KR20020097082A - Transformed yeast system for developing therapeutic hybrid protein drug by site-specific modification - Google Patents

Abstract

PURPOSE: A transformed yeast system for developing fusion glycoproteins by a site-specific modification method is provided, thereby cheaply mass producing the fusion glycoproteins having improved medical activity and titer. CONSTITUTION: Transformed yeast Saccharomyces cerevisiae MPY(KCCM-10441) has no genes which are related to the addition of sugar chain, such as OCH1(SEQ ID NO: 1), ANP1(SEQ ID NO: 2), MNN11(SEQ ID NO: 3), HOC1(SEQ ID NO: 4) and MNN2(SEQ ID NO: 5), for expression of partially sugar chain removed glycoproteins. The glycoproteins are produced by using the transformed yeast Saccharomyces cerevisiae MPY(KCCM-10441), wherein the sugar comprises N-acethylglucosamine; and the glycoproteins are α-, β-, and γ-interferon, asparaginase, arginase, adenosine deaminase, superoxide dismutase, catalase, lipase, adenosine diphosphatase, thyrosinase, glucose oxidase, glucosidase, blood factor VII, VIII and IX, immunoglobulin, cytokines, interleukins, granulocyte colony stimulating factor and granulocyte macrophage colony.

Description

Transformed yeast system for developing therapeutic hybrid protein drug by site-specific modification}

The present invention relates to genes involved in glycosylation of existing cells for regioselective protein modification, which is performed to confer or enhance the in vivo stability and biological activity of glycoproteins produced for therapeutic, research and industrial purposes. A method of expressing a glycoprotein in a form in which only part of the glycoprotein is removed through the artificial manipulation of the glycoprotein and attaching only N-acetylglucosamine using a transgenic yeast system, and more specifically The sugar chain was added in a form in which only acetylglucosamine was attached, so that the sugar chain was removed using a genetically modified transgenic yeast strain. The functional groups distinguished in the reactivity of the reaction JPY - to provide a transformed yeast systems for producing glycoproteins that only the Liberal Democratic Party-acetyl-glucoside added.

That is, the sugar chain of the existing glycoprotein (sugar chain; oligosaccharide chain) by modifying the protein produced by the oligosaccharides removed by the synthetic or natural polymer material useful to the human body and added with only acetyl glucosamine to site-specific ) To replace or supplement the role of) to increase the length of stay in vivo, such as blood, thereby making the protein chains applicable to the manufacture of protein hybrids for conferring or enhancing medical and activity, biological efficacy and activity. The functional group is to provide a transformed yeast system for producing glycoproteins in which only N-acetylglucosamine having a functional group distinguished in reactivity in a modified reaction using a polymer material is added.

To date, various attempts have been made to enhance the in vivo bioactivity of therapeutic proteins. The main purpose of these methods was to increase the size of the therapeutic protein. That is, a method of increasing the size of protein molecules by adding natural and synthetic high molecular materials to the protein chain. This method was initially used to reduce antigenicity, but is now widely used to enhance biological activity by increasing the duration of in vivo protein.

Modification of therapeutic proteins into modified substances, such as natural and synthetic polymers, offers several advantages. Most importantly, increasing the length of stay in vivo, such as plasma, increases the bioavailability of blood and tissues in vivo. Second, the modifier may mask the antigenic determinants of non-human proteins and may also alter the immunogenicity of the protein. Modification of these proteins can often change the interaction with receptors (including receptors involved in plasma clearance or immune system recognition) by forming new structures on the protein surface.

However, this conventional method binds all the amino acids with similar reactivity at positions accessible to functional groups of the modifying materials due to the nature of the chemical bonds. There is a restriction that can not be. That is, the positional specificity of the bond is either missing or very limited. In order to solve this problem of positional nonspecificity, a method of using the active structure of the protein itself has been attempted (Korean Patent Registration No. 353,392), but it is not yet generalized and cannot be applied to a formal method for all proteins. In order to solve this problem, it is necessary to introduce a functional group that is differentiated in the reactivity of the functional group with the various amino acids constituting the protein. This desired object can be achieved by applying the method proposed in the present invention.

In addition, eukaryotic cells such as yeast are capable of glycosylation reaction, but the structure of the sugar chain of the produced protein is different from the sugar chain structure found in the protein produced in Chinese hamster ovary (CHO) cells which are commonly used as therapeutic agents. Production of erythropoietin (EPO) using cells could not actually be used in commercialized production, for example, inducing antigen-antibody reactions due to differences in sugar chain structure, despite the advantage that productivity was higher than CHO cells. As described above, even in the case of producing proteins using eukaryotic cells, the structure of sugar chains added to proteins varies depending on the host cell as in the case of EPO. As a result, when the produced protein is used for medicine, it may be toxic through antigen-antibody reaction. Alternatively, due to low body titer, it is often unsuitable for therapeutic purposes and its use has been limited. Therefore, optimal production cells exist for each protein, and production in cells other than these is usually considered impossible.

In the case of proteins produced in eukaryotic cells other than CHO cells, the problem of reducing the therapeutic effect by the antigen-antibody reaction can also be improved by the method of the present invention. In other words, using a transformed strain to produce a form in which only N-acetylglucosamine is attached instead of the existing sugar chain of glycoprotein, N-acetylglucose added to the glycoprotein is a modified substance that does not induce an antigen-antibody reaction. It can be solved by site-specific modification using limine or its derivatives, and if the high productivity of these cells is used, it can be a more commercially economical production method than the conventional production method by animal cell culture.

Most therapeutic proteins, including EPO, are produced primarily in eukaryotic cells, including yeast cells, animal cells or plant cells. Most of these therapeutic proteins cannot be used for therapeutic purposes because they are produced in prokaryotic cells, such as EPO, because they lack a post-translational modification such as sugar chains. . In other words, these proteins produced in Escherichia coli lose their efficacy as a therapeutic agent because their stay in vivo is too short because they are not properly modified.

The best protein modification methods should be site specific. In other words, there is a need for a new modification method that can selectively modify proteins at desired locations. To this end, it is necessary to introduce a functional group that is different from the various functional groups in the side chain of amino acids constituting the existing protein chain in the reactivity of the modification reaction. In the present invention, for this purpose, it is intended to produce a protein in the form of attached only N-acetylglucosamine having a functional group that is distinguished in reactivity with the functional group of the amino acid. However, with the prior art developed so far, no strain has been developed that can produce this type of glycoprotein. In order to express a glycoprotein in which a part of the oligosaccharide is removed and only N-acetylglucosamine is added, a conventional gene expression system cannot achieve its purpose. Therefore, an expression system having a new sugar chain addition reaction using a new vector is required. Will be.

The technical problem to be achieved in the present invention is to remove the sugar chains that can be used to modify the protein with a modified material containing a biopolymer material only at a desired position regioselectively in order to give position selectivity in protein modification method It is to develop a novel transgenic yeast system for expressing glycoproteins with only added en-acetylglucosamine.

Therefore, an object of the present invention is the yeast genes OCH1 (SEQ ID NO: 1), ANP1 (SEQ ID NO: 2), MNN11 (SEQ ID NO. : 3), transformed yeast strain Saccharomyces cerevisiae (HOC1 (SEQ ID NO: 4), MNN2 (SEQ ID NO: 5) removed)Saccharomyces cerevisiae) It provides MPY (Accession No .: KCCM-10441).

In addition, the sugar chain is removed using the above-described transformed yeast strain (Accession No .: KCCM-10441), and the functional group of the amino acid constituting the glycoprotein in its position is different from the functional group distinguished in the reactivity of protein modification reaction. It provides a method for expressing a glycoprotein to which a sugar such as acetylglucosamine is bound.

The protein or peptide that can be produced by the present invention is not limited to a specific therapeutic protein, and any biologically active protein can be targeted. Specifically, alpha, beta, gamma interferon (?-, ??-, ??-interferon), asparaginase, arginase, adenosine deaminase, superoxide Superoxide dismutase, catalase, lipase, uricase, adenosine diphosphatase, tyrosinase, glucose oxidase, glucosidase Glucosidase, blood factors VII, VIII and IX, immunoglobulins, cytokines, ie interleukins, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), platelet derived growth factor (PDGF), lectins, lysine, tumor necrosis factor (tum or necrosis factor (TNF), transforming growth factors (TGFs), thrombopoietin (TPO) and tissue plasminogen activator (tPA).

In addition, the present invention can be applied to variants of existing therapeutic proteins such as ARANESP of Amgen, USA.

Hereinafter, the present invention will be described in more detail.

In order to perform regioselective modification only at the desired position of the protein, there is an amino acid having a functional group that is specific in reactivity with the chemicals to be used in the modified protein at the position to be modified and distinguished from other functional groups in the surroundings. shall. Proteins in which amino acids with functional groups with such specific reactivity exist only at the modified sites are naturally less likely to be used for regioselective protein modification.

Therefore, in the present invention, a sugar which can be used for regioselective protein modification, that is, the reactivity is distinct from the functional group of the amino acid in the method for regioselectively modifying the protein without affecting the amino acid of the protein chain in the protein modification (preferably Preferably only en-acetylglucosamine) is provided to provide a method for providing the added form of glycoprotein. That is, the protein is produced intracellularly by adding sugar to a specific part of the protein chain (location to be modified). That is, after the protein is expressed in a transformed yeast system in the form of monosaccharide added thereto, this site is used as a site-selective protein modification site.

When sugar sugars are used in protein modifications without the use of single sugars in protein modifications, many functional groups originating from the various sugars present in sugar chains result in the regulation and modification of modification reactions such as the number of modified substances modified in one molecule of protein. Securing the uniformity of the product is not easy, and in the case of a long sugar chain having a tertiary structure, the possibility of inducing an antigen-antibody reaction by the sugar chain is higher than that of using a monosaccharide.

In addition, sugar chains usually contain mannose or galactose, but in this case, even after modifying them with a modified substance without leaving the sugar chains contained therein, there is mannose or galactose exposed when used as a therapeutic agent. Clearance of proteins in the body, such as endocytosis, is likely to cause rapid shortening of the length of stay in the body due to the removal of proteins from the blood.

It is most preferable that the sugar is en-acetylglucosamine when the single sugar is used as a modification site for the modification reaction. In the sugar chains of most glycoproteins in nature, the first sugar in the sugar chain that binds to the protein is en-acetylglucosamine, so making a protein attached with en-acetylglucosamine is a sugar adduct with only other monosaccharides. It is easier than producing or manufacturing.

The amino acid corresponding to the position to be modified is the amino acid corresponding to the glycosylation site in the existing production cell itself, and the sugar must be attached to the amino acid when produced by the glycation reaction of the producing cell. In the present invention, the glycoprotein produced in the form in which the monosaccharide is added to the protein is referred to as "glycoside."

In the present invention, in the production of the "glycoside," using the transformed yeast strain of the present invention capable of producing glycoproteins attached with en-acetylglucosamine instead of oligosaccharide, en-acetylglucosamine instead of oligosaccharides at the existing sugar chain portion Produces glycoproteins attached only.

Hereinafter, specific examples of the therapeutic protein will be described in detail for producing EPO using the method of the present invention. However, EPO is only presented as an example of a therapeutic protein, and the present invention is not limited to EPO.

EPO is produced by removing all parts of the existing sugar chain except en-acetylglucosamine, and then modifying them with modified substances such as natural and synthetic polymers to reduce antigen-antibody reactions when administered as therapeutics. Increasing the length of time in the back can increase the biological activity. In particular, the method of removing the rest of sugar chains except specific sugars is not influenced by the glycosylation pattern inherent in the production cell, so the production strain may be freely selected without any restriction regardless of the difference in the glycosylation reaction of the production strain.

The present inventors produced EPO using the transformed yeast strain of the present invention (Accession No .: KCCM-10441). The transgenic yeast strain Saccharomyces cerevisiae MPY was deposited internationally under the Budapest Treaty with Accession No. KCCM-10441 to the Korea Microorganism Conservation Center on October 17, 2002.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are merely illustrative of the present invention and the scope of the present invention is not limited to these examples.

Example 1 Preparation of E. coli Cloning Vector for Production of Transgenic Yeast Strains

Transformed yeast of interest in the present invention is a strain that performs the sugar chain addition reaction in the form of only the en-acetylglucosamine attached to the protein produced. For the production of such a transformed strain it is necessary to remove the gene associated with the sugar chain portion. A newly produced E. coli cloning vector was used for genetic engineering to remove the yeast gene associated with the sugar chain.

The method for constructing the vector will be described in detail. The pBlueScript SK (+) (stratagen) vector is cut using a restriction enzyme Ssp I to remove a 130bp fragment located at the origin of origin, and then phenol is extracted. The remaining vector portion was purified. The purified vector portion was reacted with T4 DNA polymerase at 16 ° C. for 3 hours to reattach the cleaved portion. E. coli was transformed using this polymerase reaction solution. Recombinant vectors were isolated from the transformed Escherichia coli.

The vector prepared by the above method was removed by cleaving and removing the 448 bp DNA fragment using restriction enzyme Pvu II, and the remaining vector was purified by phenol extraction. The purified vector was reacted for 3 hours at 16 ° C. using T4 DNA polymerase to reattach the cleaved portion of the vector. E. coli was transformed using this reaction solution. Recombinant vectors were isolated from the transformed Escherichia coli. The vector produced by the above method was named pEA and subsequently used as a cloning vector for Escherichia coli during genetic manipulation for yeast gene removal.

Example 2 Preparation of Donor Vector pEA-AmyE of Yeast Gene Marker Gene

In the present invention, it is necessary to remove the yeast gene for the production of the transformed yeast strain. In order to remove the yeast gene, alpha-amylase gene (AmyE) of Bacillus subtilis strain was used as a marker gene in the present invention. In the method of obtaining the AmyE gene, a primer AM-F (5'-AAC AGC TGT TTG TAT TCA CTC TGC CAA) prepared using a chromosomal gene of the Bacillus subtilis strain as a template was prepared by DNA synthesis. AmyE gene was obtained by polymerase chain reaction using SEQ ID NO: 6) and AM-R (5'-TTC AGC TGT GCA ACA CTT CAC AAA CTT: SEQ ID NO: 7). The obtained AmyE gene was purified using phenol extraction, and then digested with restriction enzyme Pvu II at 37 ° C. for 3 hours, and the AmyE gene was purified by phenol extraction and ethanol precipitation.

The pEA prepared in Example 1 was digested at 37 ° C. for 3 hours using the restriction enzyme Pvu II, and the purified vector was purified through phenol purification and ethanol precipitation. The purified vector portion and AmyE gene digested with restriction enzyme Pvu II were subjected to ligation reaction at 16 ° C. for 3 hours using T4 DNA polymerase, and then E. coli was transformed using the reaction solution. After transformation, the recombinant vector was isolated from each strain, and the recombinant vector linked to the AmyE gene in pEA was selected and digested with restriction enzyme Pvu II to identify the AmyE gene.

The vector produced by the above method was named pEA-AmyE and then used as a donor vector of a marker gene when removing a gene from a yeast strain in the present invention.

Example 3 Isolation of Yeast Chromosome DNA

In the present invention, the yeast used to isolate the yeast gene to be removed for the production of transformed yeast strain is Saccharomyces cerevisiae YPH499 ( MATa, ade2-101, ura3-52, lys2-801, trp1- 63, his3-?? 200, leu2-?? 1, Gal + ), and the chromosomal DNA of YPH499 was isolated using conventional molecular biological methods.

To explain this in more detail, YPH499 was first inoculated in 5 ml YPD medium (1% yeast extract, 1% peptone, 2% glucose) and incubated at 28 ° C. for 16 hours. After incubation, YPH499 was centrifuged (12,000 rpm, 4 ° C., 5 minutes), and the precipitate was suspended in 0.5 ml of distilled water. The suspension was transferred to a 1.5 mL microtube and then centrifuged again (12,000 rpm, 4 ° C., 1 min) to discard the supernatant and only collected precipitated YPH499 cells. 0.2 ml of buffer A (2% Triton X-100, 1% sodium dodecyl sulfate (SDS), 100 mM sodium chloride, 10 mM tris-hydrochloric acid, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0), glass beads 200 µm in diameter, and 0.2 ml of phenol / chloroform / isoamyl alcohol mixture (volume ratio; 25/24/1), respectively, were vigorously stirred for 3 minutes, and then 0.2 ml of Tris- Further EDTA buffer (TE buffer (50 mM Tris-HCl, 10 mM EDTA, pH 8.0) was added. Centrifuge the microtube (12,000rpm, 4 ℃, 5min), transfer the supernatant to a new microtube, add the same amount of chloroform with the supernatant, mix 5-10 times, and centrifuge (12,000rpm, 4 ℃, 5). Minutes). After centrifugation, the supernatant was transferred to a new microtube. 1 ml of 100% ethanol was added thereto, the microtubes were mixed 5-10 times, and centrifuged (12,000 rpm, 4 ° C., 5 minutes) to discard the supernatant. 0.4 ml of Tris-EDTA buffer was added to the precipitate to dissolve the chromosomal gene. 10 μl of 4M ammonium acetate was added to the mixture, followed by adding 1 ml of 100% ethanol and mixed again 5-10 times. Then, the supernatant was removed by centrifugation (12,000 rpm, 4 ° C., 5 minutes), the precipitate was left to dry in air, and 50 μl of Tris-EDTA buffer was added to dissolve the chromosomal DNA.

Yeast chromosomal DNA prepared by the above method was used as a material for separating the yeast gene of interest in the present invention.

Example 4 Cloning of Yeast Gene OCH1

In the present invention, OCH1 (SEQ ID NO: 1), ANP1 (SEQ ID NO: 2), MNN11 (SEQ ID NO: 3), HOC1 (SEQ ID NO: 4) in yeast to produce EPO having only en-acetylglucosamine attached thereto. , MNN2 (SEQ ID NO: 5) gene removal is required. In order to remove the OCH1 gene, it is first necessary to obtain the OCH1 gene from yeast chromosomal DNA.

This method is described in detail. First, OCH1-F (5'-ATT GCT TAA ACT ATT TGG CCG GCC CAC), which is a primer prepared by yeast chromosome DNA isolated by the method of Example 3 and prepared by DNA synthesis, SEQ ID NO: 8 ) And OCH1-R (5'-AGA AGC ACT G CT CAC CAG TTG TCT ACG: SEQ ID NO: 9) using OCH1 gene was obtained by PCR. The OCH1 gene obtained by PCR was purified using phenol extraction, and then reacted at 12 ° C. for 1 hour using a T4 DNA polymerase to transform the OCH1 gene end into a blunt end. This was digested with the restriction enzyme Pvu II of the E. coli cloning vector pEA prepared in Example 1 and subjected to ligation reaction at 16 ° C. for 3 hours using the purified portion and T4 DNA polymerase. E. coli XL1-Blue was transformed using this reaction solution. The pEA vector to which the OCH1 gene was linked was isolated from the transformed Escherichia coli and named as pEA-OCH1, and used as a template vector to remove the OCH1 gene in the future.

Example 5 Removal of Yeast Gene OCH1

After first removing the embodiment in pEA-OCH1 vector prepared in four restriction enzyme which is located in the OCH1 gene sequence Kpn I and Eco fragment of 733bp in size to the Eco RI in the Kpn I in the OCH1 gene with the RI, the remaining vector parts Purification was carried out using the phenol extraction method.

After phenol purification, the terminal was reacted at 12 ° C. for 1 hour using T4 DNA polymerase to transform the terminal into a smooth terminal. And Example 2 Method vector pEA-AmyE the restriction enzyme was cut with Pvu II to remove the AmyE gene of 2293bp size phenol purified and the fragment to the Eco RI in the Kpn I prepared above removal pEA-OCH1 prepared in the vector portion And T4 DNA polymerase were used for 3 hours at 16 °.

E. coli XL1-Blue was transformed using the linked gene. In the transformed Escherichia coli, a recombinant vector having AmyE gene linked to a portion of the incomplete OCH1 gene was isolated and named pEA-?? OCH1 / AmyE.

The pEA-?? OCH1 / AmyE vector prepared by the above method was digested with restriction enzyme Sma I located in the AmyE gene, phenol purified, and then transformed into yeast. Transformed yeast was isolated and chromosomal DNA was isolated by the method of Example 3, and each chromosome DNA was used as a template, and primers prepared by DNA synthesis were OCH1-F (5'-ATT GCT TAA ACT ATT TGG CCG GCC CAC). : The size of the OCH1 gene was compared by PCR using SEQ ID NO: 10) and OCH1-R (5'-AGA AGC ACT G CT CAC CAG TTG TCT ACG: SEQ ID NO: 11). After a comparison with a 733bp Eco RI to remove from the Kpn I and screening a strain that OCH1 gene of 3514bp in size AmyE gene associated check MPY - was named ?? OCH1.

Example 6 Cloning of Yeast Gene ANP1

In order to remove the ANP1 gene, ANP1 gene was first obtained from yeast chromosomal DNA. In detail this method, first, the yeast chromosome DNA isolated by the method of Example 3 as a template and prepared by the DNA synthesis method ANP1-F (5'-TCA TCGTGA AAA AAT TGC GTA AGA T: SEQ ID NO: 12) ANP1 gene was obtained by PCR using ANP1-R (5'-TCG CTA TTT CTT GCC TAG CCA TAA T: SEQ ID NO: 13).

The obtained ANP1 gene was first purified using phenol extraction, and then reacted at 12 ° C. for 1 hour using a T4 DNA polymerase to transform the ANP1 gene terminal into a smooth end. This was digested with the restriction enzyme Pvu II of the E. coli cloning vector pEA prepared in Example 1, followed by ligation reaction at 16 ° C. for 3 hours using the purified portion and T4 DNA polymerase. E. coli XL1-Blue was transformed using this reaction solution. The pEA vector to which the ANP1 gene was linked was isolated from the transformed Escherichia coli and named as pEA-ANP1, and then used as a template vector to remove the ANP1 gene in the future.

Example 6 Removal of Yeast Gene ANP1

In order to produce EPO to which only en-acetylglucosamine is attached in the present invention, a yeast strain from which the ANP1 gene was removed was prepared using the yeast MPY-?? OCH1 prepared in Example 4. After first removing the Example 6 prepared by the limitation which is located in ANP1 gene sequences from pEA-ANP1 vector enzyme Pvu II and the fragment of 696bp size up to Kpn I in Pvu II in ANP1 gene using the Kpn I, the remaining vector parts Purification was carried out using the phenol extraction method.

After phenol purification, the terminal was reacted at 12 ° C. for 1 hour using T4 DNA polymerase to transform the terminal into a smooth terminal. And the Example 2 method into the prepared vector pEA-AmyE the restriction in the Pvu II purified phenol to remove the AmyE gene of 2293bp in size was cut in and prepared on the Pvu II Kpn I fragment was removed the vector portion of pEA-ANP1 with T4 DNA polymerase was used to connect at 16 ° C. for 3 hours.

E. coli XL1-Blue was transformed using the linked gene. In the transformed Escherichia coli, a recombinant vector in which the AmyE gene was linked to a portion of an incomplete ANP1 gene was isolated and named pEA-ANP1 / AmyE.

The pEA-?? ANP1 / AmyE vector prepared by the above method was digested with restriction enzyme Sma I located in the AmyE gene, phenol purified, and then transformed into yeast. Transformed yeast was isolated from each other and the chromosomal DNA was isolated by the method of Example 3, and each chromosome DNA was used as a template and prepared by DNA synthesis method ANP1-F (5'-TCA TCG TGA AAA AAT TGC GTA AGA T : The size of the ANP1 gene was compared by PCR using SEQ ID NO: 14) and ANP1-R (5′-TCG CTA TTT CTT GCC TAG CCA TAA T: SEQ ID NO: 15). As a result, the removal of up to 696bp Kpn I in Pvu II and selecting a strain that AmyE gene is a gene of 3547bp ANP1 size associated check MPY - was named ?? (OCH1 / ANP1).

Example 8 Cloning of Yeast Gene MNN11

In order to remove the MNN11 gene, MNN11 gene was first obtained from yeast chromosomal DNA. Detailed description of this method, the primer MNN11-F (5'-CGC TAG AGA GTG TAT ATA CA A ACA GA) which was prepared by yeast chromosome DNA isolated by the method of Example 3 as a template and synthesized by DNA synthesis: SEQ ID NO: 16 ) And MNN11-R (5′-ATT ATA TTT GCT TTT TCT AAT TTT T: SEQ ID NO: 17) to obtain MNN11 gene by PCR. The obtained MNN11 gene was first purified using phenol extraction, and then reacted at 12 ° C. for 1 hour using a T4 DNA polymerase to transform the MNN11 gene terminus to a smooth end. This was digested with the restriction enzyme PvuII of the E. coli cloning vector pEA prepared in Example 1, followed by ligation reaction at 16 ° C. for 3 hours using the purified portion and T4 DNA polymerase. E. coli XL1-Blue was transformed using this reaction solution. The pEA vector to which the MNN11 gene was linked was isolated from the transformed Escherichia coli and named as pEA-MNN11, and used as a template vector to remove the MNN11 gene in the future.

Example 9 Removal of Yeast Gene MNN11

In order to produce EPO to which only en-acetylglucosamine is attached in the present invention, a yeast strain from which the MNN11 gene was removed was made using the yeast MPY-?? (OCH1 / ANP1) prepared in Example 6. In the pEA-MNN11 vector prepared in Example 8, 479 bp fragments of Vsp I to Eco RI were removed from the MNN11 gene using restriction enzymes Vsp I and Eco RI located at the MNN11 gene sequence, and the remaining vector portion was phenol. Purification was performed using the extraction method.

After phenol purification, the terminal was reacted at 12 ° C. for 1 hour using T4 DNA polymerase to transform the terminal into a smooth terminal. Then, the pEA-AmyE vector prepared by the method of Example 2 was digested with restriction enzyme Pvu II, 2293 bp AmyE gene was isolated, phenol-purified, and the pEA-MNN11 vector portion and T4 from which the Eco RI fragment was removed from the prepared Vsp I. DNA polymerase was used to connect at 16 ° C. for 3 hours.

E. coli XL1-Blue was transformed using the linked gene. In the transformed Escherichia coli, a recombinant vector in which the AmyE gene was linked to a part of the incomplete MNN11 gene was isolated and named pEA-?? MNN11 / AmyE. The pEA-?? MNN11 / AmyE vector prepared by the above method was digested with restriction enzyme Sma I located in the AmyE gene, phenol purified, and transformed with yeast. Transformed yeast was isolated from each other, and chromosomal DNA was isolated by the method of Example 3, and each chromosomal DNA was used as a template and prepared by DNA synthesis. GA: SEQ ID NO: 18) and MNN11-R (5'-ATT ATA TTT GCT TTT TCT AATTTT T: SEQ ID NO: 19) was used to compare the size of the MNN11 gene by PCR. As a result, 479 bp from Vsp I to Eco RI was removed, and a strain of 3415 bp of MNN11 gene linked to AmyE gene was identified and named MPY-?? (OCH1 / ANP1 / MNN11).

Example 10 Cloning of Yeast Gene HOC1

In order to remove the HOC1 gene, HOC1 gene was first obtained from yeast chromosomal DNA. In detail, this method is based on the yeast chromosome DNA isolated by the method of Example 3 as a template and prepared by DNA synthesis method HOC1-F (5'-TCT TTC AAA TTT TAA CCT TAT GTT T: SEQ ID NO: 20) and HOC1 gene was obtained by PCR using HOC1-R (5'-CTT CCT CAC TTA TTT CTT CTT TCT C: SEQ ID NO: 21). The obtained HOC1 gene was first purified using phenol extraction, and then reacted at 12 ° C. for 1 hour using T4 DNA polymerase to transform the HOC1 gene terminal into a smooth end.

This was digested with the restriction enzyme PvuII of the E. coli cloning vector pEA prepared in Example 1, followed by ligation reaction at 16 ° C. for 3 hours using the purified portion and T4 DNA polymerase. E. coli XL1-Blue was transformed using this reaction solution. The transformed Escherichia coli isolates the pEA vector to which the HOC1 gene was linked, named it pEA-HOC1, and used it as a template vector to remove the HOC1 gene in the future.

Example 11 Removal of Yeast Gene HOC1

In the present invention, the yeast MPY-?? (OCH1 / ANP1 / MNN11) prepared in Example 9 was used to produce EPO to which only en-acetylglucosamine was attached. First, in the pEA-HOC1 vector prepared in Example 10, the HOC1 gene was cleaved using the restriction enzyme Ssp I located in the HOC1 gene nucleotide sequence, and then a fragment of 735 bp that was cleaved and isolated by Ssp I was removed. Purification was performed using the extraction method.

After cutting the vector pEA-AmyE prepared by the method of Example 2 with restriction enzyme Pvu II, 2293 bp AmyE gene was isolated and phenol purified, and the pEA-HOC1 vector portion and the T4 DNA polymerase from which the Ssp I fragment prepared above were removed. 3 hours at 16 ° C.

E. coli XL1-Blue was transformed using the linked gene. In the transformed Escherichia coli, a recombinant vector in which the AmyE gene was linked to a portion of the incomplete HOC1 gene was isolated and named pEA-HOC1 / AmyE. The pEA-?? HOC1 / AmyE vector prepared by the above method was digested with restriction enzyme Sma I located in the AmyE gene, phenol purified, and then transformed into yeast. Transformed yeast was isolated and chromosomal DNA was isolated by the method of Example 3, and each chromosomal DNA was used as a template, and primers prepared by DNA synthesis method HOC1-F (5'-TCT TTC AAA TTT TAA CCT TAT GTT T : The size of HOC1 gene was compared by PCR using SEQ ID NO: 22) and HOC1-R (5′-CTT CCT CAC TTA TTT CTT CTT TCT C: SEQ ID NO: 23). As a result, the strain 735bp fragment cut by Ssp I was removed and the strain identified as the HOC1 gene having a size of 3508bp linked to the AmyE gene was selected and named MPY-?? (OCH1 / ANP1 / MNN11 / HOC1).

Example 12 Cloning of Yeast Gene MNN2

MNN2 gene was obtained from yeast chromosomal DNA to remove MNN2 gene. This method is described in detail. First, primers MNN2-F (5'-AAT AAA TTA CGT GTA CTT GGT GTT: SEQ ID NO: 24) prepared using a yeast chromosome DNA isolated by the method of Example 3 as a template and prepared by DNA synthesis MNN2 gene was obtained by PCR using MNN2-R (5'-TAA AGT CAT TTT ATA TGG ATT GTG A: SEQ ID NO: 25). The obtained MNN2 gene was first purified using phenol extraction, and then reacted at 12 ° C. for 1 hour using T4 DNA polymerase to transform the MNN2 gene terminus to a smooth end. This was digested with the restriction enzyme Pvu II of the E. coli cloning vector pEA prepared in Example 1, and then purified by the purified portion and T4 DNA polymerase. E. coli XL1-Blue was transformed using this reaction solution. The pEA vector to which the MNN2 gene was linked was isolated from the transformed Escherichia coli, named as pEA-MNN2, and used as a template vector to remove the MNN2 gene in the future.

Example 13 Removal of Yeast Gene MNN2

In the present invention, the yeast MPY-?? (OCH1 / ANP1 / MNN11 / HOC1) prepared in Example 11 was used to produce EPO to which only en-acetylglucosamine was attached. First, use the embodiment 12 pEA-MNN2 restriction enzyme which is located in MNN2 gene sequences in the vector Bgl II and Ssp I prepared after removal of the fragment of 749bp size in MNN2 gene in the Bgl II to Ssp I phenol remaining vector parts Purification was performed using the extraction method.

After the phenol purification, the pEA-AmyE vector prepared by the method of Example 2 was digested with restriction enzyme Pvu II, and a 2293 bp size AmyE gene was isolated and phenol-purified and the fragments from Bgl II to Ssp I prepared above were removed. Part and T4 DNA polymerase were used for 3 hours at 16 ° C.

E. coli XL1-Blue was transformed using the linked gene. In the transformed Escherichia coli, a recombinant vector in which the AmyE gene was linked to a part of the incomplete MNN2 gene was isolated and named pEA-?? MNN2 / AmyE. The pEA-?? MNN2 / AmyE vector prepared by the above method was digested with restriction enzyme Sma I located in the AmyE gene, phenol purified, and transformed with yeast. Transformed yeast was isolated from each other and the chromosomal DNA was isolated by the method of Example 3, and each chromosomal DNA was used as a template and prepared by DNA synthesis method MNN2-F (5'-AAT AAA TTA CGT GTA CTT GGT GTT: The size of the MNN2 gene was compared by PCR using SEQ ID NO: 26) and MNN2-R (5'-TAA AGT CAT TTT ATA TGG ATT GTG A: SEQ ID NO: 27). As a result, the strain 749bp fragments cut from Bgl II to Ssp I were removed and a 3618bp MNN2 gene linked to the AmyE gene was identified, and the strain was named Saccharomyces cerevisiae MPY. This transformed yeast strain was deposited internationally under the Budapest Treaty with Accession No. KCCM-10441.

Yeast Saccharomyces cerevisiae MPY (Accession No .: KCCM-10441) made by the above method was used as a production strain for producing EPO to which only en-acetylglucosamine is attached in the present invention. .

Example 14 Preparation of Yeast Vector for EPO Expression

In the present invention, a new expression vector was prepared and used to express EPO from the transformed yeast. To explain the method in detail, the primer pEY-F (5'-ACA GCT GAT CAC AGG AGG TAC TAG ACT ACC TT: SEQ ID NO: 28) prepared by DNA synthesis method was prepared by preparing a pB42AD (Clontech Co.) vector used as a template. PCR was carried out with pEY-R (5'-ACA GCT GAA AGG CGG TAA TAC GGT TAT CCA CA: SEQ ID NO: 29) to obtain a fragment of 4418 bp. This was purified using phenol extraction and then reacted at 12 ° C. for 1 hour using T4 DNA polymerase. After the reaction, phenol was purified and phosphoric acid was added at the 5 'end using a T4 polynucleotide kinase. This was further purified using phenol extraction and then fragments were linked using T4 DNA polymerase. E. coli XL1-Blue was transformed using the linked fragments, and each vector was isolated from the transformed E. coli, digested with restriction enzyme Pvu II, and a vector having a size of 4418 bp was selected and named pEY. It was used as a template of the yeast vector for.

In the present invention, a promoter and terminator system of yeast alcohol dehydrogenase 1 (ADH1) was used to express EPO. In detail the preparation of the EPO expression system, first, the pGAD424 (Clontech) vector was digested with restriction enzyme Sph I to isolate a 1423 bp fragment containing the promoter and terminator of yeast alcohol dehydrogenase 1, followed by phenol extraction and ethanol. Purification via precipitation. After purification, the mixture was reacted at 12 ° C. for 3 hours using T4 DNA polymerase, and then purified through phenol extraction and ethanol precipitation. The above-described vector pEY was digested with restriction enzyme Pvu II, purified by phenol extraction and ethanol precipitation, and a 1423 bp fragment containing the promoter and terminator of yeast alcohol dehydrogenase 1 was purified using T4 DNA polymerase. After 3 hours of connection at C, E. coli was transformed using the reaction solution. The recombinant vector was isolated from the transformed strain and named pEY-ADH.

In addition, the EPO gene is a pSG5-EPO vector as a template primer EPO-F (5'-AAC ATA TGG GGG TGC ACG AAT GTC CT: SEQ ID NO: 30 and EPO-R (5'-TTC ATA TGT CA T CTG TCC CCT GTC CTG CA: SEQ ID NO: 31) was obtained by PCR, and the obtained EPO gene was recovered by phenol extraction and purification using ethanol precipitation.The recovered gene was digested with restriction enzyme Nde I for 3 hours at 37 ° . The purified EPO gene and pEY-ADH were digested with restriction enzyme Hind III, purified through phenol extraction and ethanol precipitation, and then recovered by T4 DNA polymerase. After each reaction, the cleaved portion was recovered by reaction at 12 ° C. for 3 hours, and each reaction was purified by phenol extraction and ethanol precipitation from the reaction solution, and then recovered. 3 hours connection (C) The T4 DNA polymerase was transformed with the reaction of E. coli using the solution. Transformants were isolated, each recombinant vector from E. coli to remove the vector is EPO gene linked to pEY-ADH vector and it termed pEY-EPO.

Example 15 Expression and Production of EPO with Only N-acetylglucosamine Added

Transformed yeast Saccharomyces cerevisiae MPY (Accession No .: KCCM-10441) prepared in Example 13 using the expression vector pEY-EPO prepared in Example 14 was transformed into en-acetyl Yeast strains were produced that produced only Eglucosamine added EPO. In order to produce EPO to which only N-acetylglucosamine was added, the transformed yeast strain containing the EPO gene was inoculated into YPD medium, followed by 16 hours shaking culture at 28 °. Subsequently, the cell concentration was 0.4-0.6 OD ₆₀₀ / ml (wherein OD ₆₀₀ was absorbed at a wavelength of 600 nm in a spectrophotometer) in YPD0.5 (1% yeast extract, 1% peptone, 0.5% glucose) medium used as the production medium. After the inoculation, the shaking culture was performed at 30 ° C. for 5 hours. After incubation, the culture solution was centrifuged (12,000 rpm, 4 ° C, 10 minutes) to recover the supernatant, and the antigen-antibody reaction was performed without refolding the EPO containing only N-acetylglucosamine instead of the sugar chain at the sugar chain portion. Separated using. En-acetylglucosamine attached to the EPO was cut out of the protein using enzymatic and chemical reactions and analyzed and confirmed using high performance liquid chromatography.

The present invention relates to a site-selective formula for conferring regioselectivity in protein modifications used in the manufacture of protein hybrids to confer or enhance in vivo stability and biological activity of proteins produced for therapeutic, research and industrial purposes. It is to provide a sugar adjuvant in which sugars with possible specific reactors are bound. Unlike the conventional protein modification method was not able to modify the protein regioselectively, by using the transformed yeast strain developed in the present invention in the form of the existing sugar chain portion in the form of the addition of en-acetylglucosamine instead of sugar chain at the position of protein production The protein can be regioselectively modified to include a synthetic polymer or a natural polymer by using the en-acetylglucosamine or a derivative of en-acetylglucosamine formed through a derivatization reaction so that the protein is produced. As a result, it is possible to increase the length of stay in the living body such as blood, thereby conferring or enhancing medical efficacy and activity, biological efficacy and activity.

Therefore, the present invention can be used to inexpensively mass-produce novel therapeutic proteins with improved titers with the same medical activity as many existing therapeutic proteins, and this method is applicable to all therapeutic, research and industrial proteins. Applicable

Claims (3)

Yeast genes OCH1 (SEQ ID NO: 1), ANP1 (SEQ ID NO: 2), MNN11 (SEQ ID NO: 3), HOC1 (SEQ ID NO: 4), which are related to the addition of the oligosaccharide for the expression of the glycoprotein from which a part of the sugar chain has been removed Transformed yeast strain Saccharomyces cerevisiae MPY from MNN2 (SEQ ID NO: 5) (Accession No .: KCCM-10441) En-acetylglucosamine having a functional group whose reactivity is distinguished from the functional group of the amino acid constituting the glycoprotein at the position by using the transformed yeast strain (Accession No .: KCCM-10441) of claim 1; How to express proteins with the same sugar Proteins prepared according to the method of claim 2 are alpha, beta, gamma interferon, asparaginase, arginase, adenosine deaminase, superoxide dismutase, catalase, lipase, freecase, adenosine diphosphatase, Tyrosinase, glucose oxidase, glucosidase, blood factors VII, VIII and IX, immunoglobulins, cytokines ie interleukin, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, platelet derived growth factor, lectin, lysine, At least one protein selected from tumor necrosis factor, tumor growth factor, and platelet stimulating factors