KR20150042936A - Mannosylphosphorylation Reaction Using YlMpo1 Protein - Google Patents

Abstract

The present invention relates to a mannose phosphorylation using YlMpo1 protein and, more specifically, a method for adding mannose-6-phophate to a sugar chain using recombinant microorganisms having a capacity for producing YlMpo1 and YlMpo1 and a manufacturing method of sugar protein or useful compounds having sugar chain with attached mannose-6-phophate by using the method wherein the method for adding mannose-6-phophate to the sugar chain is allowed to efficiently add mannose phosphate to the sugar protein used as an enzyme treatment agent in vitro human body and is accordingly used for manufacturing various enzyme treatment agents in an yeast whose biosynthetic paths of an animal cell or a sugar chain are redesigned compared with that of directly manufacturing an yeast treatment agent with attached mannose-6-phophate sugar chain, thereby being used for delivering useful materials to cytoplasm or nucleus except for lysosome by combining sugar proteins, peptides for escaping the mannose-6-phophate sugar chain from the inside, lipid or nanomaterials.

Description

Mannosylphosphorylation Reaction Using YlMpo1 Protein}

The present invention relates to a mannose phosphorylation reaction using YlMpo1 protein, more specifically, a method for adding mannose-6-phosphate to a sugar chain using a recombinant microorganism having YlMpo1 production ability and YlMpo1, a method for producing mannose- To a method for producing a glycoprotein or a useful compound to which the added sugar chain is attached.

Glycoproteins, which are glycosylated through glycosylation, are now leading the market, accounting for more than 60% of the global recombinant protein drug market. In particular, oligosaccharides play an important role in the therapeutic efficacy of glycoprotein drugs, persistence in the body, targeting, and immune response, and thus are becoming important factors in determining drug quality. The addition reaction in which sugar chain is largely N - O bond or a - combination glycosylation and two types of (N -linked or O -linked glycosylation) , the of the N - a sugar chain which is added via the binding glycosylation N - oligosaccharide ( N- glycan) and begins in the endoplasmic reticulum. First, Glc ₃ Man ₉ GlcNAc ₂ -PP-Dol, a glycoconjugate in the form linked to dolichol pyrophosphate (PP-Dol) present in the membrane of the endoplasmic reticulum, is formed. Oligosaccharyltransferase then transfers it to the N -glycosylation sequence of the NXS / T sequence among the proteins that are translated into ribosomal to endoplasmic reticulum by a co-translational translocation mechanism. Glucosidase and mannosidase eliminate the glucose and mannose at the end by the mechanisms responsible for the quality control of the glycoprotein folding in the endoplasmic reticulum, resulting in a Man ₈ GlcNAc ₂ structure (High-mannose type) oligosaccharide having a Golgi apparatus attached to the Golgi apparatus.

The initial biosynthetic process of the N - glycoprotein in the above - described ER is preserved in almost the same process from the eukaryotic microorganism yeast to the mammal, the higher animal. However, the oligosaccharide transferred to the Golgi is subjected to a variety of oligosaccharide modification processes specifically for each species, and as a result, completely different types of oligosaccharides are produced in yeast, insects, plants and animals. In higher animals, oligosaccharides of the glycoprotein that enter the Golgi are polished to the Man ₅ GlcNAc ₂ form by mannosidase. After N- acetylglucosaminyltransferase (GNT) I was added, GlcNAc was added, Mannocidase II was added to remove 2 mannos added to the opposite branch, and trimannosyl core (Man ₃ GlcNAc ₂ ) A mixed structure in which GlcNAc is added is made. GNT II acts on the branches where mannose has been removed, and GlcNAc is added to produce oligosaccharides with two antenna structures. After that, GNT IV and V works to form four antenna structures. In some cases, GNT VI, IX, or VB works to form six antenna structures. After the addition of GlcNAc, the β-galactosyltransferase and α-sialyltransferase present in the Golgi after action of the antenna make a complex type sugar chain structure in which galactose and sialic acid are added on GlcNAc.

In animal cells, many glycoproteins have the complex type N - oligosaccharide as described above. However, depending on the type of the glycoprotein, there are cases in which the oligosaccharide type oligosaccharide is introduced from the endoplasmic reticulum. In addition, the glycoproteins that go to lysosomes with digestion and autolysis to digest and recycle unnecessary substances are sometimes added to mannan-6-phosphate in the gonadotropic oligosaccharide. In this case, N- acetylglucosamine-1-phosphotransferase (GIcNAc-PT) recognizes the tertiary structure of glycoproteins migrating to lysosomes in Golgi, and GlcNAc-phosphate is added to the mannose residue of the oligosaccharide (FIG. Then, mannose-6-phosphate is formed in a two-step process in which an uncovering enzyme ( N- acetylglucosamine-1-phosphodiester a- N- acetylglucosaminidase) acts to remove only the outer GlcNAc residue. The mannose-6-phosphate thus formed is recognized by a cation-dependent mannose-6-phosphate receptor (CD-MPR) and is transferred to the lysosome through the endosome. However, at this time, the glycoproteins that do not bind to CD-MPR are secreted out of the cell. There is a " secretion-recapture " pathway in which these secreted glycoproteins are again transferred to lysosomes through endocytosis, and mainly consists of cation-independent mannose-6- phosphate receptor, CI-MPR) is known to function.

Lysosomal storage disease is a disease caused by accumulation of metabolites due to deficiency of the activity of lysosomal degrading enzyme due to congenital gene abnormality. Patients born with these diseases are most likely to die at a young age if they are not treated. Currently the only approved method of treatment is enzyme replacement therapy, which injects normal enzymes externally. Genzyme of the United States has developed Cerezyme for the treatment of Gaucher disease as the first enzyme treatment in 1998 and has since developed Fabrazyme, Aldurazyme and Myozyme, Which is a unique area.

Although the enzyme treatment targets a small number of patients with genetic metabolic diseases, it is a large market with a total size of over 2.5 trillion won due to the high price. However, in order to target the enzyme treatment to lysosome, Due to the high technical barriers that need to be optimized, a few companies are showing an unusual structure that monopolizes the global market.

Cerezyme, a treatment for Gaucher disease, is a human glucocerebrosidase produced in CHO (Chinese hamster ovary) cells, which is produced by sequential exoglycosidase enzymatic reactions and trimannosyl core Oligosaccharide. Glucocerebrosidase with this oligosaccharide is taken into the cell through the mannose receptor present on the surface of the trophoblastic cells in question and then transferred to the lysosome. On the other hand, other enzyme therapeutic agents except cerezyme have the above-described mannose-6-phosphate-added oligosaccharide and migrate to intracellular lysosomes through the mannose-6-phosphate receptor (Fig. 1).

That is, when a recombinase having a mannose-6-phosphate oligosaccharide is administered to patients, CI-MPR, which is a mannose-6-phosphate receptor in the above-mentioned "secretory- To metabolize the accumulated metabolites instead. Therefore, it is important in quality control to allow a large amount of mannose-6-phosphate sugar chain to be added in an enzyme therapeutic agent.

Yeast is a eukaryotic microorganism that is easy to manipulate because it is eukaryotic microorganism. It is easy to handle and can produce large amount of proteins at low cost, which not only makes it economical, but also has many advantages such that there is no possibility of contamination with viruses and plasters that infect humans. . However, the oligosaccharide structure synthesized in yeast is very different from that of human, which causes an immune response upon human body injection. The yeast has the same oligosaccharide biosynthetic process as the higher animal to the endoplasmic reticulum but has a yeast-specific oligosaccharide modification pathway in which mannose is further added after golgi migration. That is, a glycan outer chain initiation reaction occurs in which mannose is added to the Man ₈ GlcNAc ₂ oligosaccharide transferred from the endoplasmic reticulum by the OCH1 gene product through α (1,6) -binding, and α (1,6) -Bonded and? (1,2) -bonded mannose is continuously added to synthesize a sugar chain outer chain. Traditional yeast Saccharomyces ( Saccharomyces) In the case of S. cerevisiae , 50-200 mannose is continuously added to the core sugar chain, and α (1,3) -mannoscemia , which can be recognized as an antigen in the human body, is added by the MNN1 gene product . It has also been found that acidic oligosaccharides are produced by mannosylphosphorylation, in which mannose-1-phosphate is additionally added to the oligosaccharide. It is caused by mannose phosphorylation by an enzyme expressed by MNN6 gene (Wang et < RTI ID = 0.0 > al , J Biol Chem. , 272: 18117, 1997), MNN4 has been proposed as a gene expressing a protein that controls it (Odani et al ., Glycobiol ., 6: 805, 1996; Odani et al ., FEBS Lett ., 420: 1860,1997).

As described above, the oligosaccharide attached to the glycoprotein produced by the yeast is different from that of the human, so that the oligosaccharide can not be used for medicinal purposes because it causes an immune reaction upon injection of the human body. Therefore, Methods for disrupting the glycosyltransferase genes have been proposed. In particular, the yeast S. cerevisiae OCH1 gene and α (1,3) for initiating a oeswae sugar chain extension reaction - such as by crushing the MNN1 gene of adding mannose presented are methods to prevent the addition of yeast-specific sugar chain (Nakayama et al ., EMBO J., 11: 2511, 1992; Nakanishi-Shindo et al ., J Biol Chem. , 268: 26338, 1993).

In addition, the above-mentioned MNN4 And mannose-6-phosphate-1-mannose forms in which MNN6 is formed by adding mannose phosphoric acid to the mannose residue may also cause an immune response upon human body injection, For the production of glycoproteins, it must be removed. In the case of the enzyme treatment, a mannose-6-phosphate-added sugar chain is required, so that a technique for forming a mannose-6-phosphate sugar chain by removing the outer mannose from the mannose-6-phosphate-1-mannose structure has been developed.

Japan, AIST (National Institute of Advanced Industrial Science and Technology) group in order to express the enzyme treatments in yeast disrupted genes such as OCH1 and MNN1 and to overexpress MNN4 induced more mannose phosphorylation (Chiba et al., Glycobiol , 12: 821, 2002; Akeboshi et al ., Glycobiol , 19: 1002), Korean Patent No. 0888316 discloses that mannose-6-phosphate is formed by treating outer mannose by treatment with a-mannosidase derived from the culture medium of Cellulomonas bacteria . ≪ / RTI >

In this technique, the enzyme having uncapping activity to remove the outer mannose can not be identified to the molecular level, but an enzyme-active solution partially purified from the culture medium was used. However, Callewaert's team of Ghent University in Belgium reported that the outer part of the cellulosimicrobium cellulans The enzyme CcGH92_5, which can form mannose-6-phosphate, was isolated by removing the mannose (Tiels et al ., Nature Biotech , 30: 1225, 2012), recombinantly expressing it and producing Yarrowia lipolytica ) yeast to treat mannose-6-phosphate.

In addition to the traditional yeast S. cerevisiae , genes related to the addition reaction of mannose phosphate were also found in various yeasts, including the methanol-induced yeast Pichia pastoris . In Japan, Miura et al . Found the PNO1 gene of P. pstoris , which plays an important role in the addition of mannose phosphate using the MNN4 gene sequence of S. cerevisiae as a probe, and reported that the mannose phosphorylation can be controlled by disrupting the PNO1 gene Miura et al . , Gene , 324: 129, 2004). However, the US GlycoFi company found that the addition of mannose phosphate could be inhibited to some extent by the disruption of the PNO1 gene, but it could not be completely eliminated (US Patent No. 07259007). In P. pastoris , Mnn4 protein of S. cerevisiae , And named MNN4A , MNN4B and MNN4C , respectively. We also showed that double-disruption of MNN4B gene with PNO1 completely controls the addition of mannose phosphate.

The present inventors have found a gene having high sequence similarity to MNN4 of S. cerevisiae from Y. lipolytica, which is a heterozygous yeast, and discovered that the addition of mannose phosphate is completely controlled by disrupting the gene in Y. lipolytica yeast Patent No. 0915670). This gene of Y. lipolytica , which is similar in sequence to MNN4 of S. cerevisiae , was named YlMPO1 ( Y. lipolytica Mannosyl Phosphorylation of Oligosaccharide) as a novel gene that can completely control mannose phosphorylation by disruption of a single gene. Furthermore, further studies have shown that YlMPO1 plays a key role in mannose phosphorylation of O - glycosylation as well as N - glycosylation , all of which are carried out singly without the help of MNN6 gene (Park et al, Appl Env Microbiol, 77: 1187, 2011).

Therefore, the present inventors have inferred that YlMpo1 is an enzyme responsible for direct mannose phosphorylation, and as a result of efforts to perform the mannose phosphorylation reaction using the enzyme, a recombinant microorganism having YlMpo1 production ability was produced and YlMpo1 was subjected to mannose phosphorylation 6-phosphate-1-mannose was added to the oligosaccharide, and the outer mannose of the oligosaccharide to which the resulting mannose-6-phosphate-1-mannose was added was subjected to weak acid hydrolysis or uncapping (Mannose-6-phosphate) was formed when the enzyme was removed through the enzyme-catalyzed reaction.

In order to achieve the above object,

A recombinant microorganism having the ability to produce YlMpo1, wherein a gene encoding YlMpo1 or a recombinant vector containing the gene is introduced into a host cell.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, the YlMpo1 may be Yarrowia < RTI ID = 0.0 > lipolytica , wherein YlMpo1 is YlMpo1 represented by the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8.

The National Center for Biotechnology Information (NCBI) has determined the genomic sequence of the Y. lipolytica CLIB122 strain. The base and amino acid sequences corresponding to YlMpo1 are described in XM_503217 and XP_503217 and are described in the homologue of Mnn4 of Candida albicans . The present inventors found that YlMpo1 has 47 amino acid sequences (141 nucleotide sequences) at the N-terminus than the sequence described in XP_503217 of NCBI in the prior art and described in the patent (Korean Patent No. 0915670) (SEQ ID NO: 1 base sequence, SEQ ID NO: 2 amino acid sequence). In the present invention, the YlMpo1 gene was amplified from the genomic DNA of strain Y. lipolytica SMS397A using a polymerase chain reaction and the sequence was determined again. It was confirmed that the 646th and 655th bases were different from the previously known sequences (SEQ ID NO: Base sequence). Of these, the change of the 646th nucleotide sequence was accompanied by the change of the amino acid sequence, confirming that Asn was changed to His (SEQ ID NO: 4 amino acid sequence).

In the present invention, YlMpo1 represented by the amino acid sequence of SEQ ID NO: 2 is the gene represented by the nucleotide sequence of SEQ ID NO: 1, YlMpo1 represented by the amino acid sequence of SEQ ID NO: 4 is the gene represented by the nucleotide sequence of SEQ ID NO: , YlMpo1 represented by the amino acid sequence of SEQ ID NO: 6 is a gene represented by the nucleotide sequence of SEQ ID NO: 5 and YlMpo1 represented by the amino acid sequence of SEQ ID NO: 8 is encoded by the gene represented by the nucleotide sequence of SEQ ID NO: 7 .

In the present invention, the recombinant vector may be pET21-YlMpo1-dTM1, pET21-YlMpo1-dTM2 or pFN18A-YlMpo1, but is not limited thereto.

In the present invention, "vector" means a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in an appropriate host. The vector may be a plasmid, phage particle or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purpose of the present invention, it is preferable to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) a cloning start point that allows replication to be efficiently made to include several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector And (c) a restriction enzyme cleavage site into which the foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site is not present, using a synthetic oligonucleotide adapter or a linker according to a conventional method can easily ligate the vector and the foreign DNA.

The recombinant microorganism according to the present invention can be prepared by inserting the gene on a chromosome of a microorganism according to a conventional method, or introducing the recombinant vector onto a plasmid of a microorganism.

After ligation, the vector should be transformed into the appropriate host cell. In the present invention, the preferred host cells are prokaryotic cells. Suitable prokaryotic host cells include E. coli XL -1 Blue ( Stratagene ), E. coli DH5 [alpha], E. coli JM101, E. coli K12 , E. coli W3110 , E. coli X1776 , E. coli BL21 and the like. However , E. coli such as FMB101 , NM522 , NM538 and NM539 Speices and genera of strains and other prokaryotes may also be used. In addition to the E. coli, such as Agrobacterium sp, Bacillus subtilis (Bacillus subtilis), such as Agrobacterium A4 Bashile (bacilli), S. typhimurium (Salmonella typhimurium ) or Serratia margensense ( Serratia marcescens ) and various strains of the genus Pseudomonas can be used as host cells.

Transformation of prokaryotic cells was performed by Sambrook et al ., supra , using the calcium chloride method described in section 1.82. Alternatively, electroporation (Neumann et < RTI ID = 0.0 > al ., EMBO J., 1: 841, 1982 ) can also be used to transform these cells.

As a method for inserting the gene encoding the recombinant YlMpo1 into the chromosome of the host cell in the present invention, a commonly known gene manipulation method can be used. For example, microinjection (direct insertion of DNA into cells), liposome, directed DNA uptake, receptor ~ mediated DNA transfer, or DNA transport using Ca ⁺⁺ . Is widely used. Examples include retrovirus vectors, adenovirus vectors, adeno-associated viral vectors, herpes simplex virus vectors, poxvirus vectors, or lentiviral vectors. Particularly, retroviruses have a high gene transfer efficiency, gross deletion It can be used in a wide range of cells without binding by host DNA and rearrangement (resulting in alteration of host DNA function by altering the region of the host DNA similar to that of the host DNA).

A nucleic acid is "operably linked" when placed in a functional relationship with another nucleic acid sequence. This may be the gene and regulatory sequence (s) linked in such a way as to enable gene expression when a suitable molecule (e. G., Transcriptional activator protein) is attached to the regulatory sequence (s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a whole protein participating in the secretion of the polypeptide; A promoter or enhancer may be operably linked to a coding sequence if it affects the transcription of the sequence; Or the ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; Or a ribosome binding site is operably linked to a coding sequence if positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in contact and, in the case of a secretory leader, are in contact and present in the reading frame. However, the enhancer need not be in contact. The linkage of these sequences is carried out by ligation (linkage) at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.

In the present invention, the microorganism is selected from the group consisting of Agrobacterium genus, Aspergillus genus, Acetobacter genus, Aminobacter genus, Agromonas genus, Acidphilium genus, Bulleromyces genus, Buller genus, Brevundimonas genus, Cryptococcus genus, Chionosphaera genus, Candida genus, Cerinosterus genus, Escherichia genus, Exisophiala in, Exobasidium in, Fellomyces in, Filobasidium in, Geotrichum genus, Graphiola genus, Gluconobacter genus, Kockovaella in, Curtzmanomyces in, Lalaria in, Leucospoidium genus, Legionella genus, Psedozyma in, Paracoccus genus, Petromyc genus, Rhodotorula genus, Rhodosporidium in , Rhizomonas in, Rhodobium in, Rhodoplanes in, Rhodopseudomonas in, Rhodobacter genus, Sporobolomyces in, Spridobolus in, Saitoella in, Schizosaccharomyces genus, Sphingomonas genus, Sporotrichum genus, Sympodiomycopsis in, Sterigmatosporidium in, Tapharina in, Tremella genus, Trichosporon genus, Tilletiaria genus, genus Tilletia, Tolyposporium genus, genus Tilletiposis, Ustilago genus, genus Udenlomyce, Xanthophilomyces genus, genus Xanthobacter, P aceylomyces sp., Acremonium sp., Hyhomon sp., Rhizobium sp. It is preferably Escherichia coli .

In one embodiment of the invention, Yarrowia < RTI ID = 0.0 > lipolytica) YlMpo1_dTM1 (SEQ ID NO: removal of amino acid sequence predicted transmembrane region for N- terminal portion of YlMpo1 (SEQ ID NO: 1 or 3 nucleotide sequence, SEQ ID NO: 2 or 4 amino acid sequence) derived from the 5-base sequence, SEQ ID NO: 6 amino acids The nucleotide sequence of the dTM1 and dTM2 portions was amplified from the genomic DNA of Y. lipolytica yeast in order to express the protein of YlMpo1_dTM2 (SEQ ID NO: 7 sequence, SEQ ID NO: 8 amino acid sequence) which was removed together with the hydrophobic region following the sequence (Fig. 3), pET21-YlMpo1-dTM1 and pET21-YlMpo1-dTM2 vectors were prepared and E. coli was transformed to express His-tagged YlMpo1-dTM1 and His-tagged YlMpo1-dTM2. Analysis of the expressed His-tagged YlMpo1_dTM1 and His-tagged YlMpo1_dTM2 revealed that most of the His-tagged YlMpo1_dTM1 and His-tagged YlMpo1_dTM2 were observed in the insoluble portion even though the transmembrane regions were removed 5), and then purified by expressing His-tagged YlMpo1_dTM2.

In order to express Halo-tagged YlMpo1, the pFN18A-YlMpo1 vector was prepared and transformed into Escherichia coli KRX strain. As a result, as shown in Fig. 7, the predicted YlMpo1 protein of about 74 kDa was isolated, Purified.

The transformant was named KRX / pFN18A-YlMpo1 and was deposited with the Korea Research Institute of Bioscience and Biotechnology on Sep. 25, 2013, in accordance with the Budapest Treaty on the International Recognition of Microorganism Deposit under Patent Procedure , KRIBB) deposited with Korea Collection for Type Cultures (KCTC) under accession number KCTC12493BP.

(A) adding mannose-6-phosphate-1-mannose to the oligosaccharide using YlMpo1; And (b) removing the outer mannoside of the mannose-6-phosphate-1-mannose-added oligosaccharide to produce a mannose-6-phosphate-added oligosaccharide, 6-phosphate is added.

In the present invention, the YlMpo1 is represented by the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, and Yarrowia lipolytica . < / RTI >

In the present invention, the oligosaccharide of step (a) may be characterized by being a high-mannose type and a pauci-mannose type oligosaccharide. Specifically, Man _{3 -9} GlcNAc ₂ (M3-9).

In one embodiment of the invention, pFN18A-YlMpo1 vector (Halo-tagged YlMpo1) by the expression that YlMpo1 in vitro use (in with APTS through GU); vitro) from Man ₈ GlcNAc ₂ (M8) it was found that by a sugar chain as a substrate to perform a phosphorylation mannose, through a DNA sequencer analysis dextran ladder (Dextran ladder) and the glucose unit (glucose unit using The labeled oligosaccharides were compared and analyzed. As a result, the M8 oligosaccharide peak was detected at the GU 9 position, and after the mannose phosphorylation by YlMpo1, the monosaccharide peak added with mannose phosphate was detected at the GU 6 position, and the two monosaccharide added oligosaccharides were found to be GU 4 (Fig. 8). That is, YlMpo1 prepared by the method of the present invention is in vitro ( in vitro ), it was confirmed that a sugar chain having mannos-6-phosphate-1-mannose structure can be produced by adding mannose phosphoric acid to the sugar chain.

In the present invention, the process of adding mannose-6-phosphate-1-mannose to the oligosaccharide of step (a) may be characterized in that a buffer solution, a metal ion and GDP-mannose are further added .

The buffer solution may be sodium phosphate of pH 5.0 to 8.0, and the metal ion may be calcium ion or manganese ion.

Further, a surfactant may be further added to the step (a), and the surfactant may be selected from nonyl phenoxypolyethoxylethanol (NP-40), polyoxyethylene octylphenyl ether polyoxyethylene octyl phenyl ether (Triton X-100), polyoxyethylene sorbitan monolaurate (Tween-20), polyoxyethylene (23) lauryl ether (Brij-35) And 3 - [(3-cholamidopropyl) dimethylammonio] -1-propanephosphate (3 - [(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate .

To increase the mannose phosphorylation activity of the recombinant YlMpo1 of the present invention, the reaction time, YlMpo1 treatment concentration, pH change, mannose phosphorylation activity by addition of metal ion and surfactant were analyzed in order to establish optimal reaction conditions of YlMpo1. As a result of analyzing the difference of mannos phosphorylation by YlMpo1 according to the reaction time, mannose phosphorylation was increased compared to the rest of the reaction time when the reaction time was 240 minutes. However, when the reaction time was 120 minutes, In the subsequent experiments, the reaction time was 2 hours (Fig. 9A).

As the YlMpo1 concentration was increased, it was confirmed that the proportion of glucose-added oligosaccharides increased, but it was confirmed that the concentration did not increase significantly depending on the concentration (FIG. 9B). The optimum pH was confirmed. As a result, the activity was generally high when sodium phosphate buffer having a pH of 5.0 to 8.0 was used, and the highest activity was observed especially at pH 6.0, 6.5 and 7.0 (FIG. 9C).

As a result of analyzing the effect of the metal ion on the mannosylation phosphorylation of YlMpo1, the highest activity was shown when 10 mM calcium ion (CaCl ₂ ) was added and the manganese ion (MnCl ₂ ) ₂ ), the next highest activity was shown (Fig. 10A). The highest activity of CaCl ₂ was observed at 2 mM CaCl ₂ by adding 0.4 mM, 2 mM, 10 mM and 20 mM CaCl ₂ , 10B).

To investigate the effect of surfactant on the mannose phosphorylation activity in YlMpo1, we used NP-40 (nonyl phenoxypolyethoxylethanol), Triton-X100 (polyoxyethylene octylphenyl ether), Tween-20 (Polyoxyethylene Sorbitan Monolaurate) The addition of surfactant to polyoxyethylene (23) lauryl ether and CHAPS (3 - [(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate (Fig. 11A). In order to investigate the activity changes depending on the concentration of Tween-20 and CHAPS, two kinds of surfactants were 0.05% and 0.1%, respectively, %, 0.5%, and 2% concentration, the activity of Tween-20 was the highest at 0.5% and the activity at 2% was significantly decreased while the activity of CHAPS was 2% (Fig. 11B and Fig. 11C).

His-tagged YlMpo1_dTM2 was solubilized and purified. Mannos phosphorylation was performed under the above conditions. As a result, it was confirmed that mannosylphosphate was added to the M8 oligosaccharide by solubilized His-tagged YlMpo1. Mannos-6-phosphate- Structure was also generated (Fig. 12).

In the present invention, the step of removing the outer mannose of the oligosaccharide to which mannose-6-phosphate-1-mannose is added in step (a) may be characterized in that a weak acid hydrolysis or uncapping enzyme is used have. The weak acid may be sulfuric acid or hydrochloric acid, but it is preferably 0.1 to 5 M formic acid, more preferably 0.5 mM formic acid. The uncapping enzyme may be characterized as an α-mannosidase.

Since the mannose-6-phosphate-1-mannose structure added to the oligosaccharide by the YlMpo1 of the present invention is an oligosaccharide structure that does not exist in humans, it must be removed in order to produce most pharmaceutical glycoproteins. The mannose-6-phosphate-1-mannose structure can be chemically modified by weakly acidic hydrolysis, such as by removing the outer mannose from the mannose-6-phosphate-1-mannose structure, Mannosidase derived from a culture of Cellulomonas bacteria can be used using the uncapping enzyme CcGH92_5 (Tiels et al, Nature Biotech 30: 1225-1231, 2012) Patent No. 0888316).

In the examples of the present invention, the outer mannos of the oligosaccharide to which mannose-6-phosphate-1-mannose was added was removed, and in order to prepare an oligosaccharide to which mannose-6-phosphate was added, Cellulosimicrobium cellulans ) Was cloned and expressed by the enzyme activity domain (1-774) of CcGH92_5, which is the uncapping enzyme derived from the cisplatin gene (Fig. 13), and the uncapped enzyme cleaved by the above-described isolated and purified mannose-6-phosphate- As a result, it was confirmed that the mannose-6-phosphate-added oligosaccharide formed by removing the outer mannose was formed (FIG. 14).

In addition, the oligosaccharide to which mannose-6-phosphate-1-mannose was added was treated with 0.5 M formic acid to conduct weakly acidic hydrolysis. As a result, it was confirmed that an oligosaccharide added with mannose- When the oligosaccharide was treated with calf intestinal alkaline phosphatase (CIP), it was confirmed that phosphoric acid was completely removed and converted to M8 oligosaccharide (FIG. 15).

In another aspect, the present invention relates to a method for producing a glycoprotein, wherein mannose-6-phosphate is added by adding mannose-6-phosphate to the sugar chain.

In another aspect of the present invention, there is provided a method for producing a glycoprotein comprising the steps of chemically or physically binding a mannose-6-phosphate-added oligosaccharide prepared by adding mannose-6- And a method for producing the same.

The above method has the advantage that it is possible to have a much more versatile and flexible application possibility than the direct production of an enzyme therapeutic agent to which mannose-6-phosphate oligosaccharide is attached in an animal cell or a yeast whose sugar chain biosynthetic pathway has been redesigned. For example, in mannose-containing oligosaccharides ( in vitro , Mannos-6-phosphate prepared by adding mannose phosphate using YlMpo1 and uncapping the outer mannose can be chemically or physically bound to the glycoprotein. If this method is used, the enzyme protein is produced in Escherichia coli having high productivity and economical efficiency, but it can be targeted to intracellular lysosomes by attaching mannose-6-phosphate oligosaccharide thereto by the method described above It is possible to produce a glycoprotein.

In the present invention, the protein may be a pathogen protein, a lysosomal protein, a growth factor, a cytokine, a chemokine, an antibody or an antigen-binding fragment thereof -binding fragment, or a fusion protein.

In the present invention, the lysosomal protein may be a lysosomal storage disease (LSD) -related degrading enzyme.

)을 갖는 킬로미크론 보유 질병(chylomicron retention disease), 헤르만스키-푸드락 증후군(Hermansky-Pudlak syndrome), 체디아크-히가시 증후군(Chediak-Higashi syndrome), 다논병(Danon disease), 또는 겔레오피직 이형성증(Geleophysic dysplasia) 등이 포함된다.The lysosomal storage disease may be selected from the group consisting of Fabry's disease, mucopolysaccharidosis I, Farberdisease, Gaucher disease, GM1-gangliosidosis, The present invention relates to a method of treating a disease selected from the group consisting of Tay-Sachs disease, Sandhoff disease, GM2 activator disease, Krabbe disease, metachromatic leukodystrophy, Niemann-Pick disease ), Schie disease, Hunter disease, Sanfilippo disease, Morquio disease, Maroteaux-Lamy disease, hyaluronidase, (Aspartylglucosaminuria), fucosidosis, mannosidosis, Schindler's disease, sialidosis type 1, hyaluronidase deficiency, aspartylglucosaminuria, Pompe disease, Pycnodys < RTI ID = 0.0 > osteopenia, osteosynthesis, ceroid lipofuscinosis, cholesterol ester storage disease, Wolman disease, multiple sulfatase deficiency, galactosialidosis, Mucolipidosis, cystinosis, sialic acid storage disorder, Marinische-Schleglen syndrome ("

Chylomicron retention disease, Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, Danon disease, or gelo-lipid dysplasia, (Geleophysic dysplasia).

In the present invention, mannose-6-phosphate produced by adding mannose-6-phosphate-1-mannose by using YlMpo1 and uncapping the outer mannose is chemically or physically bound to the glycoprotein However, the present invention is not limited to this, and a glycoprotein to which mannose-6-phosphate-1-mannose has been added is bound to a glycoprotein, followed by uncapping the outer mannose to prepare a mannose- can do.

According to another aspect of the present invention, there is provided a method for producing a lysosomal salt by combining mannose-6-phosphate-added oligosaccharide prepared by adding mannose-6-phosphate to the oligosaccharide and a peptide, lipid or nano- To a method for preparing a transporter that transfers a useful substance into a non-cytoplasm or nucleus.

Since the mannose-6-phosphate-added oligosaccharide prepared by the method of the present invention is transferred to the intracellular lysosomes through the encapsulation process, the mannose-6-phosphate oligosaccharide is converted into the peptide for liposome escape, Or nanomaterials to transfer useful materials into the cytoplasm or nucleus rather than the lysosomes. In this way, it can be used as a tool for transferring not only transcription factors, antibodies, signal transduction proteins, etc. for cell control but also microRNA, siRNA, plasmid DNA and the like into cells.

The method for producing mannose-6-phosphate-added oligosaccharide using recombinant YlMpo1 prepared by the method of the present invention can effectively add mannose phosphate to a glycoprotein used as an enzyme therapeutic agent in vitro , Or a sugar chain biosynthetic pathway can be applied to the production of various enzyme treatments rather than directly producing an enzyme therapeutic agent having a mannose-6-phosphate oligosaccharide in a yeast that has been redesigned. In addition to the glycoprotein, Lipids or nanomaterials to transfer useful substances into the cytoplasm or nucleus rather than the lysosomes.

FIG. 1 is a schematic diagram showing a process of targeting a therapeutic enzyme to intracellular lysosomes using a mannose-6-phosphate signal.
Proteins to be transferred to lysosomes are added to GlcNAc-phosphate by N-acetylglucosamine-1-phosphotransferase (GIcNAc-PT) in Golgi, and mannose-6-phosphate is produced in the next step by removing the outer GlcNAc by uncovering enzyme. There are two types of mannose-6-phosphate receptors (MPR), cation-dependent (CD) -MPR and cation-independent (CI) -MPR, respectively. When an external recombinant enzyme is added, CI-MPR present in the cell membrane recognizes it, binds it, and encapsulates it to target lysosomes. In the drawings, symbols representing oligosaccharides conform to the format of the United States Consortium for Functional Glycomics (htpp: //www.functionalglycomics.org/), and phosphoric acid is represented by P (blue square: GlcNAc, green circle: mannose, phosphoric acid: P )
FIG. 2 is a schematic diagram of the process of forming mannose-6-phosphate, wherein (A) shows the formation of mannose-6-phosphate by GlcNAc-PT and uncovering enzyme in animal cells, 6-phosphoric acid by using an engineered yeast.
3 is a diagram showing the amino acid sequence of the recombinant YlMpo1.
The sites predicted as transmembrane domains are indicated by underlined and bold letters, and the start regions of dTM1 and dTM2 expressing except membrane permeation domain are indicated by arrows. The LicD domains found in yeast MNN4 homologues are shown in box and italics.
Fig. 4 is a schematic diagram of vectors pET21-YlMpo1_dTM1 (A) and pET21-YlMpo1-dTM2 (B) constructed to express His-tagged YlMpo1.
FIG. 5 shows the degree of expression of His-tagged YlMpo1 (1: insoluble portion before induction, 2: insoluble portion before induction, 3: insoluble portion after induction, 4: insoluble portion after induction, arrow: YlMpo1 band).
Fig. 6 shows data showing the purification process and purification purity of His-tagged YlMpo1.
(A) Purity according to the YlMpo1 purification step (1: solubilization of the insoluble portion after cell lysis in 8 M urea buffer (100 mM NaH ₂ PO ₄ , pH 8.0, 10 mM Tris-Cl, 8 M urea) (100 mM NaH ₂ PO ₄ , pH 6.3, 10 mM Tris-Cl, 8 M urea), Elution D: Buffer D (100 mM NaH _{2 PO 4, pH 5.9, 10 mM} Tris-Cl, 8 M urea) eluting a solution, elution E using: buffer _{E (100 mM NaH 2 PO 4} , pH 4.5, 10 mM Tris-Cl, 8 M urea) to ≪ / RTI >
(B) Purification of YlMpo1 using SDS-PAGE after purification (4: BSA 2 ㎍, 5: BSA 4 ㎍, 6: BSA 8 ㎍, 7: Concentrated YlMpo1 2 ㎕, 8: Concentrated YlMpo1 4: 1, 9: concentrated YlMpo1 8 [mu] l).
FIG. 7 shows a schematic diagram (A) of a Halo-tagged YlMpo1 expression vector and a data (B) obtained by SDS-PAGE analysis of the fraction obtained during purification (1: cell lysate before induction of YlMpo1 expression, 2 : Cell lysate after induction of expression, 3: flow through of the culture supernatant to the column, 4: effluent obtained by flowing the washing buffer to the column, 5: effluent obtained by flowing the washing buffer to the column, 6 : Eluate obtained by cleavage with TEV protease, elution buffer eluting with column, arrow: YlMpo1 band).
Fig. 8 is data obtained by analyzing the mannose phosphorylation reaction of YlMpo1 using a DNA sequencer using M8-APTS as a substrate (the symbol representing oligosaccharide in the figure is the same as in Fig. 1).
FIG. 9 is a graph showing changes in activity and optimal pH conditions according to the reaction time and YlMpo1 enzyme concentration conditions in the mannose phosphorylation reaction of YlMpo1 (A: assay of YlMpo1 activity according to reaction time, and activity according to the concentration of YlMpo1 enzyme Analysis, C: analysis of YlMpo1 activity according to pH).
FIG. 10 is an analysis of the effect of metal ions on the mannose phosphorylation activity of YlMpo1 (A: effect of 10 mM CaCl ₂ , FeCl ₃ , CuSo ₄ , MgCl ₂ , MnCl ₂ , ZnSO ₄ ion and EDTA, B: Mannose phosphorylation activity of YlMpo1 according to the concentration of calcium ions (0.4, 2, 10 and 20 mM).
FIG. 11 shows the effect of surfactant on the mannose phosphorylation activity of YlMpo1 (A: 0.5% NP-40, Triton-X100, Tween-20, Brij-35, mannose phosphorylation activity by CHAPS treatment, B: Mannose phosphorylation activity depending on the concentration of Tween-20, and C: Mannose phosphorylation activity depending on CHAPS concentration).
FIG. 12 shows data obtained by analyzing the mannose phosphorylation activity by the solubilized His-tagged YlMpo1 with a DNA sequencer.
13 is a schematic diagram (A) of a vector expressing the Halo-tagged uncapping enzyme and a result (B) obtained by SDS-PAGE analysis of the fraction obtained in the purification process (1: cell lysate before induction of uncapping enzyme expression , 2: an insoluble part of the cell lysate induced by expression, 3: an insoluble part of the cell lysate induced by expression, 4: an aqueous solution of the cell lysate induced expression, 5-6: a cell lysate aqueous solution flow-through, 7-8: effluent obtained by flowing wash buffer through the column, 9: effluent obtained by cleavage by TEV protease, and elution buffer eluting from the column). The arrow indicates the band of the uncapping enzyme.
Fig. 14 is a data showing that the outer capped mannose is removed by Uncapping enzyme.
(A) represents a glucose unit confirmed by using a Dextran ladder, (B) represents a glucose unit produced by mannose phosphorylation using YlMpo1 in M8 oligosaccharide, and mannose-6-phosphate-1-mannose Shows the oligosaccharides added. (C) is the result of confirming whether or not the mannose-6-phosphate-added oligosaccharide is produced by treating the uncapping enzyme to remove the outer mannose.
Fig. 15 shows an experiment for removing mannose camping by weak acid hydrolysis. (A) shows the glucose unit determined using a Dextran ladder, and (B) shows the oligosaccharide added with mannose-6-phosphate-1-mannose. (C) is obtained by removing the capped mannose by weak acid hydrolysis to form a mannose-6-phosphate-added oligosaccharide, (D) treating the oligosaccharide with calf intestinal alkaline phosphatase to remove phosphoric acid, Conversion.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Recombination YlMpo1  Production of expression vector

In the present invention, YlMpo1 derived from Yarrowia lipolytica was used. YlMpo1 has an amino acid sequence predicted as a transmembrane region at the N-terminal region, and the region except for this region was amplified by polymerase chain reaction (PCR) from the genomic DNA of Y. lipolytica SMS397A yeast ). Two types of YlMpo1_dTM1 (SEQ ID NO: 7 base sequence, SEQ ID NO: 8 amino acid sequence) in which the N-terminal membrane permeation site was removed and YlMpo1_dTM2 (amino acid sequence of SEQ ID NO: 5) and the hydrophobic region following the amino acid sequence of YlMpo1_dTM1 , And the following specific primers were used.

1-1: His - tagged YlMpo1  Production of expression vector

His-tagged YlMpo1-dTM1 used the following SEQ ID NOS: 9 and 11 and His-tagged YlMpo1-dTM2 was amplified by the conventional PCR method using SEQ ID NOS: 10 and 11. The amplified DNAs were digested with Hind III and Xho I restriction enzymes and cloned into pET21a (Life Technologies, USA) vector using conventional recombinant DNA technology to construct pET21-YlMpo1-dTM1 and pET21-YlMpo1-dTM2 vectors (Fig. 2)

YlMPO1_ Hind III_1F:

[SEQ ID NO: 9] 5'-CCCAAGCTTCGATGACCGACGTGGGCTTAGT-3 '

YlMPO1_ Hind III_2F:

[SEQ ID NO: 10] 5'-CCCAAGCTTCGAAAGCCATGGCCGGCAACTC-3 '

YlMPO1_ Xho I R:

[SEQ ID NO: 11] 5'-CCGCTCGAGCTCAAACTCCTCGCGAATCCA-3 '

1-2: Halo - tagged YlMpo1  Production of expression vector

Halo-tagged YlMpo1 was amplified by a conventional PCR method using the following SEQ ID NOs: 12 and 13 for the region corresponding to YlMpo1_dTM1 from which the N-terminal transmembrane region was removed. The amplified DNA was digested with SgfI and Bam HI restriction enzymes and cloned into pFN18A (Promega, USA) using a conventional recombinant method to prepare pFN18A-YlMpo1 vector.

YlMPO1_SgfI_F:

[SEQ ID NO: 12] 5'-ACGGCGATCGCCATGACCGACGT-3 '

YlMPO1_ Bam HI_R:

[SEQ ID NO: 13] 5'-AGCGGATCCCTACTCAAACTCCTCG-3 '

His - tagged YlMpo1 Expression, purification and antibody production

2-1: His - tagged YlMpo1 Manifestation of

His-tagged YlMpo1 proteins were expressed and purified using pET21-YlMpo1-dTM1 and pET21-YlMpo1-dTM2 vectors prepared in Example 1-1 above.

The pET21-YlMPO1-dTM1 and pET21-YlMPO1-dTM2 vectors were transformed into Escherichia coli BL21 (DE3) to express the His-tagged YlMpo1 protein. The transformant was cultured in Luria-Bertani (LB) medium containing ampicillin at 37 ° C. When the absorbance reached 0.4 at 600 nm (OD600), 1 mM IPTG was added and incubated at 37 ° C for 4 hours to induce protein expression Respectively.

After the cells were lysed by ultrasonication in the cultured cells, the soluble and insoluble portions of the proteins were extracted by centrifugation. The expression patterns of the extracted proteins were analyzed by SDS-PAGE, followed by coomassie brilliant blue).

As a result, it was confirmed that most of the His-tagged YlMpo1_dTM1 and His-tagged YlMpo1_dTM2 were observed in the insoluble portion even though the transmembrane regions were removed (FIG. 5), and then the His-tagged YlMpo1_dTM2 was expressed and purified .

2-2: His - tagged YlMpo1 Purification and Antibody Production

YlMpo1_dTM2 was expressed by the method of Example 2-1, and the cells were dissolved by ultrasonication to extract the proteins. Denaturation using an 8 M element (urea) provided by a Ni-NTA column manufacturer (Qiagen, USA) ≪ / RTI > method.

(100 mM NaH ₂ PO ₄ , pH 5.9, 10 mM Tris-Cl, 8 M urea) and E (100 mM NaH ₂ PO ₄ , pH 4.5, 10 mM Tris-Cl, M urea), and analyzed by staining with coomassie brilliant blue after SDS-PAGE (FIG. 6A).

The purified YlMpo1_dTM2 was concentrated by chromatography on Ni-NTA column, and SDS-PAGE was performed to confirm the purity. To measure the amount of purified YlMpo1_dTM2, 2, 4 and 8 μg of BSA (Fig. 6B).

As a result, it was confirmed that the recovery rate of YlMpo1_dTM2 was high when the purification was carried out using the elution buffer E.

YlMpo1_dTM2 purified by the above method was used as an antigen to obtain polyclonal anti-YlMpo1 antibody from rabbits in a conventional manner. This procedure was performed by AbFrontier (Seoul, Korea).

Halo - tagged YlMpo1 ≪ / RTI >

3-1: Expression of Halo-tagged YlMpo1

Halo-tagged YlMpo1 proteins were expressed and purified using the pFN18A-YlMpo1 vector prepared in Example 1-2.

The pFN18A-YlMpo1 vector was transformed into Escherichia coli KRX strain (Promega, USA) to express the Halo-tagged YlMpo1 protein. The transformants were cultured overnight at 37 ° C in LB medium containing ampicillin (100 mg / ml) and the next day, seeded in Terrific Broth (TB) medium and cultured at 37 ° C. When the absorbance at 600 nm reached 0.8, rhamnose was added to a final concentration of 0.1% to yield YlMPOl Gene expression was induced. After induction of expression, cells were further cultured at 15 ° C for 24 hours and centrifuged at 6,000 × g, 4 ° C for 20 minutes to harvest the cells.

Over-expression of the His-tagged YlMpo1 protein was observed in the soluble portion of the over-expressed Halo-tagged YlMpo1, unlike most of the insoluble portion.

The transformant into which the pFN18A-YlMpo1 vector was inserted was named KRX / pFN18A-YlMpo1 and was named as KRX / pFN18A-YlMpo1 in accordance with the Budapest Treaty on the International Approval of Microorganism Deposit for Patent Procedures, (KCTC12493BP) deposited in the Korea Collection for Type Cultures (KCTC) of the Korea Research Institute of Bioscience and Biotechnology (KRIBB).

3-2: Halo - tagged YlMpo1 Purification of

Purification of the Halo-tagged YlMpo1 protein was performed according to the purification method provided by the manufacturer of HaloTag Protein Purification System (Promega).

The above process is briefly described. The cell precipitate is resuspended in a purification buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM DTT, 0.5 M EDTA, 0.005% IGEPAL), the cells are lysed by sonication, The cell lysate was centrifuged at 6,000 × g at 4 ° C. for 20 minutes. The cell lysate was mixed with HaloLink resin previously equilibrated with the purification buffer, and reacted at 4 ° C. for 24 hours.

In the case of the Halo-tag, the resin bound to the HaloLink resion was covalently bound, and the resin bound to the Halo-tagged YlMpo1 protein was loaded into the column, and then washed with a buffer solution of 20 times the volume of the resin. The cleavage buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM DTT, 0.5 mM EDTA) was added in the same volume as the column volume, treated with TEV protease (Promega) for 1 hour, Of the purification buffer, and the YlMpo1 protein, which was cleaved from the Halo-tag by TEV protease, was eluted.

Then, to remove the TEV protease with His-tag attached to the eluted solution, the His-link resin was added and reacted at room temperature for 20 minutes. The resin was centrifuged at 1000 x g for 1 min to precipitate the resin, and only the supernatant was recovered. The fractions eluted at each step were analyzed by SDS-PAGE.

As a result, as shown in Fig. 7, it was confirmed that the expected YlMpo1 protein of about 74 kDa was isolated and purified. The eluted fractions were finally dialyzed in 20 mM sodium phosphate buffer (pH 7.4) and centrifuged at 6000 g using a 30 kDa centrifugal filter device (Amicon Ultra centrifugal filter device, Millipore) And stored at 80 ° C.

YlMpo1 On by Mannos  Identification of phosphorylation

It was confirmed whether YlMpo1 isolated and purified through Example 3 performed the mannose phosphorylation reaction in vitro using the Man ₈ GlcNAc ₂ (M8) oligosaccharide as a substrate.

First, M8 oligosaccharides were fluorescently labeled with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) according to the previously reported method to obtain M8-APTS (Laroy et al, Nature Protocols 1: 397-405, 2006). To briefly describe this, a 1.2 M citric acid solution in which 20 mM APTS is dissolved and a dimethyl sulfoxide in which 1 M cyanoborohydride is dissolved are mixed at a ratio of 1: 1, and then 58 mM 5 μl of M8 was dried and then 5 μl of the APTS solution prepared above was added to the dried M8 and reacted at 37 ° C. for 16 hours or longer. Unreacted APTS and salts were removed from the reaction solution and desalted using a Sephadex G10 column to obtain only M8-APTS. All the purified M8-APTS solutions were taken, dried and dissolved in 50 μl of purified water to prepare samples.

In vitro mannose phosphorylation by YlMpo1 was performed using M8-APTS prepared as described above as a substrate.

The reaction was carried out at 30 ° C for 2 hours with 100 mM Tris-HCl (pH 7.5), 10 mM MnCl ₂ , 2 mM GDP-mannose, M8-APTS and YlMpo1 (1 mg / The reaction was stopped by boiling at 5 ° C for 5 minutes and analyzed using a DNA sequencer according to the previously reported method (Laroy et al., Nature Protocols 1: 397-405, 2006). 10 μl of the reaction mixture was transferred to a DNA sequencer plate and analyzed using an ABI 3130 DNA sequencer equipped with a 36 cm capillary array filled with POP7-polyacrylamide linear polymer. DNA sequencer analysis conditions are as shown in Table 1, and data analysis was performed using GeneMapper software.

DNA sequencer analysis conditions Parameter 오빈 온도 60 ° C Prerun voltage 15 kV Prerun time 180s Infection voltage 1.2V Injection time 16s Run voltage 5 kV Run time 1,000s

As a result of comparing oligosaccharides labeled with APTS through a glucose unit (GU) using a Dextran ladder, the M8 oligosaccharide peak was detected at the GU 9 position, and after the mannose phosphorylation reaction with YlMpo1, The oligosaccharide peak added with one phosphate was detected at the GU 6 position, and the two oligosaccharide added oligosaccharide was detected at the GU 4 position (FIG. 8). That is, it was confirmed that YlMpo1 produced mannose-6-phosphate-1-mannose structure by adding mannose phosphate to M8 oligosaccharide in vitro.

YlMpo1 Optimal reaction conditions

Experiments were conducted to analyze the optimal reaction conditions of YlMpo1 isolated and purified through Example 3 above.

5-1: Mannose phosphorylation activity of YlMpo1 according to reaction time

In order to examine the difference in mannose phosphorylation reaction by YlMpo1 according to the reaction time, the conditions except for the reaction time were the same as those described in Example 4 above. The reaction time was 30 min, 60 min, 120 min and 240 min, respectively. The result of Mannos phosphorylation by YlMpo1 was analyzed using a DNA sequencer.

As a result, when the reaction time was 240 minutes, mannose phosphorylation was increased compared to the rest of the reaction time, but it was confirmed that the reaction was most efficient when the reaction time was 120 minutes. In the following experiments, the reaction time was 2 hours 9A).

5-2: YlMpo1  Depending on concentration Mannos  Phosphorylation activity

The reaction conditions according to YlMpo1 concentration were analyzed. The reaction was carried out at different concentrations of YlMpo1 to final concentrations of 0.25, 0.5, 0.75, and 1 mg / ml, and the reaction conditions were the same as in Examples 4 and 5-1.

As a result, it was confirmed that as the concentration of YlMpo1 increased, the proportion of glucose-added oligosaccharides increased, but it was not significantly increased according to the concentration (Fig. 9B).

5-3: pH In accordance YlMpo1 of Mannos  Phosphorylation activity

Experiments were carried out using various buffers with different pHs to confirm the optimum pH.

Sodium acetate was used as the buffer solution at pH 5.0 and 5.5 and sodium phosphate was used as the buffer solution at pH 5.0, 6.0, 6.5, 7.0, 7.5 and 8.0. pH 7.0, 7.5, 8.0 (Tris-HCl). The reaction was carried out by adding the above 50 mM buffer (sodium acetate, sodium phosphate or Tris-HCl), 2 mM GDP-mannose, M8-APTS and YlMpo1 (1 mg / Lt; / RTI >

As a result, when sodium phosphate buffer solution was used, the activity was generally high, especially at pH 6.0, 6.5, 7.0. On the other hand, when sodium acetate or Tris-HCl buffer was used, the activity was lower than that when sodium phosphate buffer was used (FIG. 9C).

5-4: Depending on the metal ion YlMpo1 of Mannos  Phosphorylation activity

In order to analyze the effect of metal ions on the mannose phosphorylation of YlMpo1, 10 mM EDTA was treated at 4 DEG C for 16 hours or more in YlMpo1 purified through Example 3, and then dialyzed with purified water to obtain YlMpo1 solution The following experiment was conducted.

In the same manner as in Example 5-3, 100 μl of each reaction solution was added to 100 μl of a reaction mixture containing 50 mM sodium phosphate (pH 6.5) buffer, 2 mM GDP-mannose, M8-APTS and YlMpo1 (1 mg / (CaCl ₂ , FeCl ₃ , CuSo ₄ , MgCl ₂ , MnCl ₂ or ZnSO ₄ ) or EDTA at 30 ° C for 2 hours, and the reaction was stopped by boiling at 100 ° C for 5 minutes and analyzed by DNA sequencer.

As a result, the highest activity was shown when 10 mM calcium ion (CaCl ₂ ) was added and the activity was the highest when manganese ion (MnCl ₂ ) was added (FIG. 10A). On the other hand, activity was greatly reduced when other ions or EDTA were added and reacted.

Experiments were conducted to determine the difference in activity during the reaction by treating the calcium ion (CaCl ₂ ), which has the highest activity, by concentration. 0.4 mM, 2 mM, 10 mM and 20 mM calcium ions were added, and the same procedure as described above was carried out.

As a result, it was confirmed that the highest activity was observed at 2 mM calcium ion as shown in FIG. 4B.

5-5: Surfactant ( surfactant )In accordance YlMpo1 of Mannos  Phosphorylation activity

Experiments were carried out using NP-40, Triton-X100, Tween-20, Brij-35 and CHAPS to investigate the effect of surfactant on mannosylation activity of YlMpo1.

Sodium phosphate (pH 6.5), 0.5% surfactant (NP-40, Triton-X100, Tween-20, Brij-35 or CHAPS), 2 mM CaCl ₂ , 2 mM GDP-mannose, M8-APTS, and YlMpo1 (1 ㎎ / ㎖) were added and reacted at 30 ° C for 2 hours and analyzed using a DNA sequencer.

As a result, when the surfactant was added, the activity was slightly increased overall, and it was confirmed that the activity was the highest when Tween-20 and CHAPS were present (FIG. 11A).

In order to investigate the changes in the activities of Tween-20 and CHAPS, we investigated the changes in the mannosylation activity of YlMpo1 by treatment with 0.05%, 0.1%, 0.5%, and 2% concentrations of two kinds of surfactants .

As a result, the activity of Tween-20 was the highest at 0.5% as shown in FIG. 11B, and the activity was greatly decreased at 2%, and the activity of CHAPS was increased when 2% CHAPS was applied (Fig. 5C).

His - tagged YlMpo1 Solubilization  Identify Tablets and Active

6-1: His - tagged YlMpo1 Solubilization  refine

In Example 2, the insoluble expressed His-tagged YlMpo1_dTM2 was solubilized and purified, and it was confirmed whether the mannose phosphorylation reaction could be performed using this.

YlMpo1 was expressed in the same manner as described in Example 2. The harvested cell pellet was resuspended in a lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF) The cells were then lysed by sonication. The cell lysate was centrifuged at 6,000 × g at 4 ° C. for 20 minutes. To the precipitate obtained after centrifugation was added a buffer (100 mM NaH ₂ PO ₄ , pH 8.0, 20% glycerol, 0.4 M NaCl, 1% CHAPS) Followed by solubilization at 4 ° C. for 3 hours, followed by centrifugation at 100,000 × g, 4 ° C. for 1 hour.

Then, YlMpo1 was purified from the solubilized solution by adding CHAPS (3 - [(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate as a surfactant using a Ni-NTA column (Qiagen, USA). His-tagged the YlMpo1 was washed with a buffer solution _{(50 mM NaH 2 PO 4,} pH 8.0, 300 mM NaCl, 20 mM imidazole) was attached to the Ni-NTA column, and then adsorbed to process a buffer solution containing the 250 mM imidazole The His-tagged YlMpo1 was eluted.

6 -2: His - tagged Confirmation of mannose phosphorylation by YlMpo1

It was confirmed that His-tagged YlMpo1 purified by solubilization using M8-APTS prepared by the method of Example 4 performs mannose phosphorylation reaction in vitro using Man ₈ GlcNAc ₂ (M8) oligosaccharide as a substrate.

To 100 μl of the whole reaction solution was added 50 mM sodium phosphate (pH 6.5), 0.5% Tween-20, 2 mM CaCl 2, 2 mM GDP-mannose, M8-APTS, His-tagged The reaction was stopped by adding YlMpo1 (1 mg / ml) at 30 DEG C for 2 hours, boiled at 100 DEG C for 5 minutes, and analyzed using a DNA sequencer according to the method of Example 4.

As a result of comparing and analyzing oligosaccharides labeled with APTS through a glucose unit (GU) using Dextran ladder, mannosylphosphate was added to M8 oligosaccharide by solubilized His-tagged YlMpo1 to obtain mannose-6- Phosphate-1-mannose structure was generated (Fig. 12).

Camped Mannos  Through removal Mannos -6-phosphoric acid-added Sugar chain  Produce

7-1: Expression and purification of Uncapping enzyme

A vector expressing the enzyme activity domain (1-774) of the recently known cellulosic microbium cellulans- derived uncapping enzyme CcGH92_5 (Tiels et al , Nature Biotech , 30: 1225, 2012). The DNA encoding the amino acid sequence was synthesized with DNA 2.0 (Menro Park, CA, USA) and cloned into pFN18A vector (Promega) using an EZ-cloning kit (Enzynomics, Taejon, Korea) Vector (Fig. 13A). The pFN18A-uncapping vector thus constructed expresses the uncapping enzyme activity domain (1-774) of CcGH92_5 fused with a Halo-tag at the N-terminus.

The above pFN18A-uncapping vector was transformed into Escherichia coli KRX strain (Promega) to express Halo-tagged uncapping protein. The transformant was cultured overnight at 37 ° C in LB medium containing ampicillin (100 mg / ml) and the next day, inoculated into Terrific Broth (TB) medium and cultured at 37 ° C. When the absorbance reached 0.8 at 600 nm, the expression of the uncapping enzyme gene was induced by adding rhamnose to a final concentration of 0.1%. After induction of expression, cells were further cultured at 15 ° C for 24 hours and centrifuged at 6,000 × g, 4 ° C for 20 minutes to harvest the cells.

Purification of Halo-tagged uncapping protein was performed according to the purification method provided by the manufacturer of HaloTag Protein Purification System (Promega). The above process is briefly described. The cell precipitate is resuspended in a purification buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM DTT, 0.5 M EDTA, 0.005% IGEPAL), the cells are lysed by sonication, The cell lysate was centrifuged at 6,000 × g at 4 ° C. for 20 minutes. The cell lysate was mixed with HaloLink resin previously equilibrated with the purification buffer, and reacted at 4 ° C. for 24 hours. In the case of the Halo-tag, the resin bound to the HaloLink resion was covalently bound, and the resin bound to the Halo-tagged uncapping protein was loaded into the column, and then washed with a buffer solution of 20 times the volume of the resin. The cleavage buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM DTT, 0.5 mM EDTA) was added in the same volume as the column volume, treated with TEV protease (Promega) for 1 hour, Of the purified buffer, and the uncapping protein cleaved from the Halo-tag by TEV protease was eluted. Then, to remove the TEV protease with His-tag attached to the eluted solution, the His-link resin was added and reacted at room temperature for 20 minutes. The resin was centrifuged at 1000 xg for 1 minute to precipitate the resin, and only the supernatant was recovered. The fractions eluted at each step were analyzed by SDS-PAGE. As a result, as shown in Fig. 13B, it was confirmed that the uncapping protein was isolated and purified. The eluted fractions were finally dialyzed in 20 mM sodium phosphate buffer (pH 7.4) and centrifuged at 6000 g using a 30 kDa centrifugal filter device (Amicon Ultra centrifugal filter device, Millipore) And stored at 80 ° C.

7-2: Uncapping  By enzyme Mannos -6-phosphoric acid-added oligosaccharide

Using the uncapping enzyme isolated and purified through the method of Example 7-1, the capping of the outer mannose was removed from the sugar chain to which mannose-6-phosphate-1-mannose was added to form an oligosaccharide to which mannose-6- Respectively. To this end, YlMpo1 was treated with Ml oligosaccharide at the optimal conditions established in Example 5 to form a mannose-6-phosphate-1 mannose sugar chain structure (FIG. 14B). Then, 20 μg of the uncapping enzyme isolated and purified in Example 7-1 was added to 100 μl of HEPES (pH 7.0) and 2 mM CaCl ₂ , and reacted at 30 ° C. for 16 hours, followed by DNA sequencer (Fig. 14C).

As a result, it was confirmed that a glycopeptide added with mannose-6-phosphate formed by removing the outer mannose was detected.

7-3: Using weak acid hydrolysis Mannos  Removing camping

The weak acid hydrolysis was carried out under various conditions to remove the outer mannose capping from the mannos-6-phosphate-1-mannose-added oligosaccharide.

For this purpose, YlMpo1 was treated with Ml oligosaccharide at the optimal conditions established in Example 5 to form a mannose-6-phosphate-1 mannose sugar chain structure (FIG. 15B). The whole reaction solution was treated with 0.5 M formic acid, reacted at 80 ° C for 1 hour, and analyzed using a DNA sequencer (FIG. 15C).

As a result, it was confirmed that a sugar chain peak added with mannose-6-phosphate formed by removing the outer mannose by weakly acidic hydrolysis was detected.

Further, it was confirmed that when the oligosaccharide was treated with calf intestinal alkaline phosphatase (CIP), phosphoric acid was completely removed and converted to M8 oligosaccharide (FIG. 15D).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Korea Biotechnology Research Institute KCTC12493BP 20130925

<110> korea research institute of bioscience and biotechnology <120> Mannosylphosphorylation Reaction Using YlMpo1 Protein <130> P13-B197 <160> 13 <170> Kopatentin 2.0 <210> 1 <211> 1935 <212> DNA <213> Yarrowia lipolytica YlMPO1 gene <400> 1 atggtgctgc acccgtttcg cctgctgcgc acatctcttg tgagcaagct cgtcgtcatt 60 ctcataacct gtctgatttt cggcagctta ctcaacttga ccgacaagct gcccgacgga 120 gtcaagtcac gggtcgccta catgaccgac gtgggcttag tcagcggcgg tgcccgctcg 180 aaagccatgg ccggcaactc gtctgtgcgc atcagtcacg tgccactgac gaaaatcaag 240 tttgccgaaa acgaaaagga attcaacccc aagtgggccc agaagaaggc tctggactcg 300 tcgtcggatt actcattcga gtggaaagac tgggtcgact tgaccgaggt cacgggtcta 360 ttcagagcca tcgaggtcgg ctcgtgtggc aaaaacaaac aatgccttag taagctccag 420 gtcaccggac ccccgactca ggtggagccc caggcgtttt tcaaccgggt cggaaaggtg 480 tttttggacc agaccatgcc caagccgaaa cagttgattt atctgaccga gaaagagtca 540 cgtggcaagc gggaggagcc cgtcgagata gagtcacatg ttccggtggt tcgggagcct 600 gagttgggcg actttagccg atcgcgtgac gccaagtcaa tccaaaatgc taagcgggag 660 gcgactgagg aggcgactgg tgagtcagcc aacgagggcc agtcacgtga ctccgcacga 720 gcctccgcgc gtgacattgc cttttccgag gctgaccccg tgcacgaaga ctcgtatgac 780 gacgacgaaa acggaaccat tctcgtgccg acggctgagt cagaggagga gctccaggaa 840 tacgcagaca aggacgcggc cgccaagtcg tacctgcgaa gcggctggtc acgtggccag 900 aaatatgtcg agctgccgcg cgagctgttc acttgggaca ttcacgaaga gattgacaag 960 ggactgacta agaagagcgt tgactcagac gacccgtcac gtgagcaggt tgcgcactcg 1020 cagtttctta gtcagcattg gaaacacatc aaaaagtccg gcaagcattt ctccgaagcg 1080 tgggttgtgg gcgacaccaa agcagccgga gtccattacg actggcgatt tttcagcgag 1140 ttaaacacca ttgacgagaa acgagtcatc ttgcgtaaat tggtgcgtgc gtggctcgac 1200 ttcacgtcac gtgagggcat tatcacgtgg ctggcgcatg gcacgttgct gggctggtac 1260 tggaacggcc agtctctgcc gtgggatttc gacggtgatg tccagatgcc gatccgcgaa 1320 ttcgaccggt ttgcccgcct ctataatcag tcgttggtaa ttgatgagtc agccggcggc 1380 cggtattatg tcgatgtggg tccctcctac gtcgagcgcc tccgaggcaa cggcaagaat 1440 gtcattgatg ctcggtttat cgacgttgat agtggcatgt acattgacat taccgccttg 1500 gcgtatgccg agcagcagga aaagttccac tgcaaaaact ggcatcggta tgagttggag 1560 agcgtttctc cgctgcgtcg gacgctgttt gagggcaagg aggcttacat tccaaacaat 1620 ttcgagtcca ttttgaacca ggagtacaag aaggcgccgc tggtgaacac ccggttcgag 1680 ggccacttct ggaacaagtt tatcaaaatg tgggttcagc aggaccagtg tgaaatgtta 1740 cagattgagg agaatgtgga ccagcgggca gtgagggaaa acggtgaacc cacaactttt 1800 ggcgcttgtt atcgaccgga gtatctcaag aggtatcacg agacccacaa gatgagtaag 1860 gctcatgagg gggagatgga ggcaatcagg caaaaggccg atgtttggga atggattcgc 1920 gaggagtttg agtag 1935 <210> 2 <211> 644 <212> PRT <213> Yarrowia lipolytica YlMpo1 <400> 2 Met Val Leu His Pro Phe Arg Leu Leu Arg Thr Ser Leu Val Ser Lys   1 5 10 15 Leu Val Val Ile Leu Ile Thr Cys Leu Ile Phe Gly Ser Leu Leu Asn              20 25 30 Leu Thr Asp Lys Leu Pro Asp Gly Val Lys Ser Arg Val Ala Tyr Met          35 40 45 Thr Asp Val Gly Leu Val Ser Gly Gly Ala Arg Ser Lys Ala Met Ala      50 55 60 Gly Asn Ser Ser Val Arg Ile Ser His Val Pro Leu Thr Lys Ile Lys  65 70 75 80 Phe Ala Glu Asn Glu Lys Glu Phe Asn Pro Lys Trp Ala Gln Lys Lys                  85 90 95 Ala Leu Asp Ser Ser Ser Asp Tyr Ser Phe Glu Trp Lys Asp Trp Val             100 105 110 Asp Leu Thr Glu Val Thr Gly Leu Phe Arg Ala Ile Glu Val Gly Ser         115 120 125 Cys Gly Lys Asn Lys Gln Cys Leu Ser Lys Leu Gln Val Thr Gly Pro     130 135 140 Pro Thr Gln Val Glu Pro Gln Ala Phe Phe Asn Arg Val Gly Lys Val 145 150 155 160 Phe Leu Asp Gln Thr Met Pro Lys Pro Lys Gln Leu Ile Tyr Leu Thr                 165 170 175 Glu Lys Glu Ser Arg Gly Lys Arg Glu Glu Pro Val Glu Ile Glu Ser             180 185 190 His Val Pro Val Val Arg Glu Pro Glu Leu Gly Asp Phe Ser Arg Ser         195 200 205 Arg Asp Ala Lys Ser Ile Gln Asn Ala Lys Arg Glu Ala Thr Glu Glu     210 215 220 Ala Thr Gly Glu Ser Ala Asn Glu Gly Gln Ser Arg Asp Ser Ala Arg 225 230 235 240 Ala Ser Ala Arg Asp Ile Ala Phe Ser Glu Ala Asp Pro Val His Glu                 245 250 255 Asp Ser Tyr Asp Asp Asp Glu Asn Gly Thr Ile Leu Val Pro Thr Ala             260 265 270 Glu Ser Glu Glu Glu Leu Gln Glu Tyr Ala Asp Lys Asp Ala Ala Ala         275 280 285 Lys Ser Tyr Leu Arg Ser Gly Trp Ser Arg Gly Gln Lys Tyr Val Glu     290 295 300 Leu Pro Arg Glu Leu Phe Thr Trp Asp Ile His Glu Glu Ile Asp Lys 305 310 315 320 Gly Leu Thr Lys Lys Ser Val Asp Ser Asp Asp Pro Ser Arg Glu Gln                 325 330 335 Val Ala His Ser Gln Phe Leu Ser Gln His Trp Lys His Ile Lys Lys             340 345 350 Ser Gly Lys His Phe Ser Glu Ala Trp Val Val Gly Asp Thr Lys Ala         355 360 365 Ala Gly Val His Tyr Asp Trp Arg Phe Phe Ser Glu Leu Asn Thr Ile     370 375 380 Asp Glu Lys Arg Val Ile Leu Arg Lys Leu Val Arg Ala Trp Leu Asp 385 390 395 400 Phe Thr Ser Arg Glu Gly Ile Ile Thr Trp Leu Ala His Gly Thr Leu                 405 410 415 Leu Gly Trp Tyr Trp Asn Gly Gln Ser Leu Pro Trp Asp Phe Asp Gly             420 425 430 Asp Val Gln Met Pro Ile Arg Glu Phe Asp Arg Phe Ala Arg Leu Tyr         435 440 445 Asn Gln Ser Leu Val Ile Asp Glu Ser Ala Gly Gly Arg Tyr Tyr Val     450 455 460 Asp Val Gly Pro Ser Tyr Val Glu Arg Leu Arg Gly Asn Gly Lys Asn 465 470 475 480 Val Ile Asp Ala Arg Phe Ile Asp Val Asp Ser Gly Met Tyr Ile Asp                 485 490 495 Ile Thr Ala Leu Ala Tyr Ala Glu Gln Gln Glu Lys Phe His Cys Lys             500 505 510 Asn Trp His Arg Tyr Glu Leu Glu Ser Val Ser Pro Leu Arg Arg Thr         515 520 525 Leu Phe Glu Gly Lys Glu Ala Tyr Ile Pro Asn Asn Phe Glu Ser Ile     530 535 540 Leu Asn Gln Glu Tyr Lys Lys Ala Pro Leu Val Asn Thr Arg Phe Glu 545 550 555 560 Gly His Phe Trp Asn Lys Phe Ile Lys Met Trp Val Gln Gln Asp Gln                 565 570 575 Cys Glu Met Leu Gln Ile Glu Glu Asn Val Asp Gln Arg Ala Val Arg             580 585 590 Glu Asn Gly Glu Pro Thr Thr Phe Gly Ala Cys Tyr Arg Pro Glu Tyr         595 600 605 Leu Lys Arg Tyr His Glu Thr His Lys Met Ser Lys Ala His Glu Gly     610 615 620 Glu Met Glu Ala Ile Arg Gln Lys Ala Asp Val Trp Glu Trp Ile Arg 625 630 635 640 Glu Glu Phe Glu                 <210> 3 <211> 1935 <212> DNA <213> Yarrowia lipolytica YlMPO1 gene <400> 3 atggtgctgc acccgtttcg cctgctgcgc acatctcttg tgagcaagct cgtcgtcatt 60 ctcataacct gtctgatttt cggcagctta ctcaacttga ccgacaagct gcccgacgga 120 gtcaagtcac gggtcgccta catgaccgac gtgggcttag tcagcggcgg tgcccgctcg 180 aaagccatgg ccggcaactc gtctgtgcgc atcagtcacg tgccactgac gaaaatcaag 240 tttgccgaaa acgaaaagga attcaacccc aagtgggccc agaagaaggc tctggactcg 300 tcgtcggatt actcattcga gtggaaagac tgggtcgact tgaccgaggt cacgggtcta 360 ttcagagcca tcgaggtcgg ctcgtgtggc aaaaacaaac aatgccttag taagctccag 420 gtcaccggac ccccgactca ggtggagccc caggcgtttt tcaaccgggt cggaaaggtg 480 tttttggacc agaccatgcc caagccgaaa cagttgattt atctgaccga gaaagagtca 540 cgtggcaagc gggaggagcc cgtcgagata gagtcacatg ttccggtggt tcgggagcct 600 gagttgggcg actttagccg atcgcgtgac gccaagtcaa tccaacatgc taagagggag 660 gcgactgagg aggcgactgg tgagtcagcc aacgagggcc agtcacgtga ctccgcacga 720 gcctccgcgc gtgacattgc cttttccgag gctgaccccg tgcacgaaga ctcgtatgac 780 gacgacgaaa acggaaccat tctcgtgccg acggctgagt cagaggagga gctccaggaa 840 tacgcagaca aggacgcggc cgccaagtcg tacctgcgaa gcggctggtc acgtggccag 900 aaatatgtcg agctgccgcg cgagctgttc acttgggaca ttcacgaaga gattgacaag 960 ggactgacta agaagagcgt tgactcagac gacccgtcac gtgagcaggt tgcgcactcg 1020 cagtttctta gtcagcattg gaaacacatc aaaaagtccg gcaagcattt ctccgaagcg 1080 tgggttgtgg gcgacaccaa agcagccgga gtccattacg actggcgatt tttcagcgag 1140 ttaaacacca ttgacgagaa acgagtcatc ttgcgtaaat tggtgcgtgc gtggctcgac 1200 ttcacgtcac gtgagggcat tatcacgtgg ctggcgcatg gcacgttgct gggctggtac 1260 tggaacggcc agtctctgcc gtgggatttc gacggtgatg tccagatgcc gatccgcgaa 1320 ttcgaccggt ttgcccgcct ctataatcag tcgttggtaa ttgatgagtc agccggcggc 1380 cggtattatg tcgatgtggg tccctcctac gtcgagcgcc tccgaggcaa cggcaagaat 1440 gtcattgatg ctcggtttat cgacgttgat agtggcatgt acattgacat taccgccttg 1500 gcgtatgccg agcagcagga aaagttccac tgcaaaaact ggcatcggta tgagttggag 1560 agcgtttctc cgctgcgtcg gacgctgttt gagggcaagg aggcttacat tccaaacaat 1620 ttcgagtcca ttttgaacca ggagtacaag aaggcgccgc tggtgaacac ccggttcgag 1680 ggccacttct ggaacaagtt tatcaaaatg tgggttcagc aggaccagtg tgaaatgtta 1740 cagattgagg agaatgtgga ccagcgggca gtgagggaaa acggtgaacc cacaactttt 1800 ggcgcttgtt atcgaccgga gtatctcaag aggtatcacg agacccacaa gatgagtaag 1860 gctcatgagg gggagatgga ggcaatcagg caaaaggccg atgtttggga atggattcgc 1920 gaggagtttg agtag 1935 <210> 4 <211> 644 <212> PRT <213> Yarrowia lipolytica YlMpo1 <400> 4 Met Val Leu His Pro Phe Arg Leu Leu Arg Thr Ser Leu Val Ser Lys   1 5 10 15 Leu Val Val Ile Leu Ile Thr Cys Leu Ile Phe Gly Ser Leu Leu Asn              20 25 30 Leu Thr Asp Lys Leu Pro Asp Gly Val Lys Ser Arg Val Ala Tyr Met          35 40 45 Thr Asp Val Gly Leu Val Ser Gly Gly Ala Arg Ser Lys Ala Met Ala      50 55 60 Gly Asn Ser Ser Val Arg Ile Ser His Val Pro Leu Thr Lys Ile Lys  65 70 75 80 Phe Ala Glu Asn Glu Lys Glu Phe Asn Pro Lys Trp Ala Gln Lys Lys                  85 90 95 Ala Leu Asp Ser Ser Ser Asp Tyr Ser Phe Glu Trp Lys Asp Trp Val             100 105 110 Asp Leu Thr Glu Val Thr Gly Leu Phe Arg Ala Ile Glu Val Gly Ser         115 120 125 Cys Gly Lys Asn Lys Gln Cys Leu Ser Lys Leu Gln Val Thr Gly Pro     130 135 140 Pro Thr Gln Val Glu Pro Gln Ala Phe Phe Asn Arg Val Gly Lys Val 145 150 155 160 Phe Leu Asp Gln Thr Met Pro Lys Pro Lys Gln Leu Ile Tyr Leu Thr                 165 170 175 Glu Lys Glu Ser Arg Gly Lys Arg Glu Glu Pro Val Glu Ile Glu Ser             180 185 190 His Val Pro Val Val Arg Glu Pro Glu Leu Gly Asp Phe Ser Arg Ser         195 200 205 Arg Asp Ala Lys Ser Ile Gln His Ala Lys Arg Glu Ala Thr Glu Glu     210 215 220 Ala Thr Gly Glu Ser Ala Asn Glu Gly Gln Ser Arg Asp Ser Ala Arg 225 230 235 240 Ala Ser Ala Arg Asp Ile Ala Phe Ser Glu Ala Asp Pro Val His Glu                 245 250 255 Asp Ser Tyr Asp Asp Asp Glu Asn Gly Thr Ile Leu Val Pro Thr Ala             260 265 270 Glu Ser Glu Glu Glu Leu Gln Glu Tyr Ala Asp Lys Asp Ala Ala Ala         275 280 285 Lys Ser Tyr Leu Arg Ser Gly Trp Ser Arg Gly Gln Lys Tyr Val Glu     290 295 300 Leu Pro Arg Glu Leu Phe Thr Trp Asp Ile His Glu Glu Ile Asp Lys 305 310 315 320 Gly Leu Thr Lys Lys Ser Val Asp Ser Asp Asp Pro Ser Arg Glu Gln                 325 330 335 Val Ala His Ser Gln Phe Leu Ser Gln His Trp Lys His Ile Lys Lys             340 345 350 Ser Gly Lys His Phe Ser Glu Ala Trp Val Val Gly Asp Thr Lys Ala         355 360 365 Ala Gly Val His Tyr Asp Trp Arg Phe Phe Ser Glu Leu Asn Thr Ile     370 375 380 Asp Glu Lys Arg Val Ile Leu Arg Lys Leu Val Arg Ala Trp Leu Asp 385 390 395 400 Phe Thr Ser Arg Glu Gly Ile Ile Thr Trp Leu Ala His Gly Thr Leu                 405 410 415 Leu Gly Trp Tyr Trp Asn Gly Gln Ser Leu Pro Trp Asp Phe Asp Gly             420 425 430 Asp Val Gln Met Pro Ile Arg Glu Phe Asp Arg Phe Ala Arg Leu Tyr         435 440 445 Asn Gln Ser Leu Val Ile Asp Glu Ser Ala Gly Gly Arg Tyr Tyr Val     450 455 460 Asp Val Gly Pro Ser Tyr Val Glu Arg Leu Arg Gly Asn Gly Lys Asn 465 470 475 480 Val Ile Asp Ala Arg Phe Ile Asp Val Asp Ser Gly Met Tyr Ile Asp                 485 490 495 Ile Thr Ala Leu Ala Tyr Ala Glu Gln Gln Glu Lys Phe His Cys Lys             500 505 510 Asn Trp His Arg Tyr Glu Leu Glu Ser Val Ser Pro Leu Arg Arg Thr         515 520 525 Leu Phe Glu Gly Lys Glu Ala Tyr Ile Pro Asn Asn Phe Glu Ser Ile     530 535 540 Leu Asn Gln Glu Tyr Lys Lys Ala Pro Leu Val Asn Thr Arg Phe Glu 545 550 555 560 Gly His Phe Trp Asn Lys Phe Ile Lys Met Trp Val Gln Gln Asp Gln                 565 570 575 Cys Glu Met Leu Gln Ile Glu Glu Asn Val Asp Gln Arg Ala Val Arg             580 585 590 Glu Asn Gly Glu Pro Thr Thr Phe Gly Ala Cys Tyr Arg Pro Glu Tyr         595 600 605 Leu Lys Arg Tyr His Glu Thr His Lys Met Ser Lys Ala His Glu Gly     610 615 620 Glu Met Glu Ala Ile Arg Gln Lys Ala Asp Val Trp Glu Trp Ile Arg 625 630 635 640 Glu Glu Phe Glu                 <210> 5 <211> 1794 <212> DNA <213> Yarrowia lipolytica YlMPO1 gene <400> 5 atgaccgacg tgggcttagt cagcggcggt gcccgctcga aagccatggc cggcaactcg 60 tctgtgcgca tcagtcacgt gccactgacg aaaatcaagt ttgccgaaaa cgaaaaggaa 120 ttcaacccca agtgggccca gaagaaggct ctggactcgt cgtcggatta ctcattcgag 180 tggaaagact gggtcgactt gaccgaggtc acgggtctat tcagagccat cgaggtcggc 240 tcgtgtggca aaaacaaaca atgccttagt aagctccagg tcaccggacc cccgactcag 300 gtggagcccc aggcgttttt caaccgggtc ggaaaggtgt ttttggacca gaccatgccc 360 aagccgaaac agttgattta tctgaccgag aaagagtcac gtggcaagcg ggaggagccc 420 gtcgagatag agtcacatgt tccggtggtt cgggagcctg agttgggcga ctttagccga 480 tcgcgtgacg ccaagtcaat ccaacatgct aagagggagg cgactgagga ggcgactggt 540 gagtcagcca acgagggcca gtcacgtgac tccgcacgag cctccgcgcg tgacattgcc 600 ttttccgagg ctgaccccgt gcacgaagac tcgtatgacg acgacgaaaa cggaaccatt 660 ctcgtgccga cggctgagtc agaggaggag ctccaggaat acgcagacaa ggacgcggcc 720 gccaagtcgt acctgcgaag cggctggtca cgtggccaga aatatgtcga gctgccgcgc 780 gagctgttca cttgggacat tcacgaagag attgacaagg gactgactaa gaagagcgtt 840 gactcagacg acccgtcacg tgagcaggtt gcgcactcgc agtttcttag tcagcattgg 900 aaacacatca aaaagtccgg caagcatttc tccgaagcgt gggttgtggg cgacaccaaa 960 gcagccggag tccattacga ctggcgattt ttcagcgagt taaacaccat tgacgagaaa 1020 cgagtcatct tgcgtaaatt ggtgcgtgcg tggctcgact tcacgtcacg tgagggcatt 1080 atcacgtggc tggcgcatgg cacgttgctg ggctggtact ggaacggcca gtctctgccg 1140 tgggatttcg acggtgatgt ccagatgccg atccgcgaat tcgaccggtt tgcccgcctc 1200 tataatcagt cgttggtaat tgatgagtca gccggcggcc ggtattatgt cgatgtgggt 1260 ccctcctacg tcgagcgcct ccgaggcaac ggcaagaatg tcattgatgc tcggtttatc 1320 gacgttgata gtggcatgta cattgacatt accgccttgg cgtatgccga gcagcaggaa 1380 aagttccact gcaaaaactg gcatcggtat gagttggaga gcgtttctcc gctgcgtcgg 1440 acgctgtttg agggcaagga ggcttacatt ccaaacaatt tcgagtccat tttgaaccag 1500 gagtacaaga aggcgccgct ggtgaacacc cggttcgagg gccacttctg gaacaagttt 1560 atcaaaatgt gggttcagca ggaccagtgt gaaatgttac agattgagga gaatgtggac 1620 cagcgggcag tgagggaaaa cggtgaaccc acaacttttg gcgcttgtta tcgaccggag 1680 tatctcaaga ggtatcacga gacccacaag atgagtaagg ctcatgaggg ggagatggag 1740 gcaatcaggc aaaaggccga tgtttgggaa tggattcgcg aggagtttga gtag 1794 <210> 6 <211> 597 <212> PRT <213> Yarrowia lipolytica YlMpo1 <400> 6 Met Thr Asp Val Gly Leu Val Ser Gly Gly Ala Arg Ser Ser Ays Met   1 5 10 15 Ala Gly Asn Ser Ser Val Arg Ile Ser His Val Pro Leu Thr Lys Ile              20 25 30 Lys Phe Ala Glu Asn Glu Lys Glu Phe Asn Pro Lys Trp Ala Gln Lys          35 40 45 Lys Ala Leu Asp Ser Ser Ser Asp Tyr Ser Phe Glu Trp Lys Asp Trp      50 55 60 Val Asp Leu Thr Glu Val Thr Gly Leu Phe Arg Ala Ile Glu Val Gly  65 70 75 80 Ser Cys Gly Lys Asn Lys Gln Cys Leu Ser Lys Leu Gln Val Thr Gly                  85 90 95 Pro Pro Thr Gln Val Glu Pro Gln Ala Phe Phe Asn Arg Val Gly Lys             100 105 110 Val Phe Leu Asp Gln Thr Met Pro Lys Pro Lys Gln Leu Ile Tyr Leu         115 120 125 Thr Glu Lys Glu Ser Arg Gly Lys Arg Glu Glu Pro Val Glu Ile Glu     130 135 140 Ser His Val Val Val Val Arg Glu Pro Glu Leu Gly Asp Phe Ser Arg 145 150 155 160 Ser Arg Asp Ala Lys Ser Ile Gln His Ala Lys Arg Glu Ala Thr Glu                 165 170 175 Glu Ala Thr Gly Glu Ser Ala Asn Glu Gly Gln Ser Arg Asp Ser Ala             180 185 190 Arg Ala Ser Ala Arg Asp Ile Ala Phe Ser Glu Ala Asp Pro Val His         195 200 205 Glu Asp Ser Tyr Asp Asp Asp Glu Asn Gly Thr Ile Leu Val Pro Thr     210 215 220 Ala Glu Ser Glu Glu Glu Leu Gln Glu Tyr Ala Asp Lys Asp Ala Ala 225 230 235 240 Ala Lys Ser Tyr Leu Arg Ser Gly Trp Ser Arg Gly Gln Lys Tyr Val                 245 250 255 Glu Leu Pro Arg Glu Leu Phe Thr Trp Asp Ile His Glu Glu Ile Asp             260 265 270 Lys Gly Leu Thr Lys Lys Ser Val Asp Ser Asp Asp Pro Ser Arg Glu         275 280 285 Gln Val Ala His Ser Gln Phe Leu Ser Gln His Trp Lys His Ile Lys     290 295 300 Lys Ser Gly Lys His Phe Ser Glu Ala Trp Val Val Gly Asp Thr Lys 305 310 315 320 Ala Ala Gly Val His Tyr Asp Trp Arg Phe Phe Ser Glu Leu Asn Thr                 325 330 335 Ile Asp Glu Lys Arg Val Ile Leu Arg Lys Leu Val Arg Ala Trp Leu             340 345 350 Asp Phe Thr Ser Arg Glu Gly Ile Ile Thr Trp Leu Ala His Gly Thr         355 360 365 Leu Leu Gly Trp Tyr Trp Asn Gly Gln Ser Leu Pro Trp Asp Phe Asp     370 375 380 Gly Asp Val Gln Met Pro Ile Arg Glu Phe Asp Arg Phe Ala Arg Leu 385 390 395 400 Tyr Asn Gln Ser Leu Val Ile Asp Glu Ser Ala Gly Gly Arg Tyr Tyr                 405 410 415 Val Asp Val Gly Pro Ser Tyr Val Glu Arg Leu Arg Gly Asn Gly Lys             420 425 430 Asn Val Ile Asp Ala Arg Phe Ile Asp Val Asp Ser Gly Met Tyr Ile         435 440 445 Asp Ile Thr Ala Leu Ala Tyr Ala Glu Gln Gln Glu Lys Phe His Cys     450 455 460 Lys Asn Trp His Arg Tyr Glu Leu Glu Ser Val Ser Pro Leu Arg Arg 465 470 475 480 Thr Leu Phe Glu Gly Lys Glu Ala Tyr Ile Pro Asn Asn Phe Glu Ser                 485 490 495 Ile Leu Asn Gln Glu Tyr Lys Lys Ala Pro Leu Val Asn Thr Arg Phe             500 505 510 Glu Gly His Phe Trp Asn Lys Phe Ile Lys Met Trp Val Gln Gln Asp         515 520 525 Gln Cys Glu Met Leu Gln Ile Glu Glu Asn Val Asp Gln Arg Ala Val     530 535 540 Arg Glu Asn Gly Glu Pro Thr Thr Phe Gly Ala Cys Tyr Arg Pro Glu 545 550 555 560 Tyr Leu Lys Arg Tyr His Glu Thr His Lys Met Ser Lys Ala His Glu                 565 570 575 Gly Glu Met Glu Ala Ile Arg Gln Lys Ala Asp Val Trp Glu Trp Ile             580 585 590 Arg Glu Glu Phe Glu         595 <210> 7 <211> 1755 <212> DNA <213> Yarrowia lipolytica YlMPO1 gene <400> 7 aaagccatgg ccggcaactc gtctgtgcgc atcagtcacg tgccactgac gaaaatcaag 60 tttgccgaaa acgaaaagga attcaacccc aagtgggccc agaagaaggc tctggactcg 120 tcgtcggatt actcattcga gtggaaagac tgggtcgact tgaccgaggt cacgggtcta 180 ttcagagcca tcgaggtcgg ctcgtgtggc aaaaacaaac aatgccttag taagctccag 240 gtcaccggac ccccgactca ggtggagccc caggcgtttt tcaaccgggt cggaaaggtg 300 tttttggacc agaccatgcc caagccgaaa cagttgattt atctgaccga gaaagagtca 360 cgtggcaagc gggaggagcc cgtcgagata gagtcacatg ttccggtggt tcgggagcct 420 gagttgggcg actttagccg atcgcgtgac gccaagtcaa tccaacatgc taagagggag 480 gcgactgagg aggcgactgg tgagtcagcc aacgagggcc agtcacgtga ctccgcacga 540 gcctccgcgc gtgacattgc cttttccgag gctgaccccg tgcacgaaga ctcgtatgac 600 gacgacgaaa acggaaccat tctcgtgccg acggctgagt cagaggagga gctccaggaa 660 tacgcagaca aggacgcggc cgccaagtcg tacctgcgaa gcggctggtc acgtggccag 720 aaatatgtcg agctgccgcg cgagctgttc acttgggaca ttcacgaaga gattgacaag 780 ggactgacta agaagagcgt tgactcagac gacccgtcac gtgagcaggt tgcgcactcg 840 cagtttctta gtcagcattg gaaacacatc aaaaagtccg gcaagcattt ctccgaagcg 900 tgggttgtgg gcgacaccaa agcagccgga gtccattacg actggcgatt tttcagcgag 960 ttaaacacca ttgacgagaa acgagtcatc ttgcgtaaat tggtgcgtgc gtggctcgac 1020 ttcacgtcac gtgagggcat tatcacgtgg ctggcgcatg gcacgttgct gggctggtac 1080 tggaacggcc agtctctgcc gtgggatttc gacggtgatg tccagatgcc gatccgcgaa 1140 ttcgaccggt ttgcccgcct ctataatcag tcgttggtaa ttgatgagtc agccggcggc 1200 cggtattatg tcgatgtggg tccctcctac gtcgagcgcc tccgaggcaa cggcaagaat 1260 gtcattgatg ctcggtttat cgacgttgat agtggcatgt acattgacat taccgccttg 1320 gcgtatgccg agcagcagga aaagttccac tgcaaaaact ggcatcggta tgagttggag 1380 agcgtttctc cgctgcgtcg gacgctgttt gagggcaagg aggcttacat tccaaacaat 1440 ttcgagtcca ttttgaacca ggagtacaag aaggcgccgc tggtgaacac ccggttcgag 1500 ggccacttct ggaacaagtt tatcaaaatg tgggttcagc aggaccagtg tgaaatgtta 1560 cagattgagg agaatgtgga ccagcgggca gtgagggaaa acggtgaacc cacaactttt 1620 ggcgcttgtt atcgaccgga gtatctcaag aggtatcacg agacccacaa gatgagtaag 1680 gctcatgagg gggagatgga ggcaatcagg caaaaggccg atgtttggga atggattcgc 1740 gaggagtttg agtag 1755 <210> 8 <211> 584 <212> PRT <213> Yarrowia lipolytica YlMpo1 <400> 8 Lys Ala Met Ala Gly Asn Ser Ser Val Arg Ile Ser His Val Leu   1 5 10 15 Thr Lys Ile Lys Phe Ala Glu Asn Glu Lys Glu Phe Asn Pro Lys Trp              20 25 30 Ala Gln Lys Lys Ala Leu Asp Ser Ser Ser Asp Tyr Ser Phe Glu Trp          35 40 45 Lys Asp Trp Val Asp Leu Thr Glu Val Thr Gly Leu Phe Arg Ala Ile      50 55 60 Glu Val Gly Ser Cys Gly Lys Asn Lys Gln Cys Leu Ser Lys Leu Gln  65 70 75 80 Val Thr Gly Pro Pro Thr Gln Val Glu Pro Gln Ala Phe Phe Asn Arg                  85 90 95 Val Gly Lys Val Phe Leu Asp Gln Thr Met Pro Lys Pro Lys Gln Leu             100 105 110 Ile Tyr Leu Thr Glu Lys Glu Ser Arg Gly Lys Arg Glu Glu Pro Val         115 120 125 Glu Ile Glu Ser His Val Val Val Val Arg Glu Pro Glu Leu Gly Asp     130 135 140 Phe Ser Arg Ser Ser Asp Ala Lys Ser Ile Gln His Ala Lys Arg Glu 145 150 155 160 Ala Thr Glu Glu Ala Thr Gly Glu Ser Ala Asn Glu Gly Gln Ser Arg                 165 170 175 Asp Ser Ala Arg Ala Ser Ala Arg Asp Ile Ala Phe Ser Glu Ala Asp             180 185 190 Pro Val His Glu Asp Ser Tyr Asp Asp Asp Glu Asn Gly Thr Ile Leu         195 200 205 Val Pro Thr Ala Glu Ser Glu Glu Glu Leu Gln Glu Tyr Ala Asp Lys     210 215 220 Asp Ala Ala Lys Ser Tyr Leu Arg Ser Gly Trp Ser Arg Gly Gln 225 230 235 240 Lys Tyr Val Glu Leu Pro Arg Glu Leu Phe Thr Trp Asp Ile His Glu                 245 250 255 Glu Ile Asp Lys Gly Leu Thr Lys Lys Ser Val Asp Ser Asp Asp Pro             260 265 270 Ser Arg Glu Gln Val Ala His Ser Gln Phe Leu Ser Gln His Trp Lys         275 280 285 His Ile Lys Lys Ser Gly Lys His Phe Ser Glu Ala Trp Val Val Gly     290 295 300 Asp Thr Lys Ala Ala Gly Val His Tyr Asp Trp Arg Phe Phe Ser Glu 305 310 315 320 Leu Asn Thr Ile Asp Glu Lys Arg Val Ile Leu Arg Lys Leu Val Arg                 325 330 335 Ala Trp Leu Asp Phe Thr Ser Arg Glu Gly Ile Ile Thr Trp Leu Ala             340 345 350 His Gly Thr Leu Leu Gly Trp Tyr Trp Asn Gly Gln Ser Leu Pro Trp         355 360 365 Asp Phe Asp Gly Asp Val Gln Met Pro Ile Arg Glu Phe Asp Arg Phe     370 375 380 Ala Arg Leu Tyr Asn Gln Ser Leu Val Ile Asp Glu Ser Ala Gly Gly 385 390 395 400 Arg Tyr Tyr Val Asp Val Gly Pro Ser Tyr Val Glu Arg Leu Arg Gly                 405 410 415 Asn Gly Lys Asn Val Ile Asp Ala Arg Phe Ile Asp Val Asp Ser Gly             420 425 430 Met Tyr Ile Asp Ile Thr Ala Leu Ala Tyr Ala Glu Gln Gln Glu Lys         435 440 445 Phe His Cys Lys Asn Trp His Arg Tyr Glu Leu Glu Ser Val Ser Pro     450 455 460 Leu Arg Arg Thr Leu Phe Glu Gly Lys Glu Ala Tyr Ile Pro Asn Asn 465 470 475 480 Phe Glu Ser Ile Leu Asn Gln Glu Tyr Lys Lys Ala Pro Leu Val Asn                 485 490 495 Thr Arg Phe Glu Gly His Phe Trp Asn Lys Phe Ile Lys Met Trp Val             500 505 510 Gln Gln Asp Gln Cys Glu Met Leu Gln Ile Glu Glu Asn Val Asp Gln         515 520 525 Arg Ala Val Arg Glu Asn Gly Glu Pro Thr Thr Phe Gly Ala Cys Tyr     530 535 540 Arg Pro Glu Tyr Leu Lys Arg Tyr His Glu Thr His Lys Met Ser Lys 545 550 555 560 Ala His Glu Gly Glu Glu Ala Ile Arg Gln Lys Ala Asp Val Trp                 565 570 575 Glu Trp Ile Arg Glu Glu Phe Glu             580 <210> 9 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> YlMPO1 HindIII 1F <400> 9 cccaagcttc gatgaccgac gtgggcttag t 31 <210> 10 <211> 31 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > YlMPO1 HindIII 2F <400> 10 cccaagcttc gaaagccatg gccggcaact c 31 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> YlMPO1 XhoI R <400> 11 ccgctcgagc tcaaactcct cgcgaatcca 30 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> YlMPO1 SgfI F <400> 12 acggcgatcg ccatgaccga cgt 23 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> YlMPO1 BamHI R <400> 13 agcggatccc tactcaaact cctcg 25

Claims (22)

A recombinant microorganism having the ability to produce YlMpo1, characterized in that a gene encoding YlMpo1 or a recombinant vector containing the gene is introduced into a host cell.
2. The method of claim 1, wherein YlMpo1 is Yarrowia &lt; RTI ID = 0.0 &gt; lipolytica . &lt; / RTI &gt;
2. The recombinant microorganism according to claim 1, wherein YlMpo1 is YlMpo1 represented by the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO:
4. The method according to claim 3, wherein YlMpo1 represented by the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 is a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: Wherein the recombinant microorganism is encoded by a gene to be expressed.
2. The recombinant microorganism according to claim 1, wherein the recombinant vector containing the gene encoding YlMpo1 is pET21-YlMpo1-dTM1, pET21-YlMpo1-dTM2 or pFN18A-YlMpo1.
The method of claim 1, wherein the host cell is Escherichia coli wherein the recombinant microorganism is E. coli .
The recombinant microorganism according to claim 1, wherein the recombinant microorganism into which the recombinant vector pFN18A-YlMpo1 containing the gene encoding YlMpo1 is introduced into the host cell is KCTC12493BP.
A method for adding mannose-6-phosphate to an oligosaccharide using a YlMpo1 protein comprising the steps of:
(a) adding mannose-6-phosphate-1-mannose to the oligosaccharide using YlMpo1; And
(b) removing the outer mannoside of the mannose-6-phosphate-1-mannose-added oligosaccharide to produce a mannose-6-phosphate-added oligosaccharide.
[Claim 9] The method according to claim 8, wherein the YlMpo1 is derived from Yarrowia lipolytica , which is represented by the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 6-phosphoric acid is added.
The method according to claim 8, wherein the oligosaccharide of step (a) is Man _{3 -9} GlcNAc ₂ (M3-9).
9. The method according to claim 8, wherein the step of adding mannose-6-phosphate-1-mannose to the sugar chain of step (a) further comprises adding a buffer solution, a metal ion and GDP- mannose. 6-phosphoric acid is added to mannose-6-phosphate.
12. The method of claim 11, wherein the buffer solution is sodium phosphate at a pH of from 5.0 to 8.0.
12. The method according to claim 11, wherein the metal ion is calcium ion or manganese ion.
12. The method according to claim 11, wherein the step of adding mannose-6-phosphate-1-mannose to the sugar chain of step (a) further comprises adding a surfactant. .
15. The method of claim 14, wherein the surfactant is selected from the group consisting of nonyl phenoxypolyethoxylethanol (NP-40), polyoxyethylene octyl phenyl ether (Triton X-100), polyoxyethylene Polyoxyethylene sorbitan monolaurate (Tween-20), polyoxyethylene (23) lauryl ether (Brij-35) and 3 - [(3-cholamidopropyl) (3 - [(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate (CHAPS).
[9] The method according to claim 8, wherein the step of removing the outer mannoside of the oligosaccharide to which mannose-6-phosphate-1-mannose is added is performed using a weak acid hydrolysis or uncapping enzyme And adding mannose-6-phosphate to the sugar chain.
17. The method of claim 16, wherein the weak acid is a formic acid of 0.1 to 5 M, and the uncapping enzyme is a-mannosidase. -6-phosphoric acid.
17. A method for producing a glycoprotein, which comprises adding mannose-6-phosphate by the method according to any one of claims 8 to 17.
17. A method for producing a glycoprotein, which comprises chemically or physically bonding a mannose-6-phosphate-added oligosaccharide prepared by the method of any one of claims 8 to 17 to a protein.
The method of claim 18 or 19, wherein the protein is selected from the group consisting of pathogen protein, lysosomal protein, growth factor, cytokine, chemokine, An antigen-binding fragment thereof, or a fusion protein. &Lt; Desc / Clms Page number 20 &gt;
The method according to claim 20, wherein the lysosomal protein is a lysosomal storage disease-related degradation enzyme.
17. A method for producing a mannose-6-phosphate-added oligosaccharide, which comprises combining a mannose-6-phosphate-added oligosaccharide prepared by the method of any one of claims 8 to 17 with a peptide, lipid or nanomaterial for encapsulation, Wherein the method comprises the steps of:

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