a yeast.   The method according to claim 16, wherein the microorganism is a bacterium belonging to the family Enterobacteriaceae or a coryneform bacterium.   The method according to claim 16, wherein the microorganism is Escherichia coli or Corynebacterium glutamicum. The method according to any one of claims 8 to 16, wherein the microorganism is Saccharomyces cerevisiae or Pichia pastoris.   The method according to any one of claims 8 to 19, comprising constructing laminin E8 from the α chain E8, β chain E8, and γ chain E8.   21. The method of claim 20, comprising obtaining the β-chain E8 and the γ-chain E8 as heterodimers, and constituting laminin E8 from the α-chain E8 and the heterodimer.   The composition containing the laminin E8 of any one of Claims 1-7.   A composition comprising laminin E8 produced by the method according to any one of claims 8 to 21.   24. The composition of claim 22 or claim 23, which is a cell scaffold material.   25. The composition of claim 24, wherein the cell scaffold material is for stem cell culture.   26. The composition according to claim 25, wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell.   24. The composition according to claim 22 or 23, which is a coating agent for cell culture containers.   28. The composition of claim 27, wherein the cell culture container is for stem cell culture.   The composition according to claim 28, wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell.   An instrument comprising laminin E8 according to any one of claims 1-7.   An instrument comprising laminin E8 produced by the method of any one of claims 8-21.   32. A device according to claim 30 or claim 31 which is a medical or research device.   The device according to claim 30, 31 or 32, which is a cell culture container.   33. A device according to claim 30, 31 or 32 which is a cell scaffold material.   34. The device of claim 33, wherein a cell culture surface is coated with the laminin E8.   The device according to any one of claims 30 to 35, which is used for culturing stem cells.   The device according to claim 36, wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell.   A method for producing a cultured cell, comprising culturing the cell with the device according to any one of claims 30 to 37.   40. The method of claim 38, wherein the cultured cells are stem cells.   40. The method of claim 38, wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell.

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