JP2019013176A - Method for producing glycoprotein - Google Patents

Abstract

To provide a technique that improves productivity of glycoprotein.SOLUTION: A fusion protein of a structure in which a peptide, found as a common sequence of a blood coagulation factor V and a blood coagulation factor VIII, is linked to a carboxy terminal side of a desired glycoprotein is expressed in a cultured mammalian cell, and a fusion protein is recovered from a culture medium.SELECTED DRAWING: None

Description

  The present invention relates to glycoprotein production technology. Specifically, the present invention relates to a production method for improving the production amount of glycoprotein and its use.

  In animal cells, most of the proteins localized on the surface and secreted extracellularly are glycoproteins. The sugar component (sugar chain) of glycoprotein is involved in various physiological phenomena such as cell proliferation, cell adhesion, antigen-antibody reaction, infection defense and the like. Thus, glycoproteins important in living organisms are used as pharmaceuticals, research reagents, and the like. Regarding the production of glycoproteins, production methods specialized for specific glycoproteins have been developed in addition to general-purpose production methods (see, for example, Patent Documents 1 to 3).

JP 2010-248228 A JP2014-156409A JP-A-2016-192936

Proc Natl Acad Sci U S A. 2010 Mar 2; 107 (9): 4034-9.doi: 10.1073 / pnas.0908526107. Epub 2010 Feb 8. Blood. 2004 May 1; 103 (9): 3412-9. Epub 2004 Jan 15.Bioengineering of coagulation factor VIII for improved secretion.

  In order to expand the use of glycoproteins that have important physiological functions and are important as pharmaceuticals, it is desirable to provide glycoproteins at low cost. The main object of the present invention is to provide a technique for improving the productivity of glycoprotein in order to meet such a demand.

While proceeding with research in view of the above problems, the present inventors focused on ERGIC-53 and MCFD2, which have been identified as causative gene products of blood coagulation factor V factor VIII deficiency (FVFVIIID). These molecules are known to function as cargo receptors that transport FV and FVIII from the endoplasmic reticulum to the Golgi apparatus by forming a complex (Non-patent Document 1). FV and FVIII transported to the ERGIC-53 / MCFD2 complex are glycoproteins having a common domain structure and modified to many N-linked sugar chains (FIG. 1). In addition to this, ERGIC-53 / MCFD2 complex has been reported to be FV due to the fact that 226 residues in the N-terminal region of the B domain are important for the secretion of FVIII (Non-Patent Document 2). Therefore, we tried to identify the common sequence (motif) from the N-terminal region of the B domain. As a result, a characteristic consensus sequence (SDLL (− / M) LL (K / R) QS) (SEQ ID NO: 2) of about 10 residues was found. The consensus sequence was predicted to be a part to which the ERGIC-53 / MCFD2 complex binds, and an experiment was conducted to demonstrate this. After detailed examination, it was found that the ERGIC-53 / MCFD2 complex bound to the sequence. On the other hand, it was revealed that when the glycoprotein is expressed in cultured cells so that the sequence is added to the C-terminal side, the secretion amount of the glycoprotein is remarkably increased. In addition, when a part of the sequence was changed, the same effect was observed, and further regularity of the sequence effective for improving the secretion amount (productivity) was found. As a result of further studies, when a glycoprotein was expressed using the sequence, a surprising phenomenon was observed in which not only the amount of secretion increased but also the amount of sialylated sugar chains increased. It was. Moreover, when another glycoprotein was expressed, the same effect was recognized, indicating that the sequence is highly versatile.
[1] A glycoprotein production method comprising the following steps (1) and (2):
(1) Peptide consisting of S (D / A) LL (-/ M) LL (K / R / A) QS (SEQ ID NO: 1) on the carboxy-terminal side of the target glycoprotein (however, (D / A) is D (Aspartic acid) or A (alanine), (-/ M) represents no amino acid residue or M (methionine), (K / R / A) represents K (lysine), Expressing a fusion protein having a structure in which R (arginine) or A (alanine) is linked in cultured mammalian cells;
(2) A step of recovering the fusion protein from the culture solution.
[2] The production method according to [1], wherein the sequence of the peptide is SDLLMLLRQS (SEQ ID NO: 2), SALLMLLRQS (SEQ ID NO: 3), SDLLMLLAQS (SEQ ID NO: 4), or SDLLLLKQS (SEQ ID NO: 5).
[3] The production method according to [1] or [2], wherein a protease recognition sequence is interposed between the target glycoprotein and the peptide.
[4] The production method according to [3], further comprising the following step (3):
(3) A step of treating the fusion protein after recovery with a protease.
[5] a promoter for mammalian cells;
And having a gene encoding the target glycoprotein,
Downstream of the gene is a peptide comprising S (D / A) LL (-/ M) LL (K / R / A) QS (SEQ ID NO: 1), where (D / A) is D (aspartic acid) Or A (alanine), (-/ M) represents no amino acid residue or M (methionine), (K / R / A) represents K (lysine), R (arginine) Or a sequence encoding A (alanine) is arranged,
A vector for producing glycoproteins.
[6] The vector according to [5], wherein a sequence encoding a protease recognition sequence is arranged between the gene and a sequence encoding the peptide.
[7] a promoter;
A cloning site for the gene encoding the target glycoprotein,
A peptide consisting of S (D / A) LL (-/ M) LL (K / R / A) QS (SEQ ID NO: 1) (provided that (D / A) is D (aspartic acid) downstream of the cloning site. ) Or A (alanine), (-/ M) represents no amino acid residue or M (methionine), (K / R / A) represents K (lysine), R (arginine) ) Or A (representing that it is alanine))
A vector for producing glycoproteins.
[8] The vector according to [7], wherein a sequence encoding a protease recognition sequence is arranged between the cloning site and a sequence encoding the peptide.
[9] A mammalian cell carrying the vector according to [5] or [6].
[10] The mammalian cell according to [9], wherein the mammalian cell is a human, mouse, rat or hamster cell.
[11] A kit for producing a glycoprotein, comprising the vector according to any one of [5] to [8].
[12] A peptide consisting of S (D / A) LL (-/ M) LL (K / R / A) QS (SEQ ID NO: 1) on the carboxy terminal side of the glycoprotein (provided that (D / A) is D ( Aspartic acid) or A (alanine), (-/ M-) represents no amino acid residue or M (methionine), (K / R / A) represents K (lysine), A gene encoding a fusion protein having a structure in which R (arginine) or A (alanine) is linked.
[13] The gene according to [12], wherein a sequence encoding a protease recognition sequence is interposed between a sequence encoding the target glycoprotein and a sequence encoding the peptide.
[14] A peptide consisting of S (D / A) LL (-/ M) LL (K / R / A) QS (SEQ ID NO: 1) on the carboxy terminal side of the glycoprotein (provided that (D / A) is D ( Aspartic acid) or A (alanine), (-/ M-) represents no amino acid residue or M (methionine), (K / R / A) represents K (lysine), A fusion protein having a structure in which R (arginine) or A (alanine) is linked.

Domain structure of blood coagulation factor 5 (FV) and factor 8 (FVIII). ¹ H- ¹⁵ N HSQC spectrum of [ ¹⁵ N] -FVIII peptide with addition of ERGIC-53 / MCFD2 complex. Comparison of NMR signal intensity before and after addition of ERGIC-53 / MCFD2 complex. The signal derived from the FVIII peptide showed a marked decrease in intensity before and after the addition of the ERGIC-53 / MCFD2 complex. Proline residues are indicated by P, and unassigned residues are indicated by *. Comparison of FVIII secretion. The number of specimens was 3, and a Dunnett's test with the FVIII secretion level in WT (wild type) as a control group was indicated by * for the group with p <0.01. The structure of the expressed protein. Erythropoietin (EPO) and EPO with added binding sequences were expressed. EPO-motif artificial gene base sequence. HindIII restriction enzyme site, gene encoding residues 28 to 193 of EPO, gene encoding TEV protease cleavage site, base sequence encoding MCFD2 binding sequence (SEQ ID NO: 3) of FVIII, stop codon, XbaI restriction enzyme site Arranged in order. Comparison of EPO secretion. The number of specimens was 3. The Dunnett's test using EPO secretion in WT (wild type) as a control group was indicated by * for the group with p <0.01. The structure of the expressed protein. EPO added with a binding sequence (SEQ ID NO: 3 to 6) and EPO added with a binding sequence (SEQ ID NO: 7) incorporating a mutation were expressed. Comparison of secretion amount of EPO-motif mutant. The control is wild-type EPO with no MCFD2 binding sequence added. SDLLMLLRQS (SEQ ID NO: 3) is a sequence derived from FVIII. In the sequence incorporating the mutation (SEQ ID NOs: 4 to 7), the mutation portion is underlined. The number of samples was 3, and the group of p <0.01 was indicated by * by Dunnett's test using the control as a control group. Comparison of secretion amount of sFcγRIIIa. The amount of protein obtained from the medium of HCT116 was evaluated by Western Butt. Results of Western blot and lectin blot. Results of Western blot and lectin blot.

In this specification, according to the convention, each amino acid is represented by one letter as follows.
Methionine: M, Serine: S, Alanine: A, Threonine: T, Valine: V, Tyrosine: Y, Leucine: L, Asparagine: N, Isoleucine: I, Glutamine: Q, Proline: P, Aspartate: D, Phenylalanine : F, glutamic acid: E, tryptophan: W, lysine: K, cysteine: C, arginine: R, glycine: G, histidine: H

1. TECHNICAL FIELD The present invention provides a method for producing a glycoprotein. The production method of the present invention enables efficient production of the target glycoprotein. The production method of the present invention is also useful as a means for obtaining a target glycoprotein having a high content of sialylated sugar chains. A sialylated sugar chain is a sugar chain having a monosaccharide having a carboxyl group at the 1-position, and is important in terms of pharmacokinetics of the glycoprotein and blood half-life. Glycoproteins with a high content of sialylated sugar chains are highly valuable or useful, for example, when they can be expected to enhance their efficacy when used as active ingredients in pharmaceuticals.

In the production method of the present invention, the following steps (1) and (2) are performed. In addition, the production method of the present invention is not a cell constituting an individual, but a cell isolated from an individual or a cell obtained by culturing, processing or the like (passage cell, Cell line etc.) and performed in vitro (ie in vitro).
(1) Peptide consisting of S (D / A) LL (-/ M) LL (K / R / A) QS (SEQ ID NO: 1) on the carboxy-terminal side of the target glycoprotein (however, (D / A) is D (Aspartic acid) or A (alanine), (-/ M) represents no amino acid residue or M (methionine), (K / R / A) represents K (lysine), A step of expressing in a cultured mammalian cell a fusion protein having a structure in which R (arginine) or A (alanine) is linked (2) a step of recovering the fusion protein from the culture solution

  Step (1) is a step of expressing a protein having a characteristic structure in a cultured cell, and uses a peptide (hereinafter referred to as “binding sequence peptide”) found by the study of the present inventors. The binding sequence peptide is a peptide of 9 or 10 amino acid residues consisting of S (D / A) LL (− / M) LL (K / R / A) QS (SEQ ID NO: 1). However, in the above sequence, (D / A) represents D (aspartic acid) or A (alanine), (-/ M) represents no amino acid residue or M (methionine), (K / R / A) represents K (lysine), R (arginine) or A (alanine).

Specific examples (Examples 1 to 4) of the binding sequence peptides are shown below. Example 1 is a peptide consisting of a sequence found in the B domain of FVIII (MCFD2 binding sequence). Examples 2 and 3 are peptides that have the same effect as the MCFD sequence, comprising a sequence obtained by mutating the MCFD2 binding sequence. Example 4 is a peptide consisting of a sequence found in the B domain of FV.
Example 1: SDLLMLLRQS (SEQ ID NO: 3). S (serine), D (aspartic acid), L (leucine), L (leucine), M (methionine), L (leucine), L (leucine), R (arginine), Q (glutamine) and S (serine) Are peptides linked in this order from the N-terminal side to the C-terminal side.
Example 2: SALLMLLRQS (SEQ ID NO: 4). S (serine), A (alanine), L (leucine), L (leucine), M (methionine), L (leucine), L (leucine), R (arginine), Q (glutamine) and S (serine) A peptide linked in this order from the N-terminal side to the C-terminal side.
Example 3: SDLLMLLAQS (SEQ ID NO: 5). S (serine), D (aspartic acid), L (leucine), L (leucine), M (methionine), L (leucine), L (leucine), A (alanine), Q (glutamine) and S (serine) Are peptides linked in this order from the N-terminal side to the C-terminal side.
Example 4: SDLLLLKQS (SEQ ID NO: 6). S (serine), D (aspartic acid), L (leucine), L (leucine), L (leucine), L (leucine), K (lysine), Q (glutamine) and S (serine) from the N-terminal side Peptides linked in this order toward the C-terminal side.

  A plurality of binding sequence peptides may be used (for example, 2 to 5 binding sequence peptides are linked in tandem). In this case, binding sequence peptides having different sequences may be used in combination.

  In step (1), a fusion protein having a structure in which a binding sequence peptide is linked to the carboxy terminus (C terminus) of the target glycoprotein is expressed. The target glycoprotein is a glycoprotein intended to be produced by the production method of the present invention, that is, a target product (expression product). Various glycoproteins (for example, those useful as components of pharmaceuticals, foods, research reagents, etc.) can be used as the target glycoprotein. Examples of target glycoproteins include cytokines (interferon (IFN), interleukin (IL)), hormones (eg erythropoietin (EPO), human chorionic gonadotropin (hCG), luteinizing hormone, follicle stimulating hormone, thyroid stimulating hormone) ), Enzymes (tissue-type plasminogen activator (t-PA), α-galactosidase, glucocerebrosidase), antibodies, antibody fragments, lactoferrin, ovalbumin, and thrombomodulin.

  In the fusion protein expressed in step (1), the C-terminus of the target protein and the binding sequence peptide are directly or indirectly linked. In the latter case, typically, one or more (for example, 2 to 31) amino acid residues are interposed between the target protein and the binding sequence peptide. As a mode in which a plurality of amino acid residues, that is, peptides are interposed, those in which the target protein and the binding sequence peptide are linked by a peptide linker, those in which a protease recognition sequence is provided between the target protein and the binding sequence peptide, the target protein and the binding sequence 1 to several amino acid residues (for example 1, 2, 3, 4, 5, 6, 7, 8, 9) that have no special function between peptides And those that intervene that do not impair the function / effect expected of the binding sequence peptide). A peptide linker is a linker consisting of a peptide in which amino acids are linked in a straight chain. A typical example of a peptide linker is a linker composed of glycine and serine (GGS linker or GS linker). On the other hand, the protease recognition sequence is an amino acid sequence that is recognized by a specific protease and necessary for cleavage of the protein by the protease. For example, sortase recognition sequence, HRV3C recognition sequence, TEV protease recognition sequence, thrombin recognition sequence, PreScission Protease recognition sequence, etc.) can be used.

  Unless the function / effect expected of the binding sequence peptide is impaired, the structure of the fusion protein may be designed so that one or a plurality of amino acid residues are linked to the C-terminus of the binding sequence peptide. In this case, apparently, a peptide having a structure in which another amino acid residue (for example, 1 to 22 amino acid residues) is linked to the C-terminus of the binding sequence peptide is linked to the target protein.

In order to express a fusion protein in a cultured mammal, typically, a host (mammalian cell) transformed with an expression vector carrying a gene encoding the fusion protein (fusion protein gene) is cultured. Specifically, for example, the fusion protein is expressed by the following steps (i) to (iii).
(I) a step of preparing an expression vector holding a gene encoding a fusion protein (ii) a step of introducing the expression vector into a host cell (iii) a transformant having the expression vector introduced therein is cultured and the fusion Protein expression step

  Step (i) is a step characteristic of the present invention, and an expression vector that enables expression of the fusion protein in a host cell is prepared. The expression vector incorporates a promoter that functions in the host cell so that the fusion protein can be expressed in the host cell. Examples of promoters include CMV-IE (cytomegalovirus early gene-derived promoter), SV40, EF1α, RSV, SRα, β actin promoter, and the like. A promoter is operably linked to the fusion protein gene. Here, “the promoter operably links the fusion protein gene” is synonymous with “the fusion protein gene is placed under the control of the promoter” and is usually on the 3 ′ end side of the promoter. The fusion protein gene will be linked directly or via other sequences. A poly A addition signal sequence is arranged downstream of the fusion protein gene. Transcription is terminated by the use of a poly A addition signal sequence. As the poly A addition signal sequence, SV40 poly A addition sequence, bovine-derived growth hormone gene poly A addition sequence, and the like can be used.

  The expression vector may contain a detection gene (reporter gene, cell or tissue-specific gene, selection marker gene, etc.), enhancer sequence, WRPE sequence, and the like. The detection gene is used for determination of success or efficiency of the introduction of the fusion protein gene, detection of fusion protein expression or determination of expression efficiency, selection and sorting of cells in which the fusion protein gene is expressed, and the like. On the other hand, the use of an enhancer sequence can improve the expression efficiency. The detection gene includes a neo gene conferring resistance to neomycin, an npt gene conferring resistance to kanamycin and the like (Herrera Estrella, EMBO J. 2 (1983), 987-995) and nptII gene (Messing & Vierra. Gene 1 9: 259-268 (1982)), hph gene conferring resistance to hygromycin (Blochinger & Diggl mann, Mol Cell Bio 4: 2929-2931), dhfr gene conferring resistance to metatrexate (Bourouis et al. , EMBO J.2 (7)), etc. (above, marker gene), luciferase gene (Giacomin, P1. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), β -Glucuronidase (GUS) gene, GFP (Gerdes, FEBS Lett. 389 (1996), 44-47) and its variants (EGFP, d2EGFP, etc.) and other fluorescent protein genes (reporter genes), intracellular domains Using genes such as the epidermal growth factor receptor (EGFR) gene You can. The detection gene can be linked to the fusion protein gene via, for example, a bicistronic regulatory sequence (for example, a ribosome internal recognition sequence (IRES)) or a sequence encoding a self-cleaving peptide. An example of a self-cleaving peptide is 2A peptide (T2A) derived from Thosea asigna virus, but is not limited thereto. Known as self-cleaving peptides are 2A peptide (F2A) from horseshoe disease virus (FMDV), 2A peptide (E2A) from equine rhinitis virus A (ERAV), 2A peptide (P2A) from porcine teschovirus (PTV-1), etc. It has been.

  Fusion protein genes are roughly divided into a portion encoding a target glycoprotein and a portion encoding a binding sequence peptide. As the sequence encoding the binding sequence peptide, various sequences can be adopted as long as it encodes the binding sequence peptide. For example, when the binding sequence peptide is SDLLMLLRQS (SEQ ID NO: 3), the agcgatctgctgatgctgctgagacagagc sequence (SEQ ID NO: 8) or the like can be used.

  The sequence encoding the target glycoprotein and the sequence encoding the binding sequence peptide are linked in-frame directly or via another sequence (for example, a sequence encoding a peptide linker or a sequence encoding a protease recognition sequence).

The expression vector having the above-described configuration is constructed by, for example, any one of the following methods (a) to (c).
(A) Method for inserting the fusion protein gene of the present invention into a mammalian cell expression vector (b) For a mammalian cell expression vector in which a sequence encoding the binding sequence peptide of the present invention is incorporated A method of inserting a sequence encoding the target glycoprotein in frame upstream of the sequence; (c) a mammalian cell expression vector retaining a sequence encoding the target glycoprotein; Method for inserting the sequence encoding the binding sequence peptide of the invention in frame

  The construction method (a) is a construction method using a vector having a cloning site in addition to elements necessary for expression such as a promoter. The cloning site is used for insertion of a gene encoding the target glycoprotein. A multi-cloning site may be provided as a cloning site. By inserting the fusion protein gene of the present invention into the cloning site, an expression vector used in the production method of the present invention is completed. One of the advantages of this construction method is that a commercially available general-purpose vector can be used.

  In the construction method (b), a vector in which a sequence encoding the binding sequence peptide of the present invention is incorporated in advance is used. A cloning site for inserting a sequence encoding the target glycoprotein in frame is provided upstream (5 ′ end side) of the sequence encoding the binding sequence peptide of the present invention. An expression vector used in the production method of the present invention is completed by inserting a sequence encoding the target glycoprotein into the cloning site. In the case of this construction method, it is not necessary to prepare a sequence encoding the target glycoprotein linked with the binding sequence peptide of the present invention (that is, the sequence encoding the target glycoprotein can be used as it is). Therefore, it can be said to be particularly effective when, for example, various types of target glycoproteins are expressed.

  Unlike the above two construction methods, the construction method (c) utilizes a vector that holds a sequence encoding the target glycoprotein in advance. In this construction method, the expression vector used in the production method of the present invention is completed by inserting the sequence encoding the binding sequence peptide of the present invention in-frame downstream of the sequence encoding the target glycoprotein. The insertion of the sequence encoding the binding sequence peptide of the present invention allows the sequence encoding the target glycoprotein and the sequence encoding the binding sequence peptide to be linked in frame. This construction method is used when a vector carrying a sequence encoding a glycoprotein of interest is available (for example, when it is already owned (constructed or obtained) or easily available). It is particularly effective.

  In the construction method (a), a fusion protein gene in which a sequence encoding a protease recognition sequence is interposed between a sequence encoding a target glycoprotein and a sequence encoding a binding sequence peptide of the present invention is used as an insertion sequence. Thus, in the construction method of (b), for example, a mammalian cell in which a sequence encoding a protease recognition sequence is arranged upstream of the sequence encoding the binding sequence peptide of the present invention (the cloning site is provided further upstream). By using the expression vector, in the construction method (c), for example, by inserting a sequence encoding a protease recognition sequence together with the binding sequence peptide of the present invention, the sequence encoding the target glycoprotein and the binding sequence An expression product in which a protease recognition sequence is interposed between the peptide coding sequence and The order of the expression vector.

  Many expression vectors that can be used in mammalian cell expression systems have been developed, and the expression vector of the present invention can be constructed using existing expression vectors.

  The expression vector prepared in step (i) is introduced into a host mammalian cell (step (ii)). The introduction operation may be performed by a conventional method. Various gene transfer methods can be used for introducing the expression vector into the host mammalian cell. For example, infection (when using viral vectors), electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984)), lipofection (Felgner, PL et al , Proc. Natl. Acad. Sci. USA 84, 7413-7417 (1987)) and the like.

The host mammalian cell is not particularly limited as long as it is a mammalian cell capable of expressing a secreted protein. For example, CHO cell, BHK cell, COS-7 cell, HeLa cell, Namalva cell, HEK293 cell, HCT116 cell, Jurkat Cells, HL-60 cells, PC-12 cells, A431 cells, U2OS cells, K-562 cells, Expi293F ^™ cells and the like can be used. The animal species of the host mammalian cell is, for example, human, mouse, rat, hamster.

  In step (iii) following step (ii), the transformed mammalian cell (transformant) into which the expression vector has been introduced is cultured to express the fusion protein. The culture conditions are not particularly limited as long as the transformant grows and the fusion protein can be expressed. Corrections may be made as necessary based on standard culture conditions. Moreover, it is possible to set appropriate culture conditions by preliminary experiments.

  The composition of the medium is not particularly limited. For example, maltose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses, organic acid and the like can be used as the carbon source of the medium. Further, as the nitrogen source, for example, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, peptone, yeast extract, corn steep liquor, casein hydrolyzate, bran, meat extract and the like can be used. As the inorganic salt, potassium salt, magnesium salt, sodium salt, phosphate, manganese salt, iron salt, zinc salt and the like can be used. In order to promote the growth of transformants, a medium supplemented with vitamins, amino acids and the like may be used.

  After expressing the fusion protein, the fusion protein is recovered from the culture solution (step (2)). For example, after removing insolubles by filtering, centrifuging the culture supernatant, etc., separation and purification are performed by appropriately combining concentration with an ultrafiltration membrane, salting out such as ammonium sulfate precipitation, dialysis, and various chromatography. As a result, a fusion protein can be obtained.

  According to the production method of the present invention, a fusion protein having a characteristic structure in which the binding sequence peptide of the present invention is linked to the C-terminal side of the target glycoprotein can be obtained. In one embodiment, the expression product in a state in which the target glycoprotein and the binding sequence peptide of the present invention are directly linked (that is, no other amino acid / amino acid sequence is interposed between the sequence of the target glycoprotein and the binding sequence peptide of the present invention). Is obtained. When a protease cleavage site is used (that is, when an expression vector incorporating a sequence encoding a protease recognition sequence is used), the protease recognition sequence is between the target glycoprotein sequence and the binding sequence peptide of the present invention. An expression product in a state mediated by is obtained. In this embodiment, the target glycoprotein from which the binding sequence peptide has been cleaved (separated) can be recovered by subjecting the expression product to prosthesis. For protease treatment, a protease corresponding to the protease cleavage site used is used.

2. Expression Vectors and Kits Expression vectors (particularly those having a cloning site) used in the production method of the present invention are excellent in versatility and have high utility and utility value. Then, this invention provides the expression vector which can be used for the production method of this invention as another situation. The structure of the expression vector of the present invention is as described above, and comprises elements necessary for the expression of the target glycoprotein in a mammalian cell expression system.

  The present invention further provides a kit for producing a target glycoprotein comprising the expression vector of the present invention. According to the kit, the target glycoprotein can be more easily produced using the production method of the present invention. The kit of the present invention contains the above expression vector as a main component. Other reagents, containers / instruments, culture media and the like necessary for carrying out the expression method of the present invention may be included in the kit of the present invention. Usually, an instruction manual is attached to the kit of the present invention.

3. Recombinant protein According to the production method of the present invention, a fusion protein having a characteristic structure is obtained. As described above, glycoproteins to which the production method of the present invention can be applied are not particularly limited, and various target glycoproteins can be produced. If a protease recognition site is incorporated between the target glycoprotein and the binding sequence peptide, it is possible to cleave (separate) the binding sequence peptide portion by, for example, purifying, or fractionating or fractionating the protease. is there.

  ERGIC-53 and MCFD2, which have been identified as the causative gene product of blood coagulation factor V factor VIII deficiency (FVFVIIID), form a complex, and as a cargo receptor that transports FV and FVIII from the endoplasmic reticulum to the Golgi apparatus It is functioning. FV and FVIII transported to the ERGIC-53 / MCFD2 complex are glycoproteins having a common domain structure and modified to many N-linked sugar chains (FIG. 1). The ERGIC-53 / MCFD2 complex is a sequence common to FV and FVIII because it is reported that 226 residues in the N-terminal region of the B domain are important for the secretion of FVIII (Non-patent Document 2). May have recognized. Thus, when the N-terminal region of the B domain was searched in detail, a characteristic common sequence (SDLL (-/ M) LL (K / R) QS) (SEQ ID NO: 2) of about 10 residues was found. . The consensus sequence was predicted to be a part to which the ERGIC-53 / MCFD2 complex binds, and the following experiment was conducted.

1. Binding of ERGIC-53 / MCFD2 complex to common sequence ERGIC-53 against ¹⁵ N-labeled factor VIII peptide NATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDD (SEQ ID NO: 9) containing the targeted 10-residue peptide SDLLMLLRQS (SEQ ID NO: 3) / MCFD2 complex was added and contacted. The NMR spectrum before and after the addition is shown in FIG. When the signal intensity before and after the addition of the ERGIC-53 / MCFD2 complex was compared, the signal intensity derived from the 10 target residues was significantly reduced (FIG. 3). In other words, it was revealed that the ERGIC-53 / MCFD2 complex mainly binds to these 10 residues.

2. Effect of loss of binding sequence (common sequence) on transport by ERGIC-53 / MCFD2 complex HCT116 wild-type (WT), ERGIC-53-deficient cells (ΔERGIC-53) and MCFD2-deficient cells of colon cancer cells (ΔMCFD2) is overexpressed with a deletion mutant (FVIIIΔmotif) obtained by removing SDLLMLLRQS (SEQ ID NO: 3) which is MCFD2 binding sequence from FVIII or FIII, and using VisuLize ^™ FVIII Antigen Kit (Affinity Biologicals Inc., Canada) The amount of FVIII secreted into the medium was evaluated. As a result, in ERGIC-53 and MCFD2 knockout cells, the amount of FVIII secretion decreased (Fig. 4). This result has already been reported. EGFIC secretion requires ERGIC-53 and MCFD2. This is consistent with the fact that Moreover, the secretion amount of FVIIIΔmotif decreased in WT cells compared with FVIII. The decrease in the secretion amount accompanying this MCFD2-binding sequence deficiency is in good agreement with the decrease in the secretion amount of FVIII in each knockout cell. This suggests that the transport by the ERGIC-53 / MCFD2 complex became impossible due to the lack of the MCFD2 binding sequence.

3. Changes in secretory glycoprotein secretion by adding binding sequences
If ERGIC-53 and MCFD2 recognize and transport N-linked sugar chains and MCFD2 recognition sequences, respectively, add a common sequence to other glycoproteins with N-linked sugar chains. We thought that it could be transported by -53 / MCFD2 complex. Therefore, erythropoietin (EPO) was used as a model glycoprotein to verify this possibility. Specifically, EPO or EPO-motif (Fig. 5) is overexpressed in HCT116 wild type, ERGIC-53 deficient strain (ΔERGIC-53), MCFD2 deficient strain (ΔMCFD2) cells, and EPO is added to the culture medium by ELISA. The amount of secretion was measured. The outline of the experimental method is shown below.

  A DNA fragment (FIG. 6, SEQ ID NO: 10) of EPO and MCFD2-binding sequence fusion EPO (EPO-motif) was prepared using a gene encoding residues 28 to 193 of EPO as a template, and N-terminal 3 × Flag Each expression plasmid was constructed by incorporating the restriction enzyme HindIII / XbaI into a CMV9 vector (Sigma-Aldrich Co.). The expression plasmid was transfected into HCT116 cells. After culturing for 48 hours, the medium was collected.

  Interestingly, in WT cells, the amount of EPO-motif was increased compared to EPO. Moreover, in each knockout cell, even when the MCFD2 binding sequence was added, the increase in the secreted amount of EPO in the WT cell was not observed (FIG. 7). From this, it is assumed that by adding the MCFD2 binding sequence, EPO was transported by the ERGIC-53 / MCFD2 complex and the secretion amount was increased.

4). Examination of sequence strictness / regularity of connected sequences
Three types of sequences in which mutations were incorporated into the MCFD2-binding sequence were prepared, EPO with these added was overexpressed in HCT116 cells, and the amount secreted into the medium was measured by ELISA. By using the EPO-motif expression plasmid prepared in the above experiment as a template, an EPO (EPO-motif variant 1, 2, 3) expression plasmid added with a sequence incorporating a mutation into the MCFD2-binding sequence was used. It was constructed.

  In the case of EPO (FIG. 8) to which SALLMLLRQS (SEQ ID NO: 4) or SDLLMLLAQS (SEQ ID NO: 5) was added, an increase in the secreted amount of EPO was observed as in the case where the MCFD2-binding sequence of FVIII was added. On the other hand, in the case of EPO to which SDAAAAARQS (SEQ ID NO: 7) was added (FIG. 8), an increase in secretion amount was not observed as compared with wild-type EPO to which MCFD2-binding sequence was not added (FIG. 9). ).

5. Difference in effect depending on the position of the binding sequence MCFD2 binding sequence (SEQ ID NO: 3) or the region containing MCFD2 binding sequence (786-838) (SEQ ID NO: 11) for soluble FcγRIIIa (sFcγRIIIa) An expression plasmid linked to the ends was prepared. When a series of expression plasmids were expressed in HCT116 cells, an increase in the expression level was observed when MCFD2 binding sequence or a region containing MCFD2 binding sequence (786-838) was added to the C-terminus of sFcγRIIIa (FIG. 10). ). From these results, it is considered that the addition of MCFD2 binding sequence to the C-terminal side is necessary for increasing the expression level.

6). Effect of Binding Sequence on Glycosylation As described above, it was shown that the addition of MCFD2 binding sequence increases EPO secretion. It is thought that the secretion amount was increased by transporting EPO to the ERGIC-53 / MCFD2 complex. In order to further investigate the function of the MCFD2-binding sequence, the effect on glycosylation was examined. Specifically, we focused on the N-linked sugar chain of EPO and performed a sugar chain analysis.

Western blotting and lectin blotting were performed on the purified protein by overexpressing EPO or EPO (EPO-motif) added with an MCFD2-binding sequence in Expi293F ^TM cells. Western blotting using an anti-FLAG antibody confirmed that EPO and EPO-motif were expressed. In the lectin blot using Con A, almost the same signal intensity was detected, indicating that the addition of the MCFD2 binding sequence had little effect on the high-mannose sugar chain. In the lectin blot using MAM, the EPO-motif detected a significantly stronger signal compared to EPO (FIG. 11). Since MAM has a high affinity for the sugar chain to which sialic acid is added, it was suggested that the addition of the MCFD2 binding sequence increased the number of sialylated sugar chains. That is, it has been clarified that the addition of the MCFD2 binding sequence brings not only an increase in glycoprotein expression but also an unexpected effect such as an increase in sialylated sugar chains. Similar effects were observed for FcR (FIG. 12).

  According to the present invention, glycoproteins used as medicines, foods, research reagents and the like can be efficiently produced. In particular, the present invention is expected to be used and applied to the production of glycoproteins containing sialylated sugar chains.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

SEQ ID NO: 1: description of artificial sequence: binding sequence peptide SEQ ID NO: 2: description of artificial sequence: common sequence peptide SEQ ID NO: 4: description of artificial sequence: binding sequence peptide SEQ ID NO: 5: description of artificial sequence: binding sequence peptide sequence number 7: description of artificial sequence: mutation motif SEQ ID NO: 8: description of artificial sequence: sequence encoding binding sequence peptide SEQ ID NO: 10: description of artificial sequence: artificial gene of EPO-motif

Claims (14)

A method for producing a glycoprotein comprising the following steps (1) and (2):
(1) Peptide consisting of S (D / A) LL (-/ M) LL (K / R / A) QS (SEQ ID NO: 1) on the carboxy-terminal side of the target glycoprotein (however, (D / A) is D (Aspartic acid) or A (alanine), (-/ M) represents no amino acid residue or M (methionine), (K / R / A) represents K (lysine), Expressing a fusion protein having a structure in which R (arginine) or A (alanine) is linked in cultured mammalian cells;
(2) A step of recovering the fusion protein from the culture solution.   The production method according to claim 1, wherein the sequence of the peptide is SDLLMLLRQS (SEQ ID NO: 2), SALLMLLRQS (SEQ ID NO: 3), SDLLMLLAQS (SEQ ID NO: 4) or SDLLLLKQS (SEQ ID NO: 5).   The production method according to claim 1 or 2, wherein a protease recognition sequence is interposed between the target glycoprotein and the peptide. The production method according to claim 3, further comprising the following step (3):
(3) A step of treating the fusion protein after recovery with a protease. A promoter for mammalian cells;
A gene encoding a target glycoprotein,
Downstream of the gene is a peptide comprising S (D / A) LL (-/ M) LL (K / R / A) QS (SEQ ID NO: 1), where (D / A) is D (aspartic acid) Or A (alanine), (-/ M) represents no amino acid residue or M (methionine), (K / R / A) represents K (lysine), R (arginine) Or a sequence encoding A (alanine) is arranged,
A vector for producing glycoproteins.   The vector according to claim 5, wherein a sequence encoding a protease recognition sequence is arranged between the gene and a sequence encoding the peptide. A promoter for mammalian cells;
A cloning site for the gene encoding the target glycoprotein,
A peptide consisting of S (D / A) LL (-/ M) LL (K / R / A) QS (SEQ ID NO: 1) (provided that (D / A) is D (aspartic acid) downstream of the cloning site. ) Or A (alanine), (-/ M) represents no amino acid residue or M (methionine), (K / R / A) represents K (lysine), R (arginine) ) Or A (representing that it is alanine))
A vector for producing glycoproteins.   The vector according to claim 7, wherein a sequence encoding a protease recognition sequence is arranged between the cloning site and a sequence encoding the peptide.   Mammalian cells carrying the vector according to claim 5 or 6.   The mammalian cell according to claim 9, wherein the mammalian cell is a human, mouse, rat or hamster cell.   A kit for producing a glycoprotein comprising the vector according to any one of claims 5 to 8.   Peptide consisting of S (D / A) LL (-/ M) LL (K / R / A) QS (SEQ ID NO: 1) on the carboxy terminal side of glycoprotein (However, (D / A) is D (aspartic acid) Or A (alanine), (-/ M-) represents no amino acid residue or M (methionine), (K / R / A) represents K (lysine), R (arginine) ) Or A (representing that it is A (alanine))).   The gene according to claim 12, wherein a sequence encoding a protease recognition sequence is interposed between a sequence encoding the target glycoprotein and a sequence encoding the peptide.   Peptide consisting of S (D / A) LL (-/ M) LL (K / R / A) QS (SEQ ID NO: 1) on the carboxy terminal side of glycoprotein (However, (D / A) is D (aspartic acid) Or A (alanine), (-/ M-) represents no amino acid residue or M (methionine), (K / R / A) represents K (lysine), R (arginine) ) Or A (representing that it is alanine)).

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