KR100545981B1 - Heteropolymerized Parritin Production Method - Google Patents

Abstract

 The present invention relates to a method for producing heteropolymeric paritin. In particular, the present invention relates to a method for producing a parritin capable of storing a large amount of iron in a highly bioavailable form, and (a) designed to express a high molecular ratio of the parine light chain in a single host cell compared to the parine heavy chain. One parity light chain expression vector I and one parity heavy chain expression vector II are sequentially introduced, (b) the transformants to which both vectors are introduced are selected, and (c) the transformants are cultured. It provides a heteropolymer-type parine production method comprising expressing heteropolymer-type parine.

Parine, Iron, Heteropolymer

Description

Heteropolymer Type Paritine Production Method {METHOD OF PRODUCING HETEROPOLYMERIC FERRITIN}

1 shows an expression cassette and a restriction enzyme map thereof comprising a heavy chain and a light chain of human parine, respectively,

Figure 2 is an electrophoresis picture of PCR amplification of the pep4 :: HIS3 allele of the posterior 19 follicle spores,

Figure 3 confirms the phytase expression rate in the parent strain and later strains,

FIG. 4 is a Prussian blue staining photograph for each transformant cell extract, A is a transformant cell extract pretreated with 1 mM ferrous ammonium sulfate, and then stained with Prussian blue by non-denatured PAGE, and B is sulfuric acid Unmodified PAGE without ferrous ammonium treatment and stained with Coomassie Blue, C is Coomassie blue stained SDS-PAGE of heteropolymerized parine separated by gel purification method,

FIG. 5 is a graph showing iron absorption rates of homopolymerized apopatini and heteropolymerized apopatini. FIG.

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a method for producing heteropolymeric paritin, and more particularly, to sequentially express each vector designed to express a paritin light chain and a heavy chain in a molecular ratio of 5: 1 to 10: 1 in a single host cell. The present invention relates to a method for introducing and producing heteropolymeric parity therefrom.

[Private Technology]

Iron is generally essential for biological activity. Iron (III) has low solubility and is limited in bioavailability. Intracellular free iron may generate toxic radicals, but when ingested, it is stored in the cell in the form of water-soluble nontoxic bioavailability (Harrison, PM). , and TH Lilley. 1989. Ferritin, p. 123-138.In TM Loehr (ed.), Iron carriers and iron proteins.VCH, New York .; Ragland, M., et al. 1990. J. Biol. Chem 265: 18339-18344).

Parritin is a storage protein in most organisms that accumulates iron, and the form of iron binding transfer is called apoferritin (Theil, EC 1987. Ann. Rev. Biochem . 56: 289-315). Parritin is a spherical macromolecule surrounded by 24 structurally equivalent subunits, with an oxyhydroside polymer in the core and accumulating 4500 iron atoms (heil, EC 1990. J. Biol. Chem. 265: 4771-4774 ). The main action of parritin is to provide iron in the synthesis of iron-containing proteins and to prevent the risk of free radicals produced by the iron / dioxine interaction (Bui, K., and P. Galzy. 1990. Food yeast , p. 241-265.In JFT Spencer, and DM Spencer (ed.), Yeast technology.Springer, Berlin Heidelberg New York .; Theil, EC, JW Burton, and JL Beard. 1997. J. Clin.Nutr . : S28-S31).

The important subunits that make up parritin are the heavy chain and the light chain, and various combinations of the two subunits form isoferritins. In general, large amounts of light chains, parritin, are characteristic of organs with very high iron accumulation and store more than 1,500 iron atoms per molecule. Heavy chains of parritin are present in organs with low iron content and store less than 1,000 iron atoms per molecule. Human serum parine lacks a heavy chain, and natural heavy chain homopolymers have not yet been isolated (Arosio, P., M. Yokota, and JW Drysdale. 1977. Br. J. Haematol . 36: 199- 207), the light chain homopolymer fraction has been extracted from parine (Ishitani, K., and I. Listowsky. 1975. J. Biol. Chem . 250: 5446-5449).

Heavy chain homopolymers are in the form of relatively insufficient iron cores, and light chain homopolymers lack paroxides centers therein. Therefore, heteropolymeric parritin is very effective in storing and releasing iron as compared to homopolymers. Natural isoparatin are mostly heteropolymers consisting of a combination between two subunits. Subunits can be combined in various ratios, and heavy chain ratio limits are required to maximize iron absorption (Santambrogio, P., S. Levi, A. Cozzi, E. Rovida, A. Albertini, and P. Arosio. 1993. J. Biol. Chem . 268: 12744-12748).

Saccharomyces cerevisiae, a food yeast, is a well-established organism and is used as feed for fish, poultry and furry animals and also as a food supplement (Bui, K., and P. Galzy. 1990. Food yeast , p. 241-265.In JFT Spencer, and DM Spencer (ed.), Yeast technology.Springer, Berlin Heidelberg New York). Saccharomyces cerevisiae reduces extracellular trivalent iron to water-soluble divalent iron using Fe ^{3+ -reducing} agent present in the plasma membrane and divalent iron through two Fe ²⁺ -specific delivery systems. Move into cells (Dix, D., J. Bridgham, M. Broderius, and D. Eide. 1997. J. Biol. Chem . 272: 11770-11777). The parine-based protein of yeast has low affinity for cellular iron and inhibits iron utilization by yeast.

In order to solve the problems of the prior art, an object of the present invention is to provide a method for producing heteropolymers by expressing the parine heavy chain and the light chain in different ratios in a single yeast, respectively.

It is another object of the present invention to provide a method for producing heteropolymeric paritin excellent in iron absorption and storage.

In addition, an object of the present invention is to provide a transformant expressing the parine heavy chain and the light chain in different ratios.

In addition, an object of the present invention is to provide a heteropolymer-type apoperytin in which the parine heavy chain and the light chain are combined in different ratios.

It is another object of the present invention to provide a heteropolymeric paritin containing a large amount of iron.

It is also an object of the present invention to provide a yeast comprising a heteropolymeric paritin in which a large amount of iron is stored.

In addition, an object of the present invention is to provide a method for utilizing a yeast comprising a heteropolymer-type parine stored in a large amount of iron as food, food additives, feed or feed additives.

In order to achieve the above object, the present invention provides a method for producing heteropolymeric paritin in yeast, wherein the method is designed to express a high proportion of parine light chains in a single host cell compared to heavy chains. A method comprising introducing expression vector I and parity heavy chain expression vector II, preparing transformants transformed with the two vectors, and culturing the transformants to express heteropolymeric paritin To provide.

In another aspect, the present invention provides a heteropolymeric parritin produced by the above method.

In addition, the present invention introduces a single host cell, parity light chain expression vector I and parity light chain expression vector I designed to express a high proportion of the parine light chain compared to the heavy chain, and transduced with the two vectors It provides a method for producing a heteropolymeric paritin producing strain comprising the production of a transformed transformant.

In addition, the present invention (a) by introducing a parritin light chain expression vector I and parine heavy chain expression vector II designed to express the paritin light chain in a high molecular ratio compared to the parine heavy chain in a single host cell It provides a method for producing a strain stored iron, comprising the step of preparing a transformant to which both vectors are introduced and (b) culturing the transformant in a medium containing iron.

In another aspect, the present invention provides a strain stored iron prepared by the above method.

The present invention also provides a polynucleotide comprising (a) an ADH2-GPD hybrid promoter, (b) a base sequence encoding a paritin light chain, between the EcoRI and HindIII restriction enzyme sites of the YEp352 vector, and (c) galactose-1-P. Provided are a vector for expression of a parine light chain comprising a uridil transferase termination site.

The present invention also provides a polynucleotide comprising (a) an ADH2-GPD hybrid promoter, (b) a base sequence encoding a paritin heavy chain, between the EcoRI and HindIII restriction enzyme sites of the YIplac128 vector, and (c) galactose-1-P. It provides a vector for the expression of paritin heavy chain containing the uridil transferase termination site.

Hereinafter, the present invention will be described in detail.

The inventors have noted that heteropolymers of light chains and heavy chains have superior iron storage properties as compared to monopolymeric parine, and simultaneously express the two subunits in a single host cell to produce heteropolymeric parine. When the light chain and the heavy chain were expressed in different ratios during the study and the combination of the heteropolymeric paritin, the iron storage property was remarkably improved to complete the present invention.

Heteropolymerized parine of the present invention is a combination of a parine light chain and a heavy chain, preferably a light chain is combined at a higher ratio than the heavy chain. More preferably, the heavy chain: light chain is 1: 5 to 1:10. If it is out of the ratio range, the iron absorption rate and storage rate may be significantly reduced.

The heteropolymer production method of the heteropolymers of the present invention comprises: (a) Parity light chain expression vector I and partin heavy chain expression vector designed to express a high ratio of parine light chains to a single chain in a single host cell. II is introduced, a transformant transformed with the two vectors is prepared, and (b) the transformant is cultured to express heteropolymerized parine.

The host cell has at least two auxotrophic phenotypes so as to confirm the transformation. In the present invention, for example, the uracil and leucine trophic phenotypes are described. In addition, the host cell is preferably a protease deficient strain to eliminate the risk of denaturing foreign protein expression. In one example, the PEP4 allele may be inactivated.

Preferred host cells cross Saccharomyces cerevisiae 2805 ( MATαpep4 :: HIS3 prb1-δCan1 GAL2 his3 ura3-52 ) and Saccharomyces cerevisiae 15Dau ( MAT ade1 his2 leu2-3,112 trp1-1 ura3Δns ) Scavenger spore produced, Saccharomyces cerevisiae 2805-a1 to 19. Saccharide in the My process three Levy Zia 2805-a1 and 19 are two selection markers has a, pep4 (ura ^{- -} and leu) ^- is the genotype.

In order to express the parine light chain and the heavy chain at different ratios, each subunit can be cloned into a vector with different plasmid copy numbers. Preferably, the parine light chain is cloned into a high copy vector, the heavy chain is cloned into a low copy vector, and both subunits are expressed under the same promoter to strictly control the expression. The promoter is a known promoter that can be expressed in yeast.

As a specific example, the paritin heavy chain and the light chain are each composed of the ADH2-GPD ((alcohol dehydrogenase2)-(glyceraldehyde-3-phosphate dehydrogenaseglyceraldehyde-3-phosphate dehydrogenase)) hybrid promoter and galactose-1-P uridil transferase termination site. Each expression cassette is prepared by inserting in between. The light chain expression cassette was cloned into a high-copy episomal vector to prepare a light chain expression vector I, and the heavy chain expression cassette was cloned into a low-copy integrative vector. Prepare heavy chain expression vector II.

The high copy episomal vector is preferably the YEp352 vector, and the low copy integrating vector is preferably YIplac128.

Transformation is selected by introducing a parritin light chain expression vector into a single host cell, and subsequently introducing the paritin heavy chain expression vector. Transformants are selected by culturing in a selection medium, and cultured in the same manner as the yeast culture method to express the light chain and heavy chain. The expressed light chains and heavy chains combine with each other to form a heteropolymeric paritin. In this case, the heteropolymer-type parine is preferably contained in a heavy chain-to-light chain ratio of 1: 5 to 1:10.

Heteropolymerized parine can be purified from the cytoplasm of the cultured transformant, and the purification method may be gel filtration or size-fractionation.

In addition, the transformant may be cultured in a medium containing iron, and then purified to produce heteropolymerized parine stored with iron, or heteropolymerized parine stored with iron by reacting iron with purified heteropolymerized parine. Can produce

In another aspect, the present invention provides a method for producing a strain producing heteropolymeric paritin. As described above, the above-mentioned strain was introduced into a single host cell by introducing a parine light chain expression vector I and a partin heavy chain expression vector II designed to express a high ratio of the parine light chain compared to the heavy chain, Both vectors can be prepared by a method for preparing a transformant introduced therein. The strain is preferably yeast, more preferably Saccharomyces cerevisiae, an edible yeast. In the present invention, as an example, a transgenic strain was produced, and the saccharomyces cerevisiae TYFHAG1 was established on March 27, 2003 at the Korea Agricultural Microbiological Conservation Center (KACC) of the National Institute of Agricultural Biotechnology (NIAB) located in Suwon, Gyeonggi-do, Korea. Was deposited and received accession number KACC93006.

The present invention also provides a method for producing a strain containing a large amount of iron. The method for preparing a strain containing a large amount of iron is (a) a parine light chain expression vector I and a partin heavy chain expression vector II designed to express a high ratio of parine light chains to a single chain in a single host cell. Introducing a transformant to which both vectors are introduced, and (b) culturing the transformant in a medium containing iron.

In one embodiment of the present invention, a transformant is selected by introducing a YEp352 vector containing a light chain expression cassette into Saccharomyces cerevisiae 2805-a7, followed by introducing a YIplac128 vector containing a heavy chain expression cassette. It was. Selected transformants showed the same growth curve as the natural type even in the iron-containing medium, it was confirmed that the iron-resistant not inhibited growth by iron. The transformants can also store more than 1004 ug / g (dry cell weight) of iron, a significant improvement over light chain homopolymers and heavy chain homopolymers of 760 and 500 ug / g (cell dry weights), respectively. It can be seen that the method of the present invention can produce a large amount of ferritin and strain containing iron.

The method of the present invention is remarkably superior to the iron absorption rate and storage capacity compared to the conventional homopolymer production method, and is an effective method compared to the method for producing heteropolymers by simultaneously expressing two subunits in one existing vector for the following reasons. .

First, the method of expressing two genes in one vector can facilitate homopolymer formation between homogeneous molecules. However, the method of the present invention, as shown in Figure 4, all form only the heteropolymer, but does not form a homopolymer. Second, when two subunits are sequentially arranged and transcribed and translated in one vector, the entire long two subunits cannot be progressed in the transcription or translation process, and only the 5 'terminal portion is intensively transcribed or translated. Can be. In this case, it is difficult to expect sufficient iron storage activity in the expressed subunits, and the expression ratio between the subunits depends on the cell growth. Finally, it is difficult to control the expression rate of two genes in one vector. However, the method of the present invention has the advantage that by using the same promoter for both vectors, once the initial expression rate is determined, the expression rate is kept constant during the culture regardless of intracellular or extracellular environmental changes.

Since the iron-containing parritin prepared by the method of the present invention has excellent bioavailability and contains iron in a harmless form, it can be used as a drug, food additive or feed additive to prevent and treat diseases caused by iron deficiency. Can be. How to use can be appropriately adjusted according to the use form or purpose, it is good to use as a parritin prescription method commonly used.

The present invention also provides a composition comprising a parritin producing strain or an iron containing parritin produced from said strain. The composition may be a food additive or feed additive. The parine-producing strain may be included in the composition after incubation for 48 to 72 hours after the iron addition to the medium, or used as a live bacterium, or after heat treatment to be included in the composition. The heat treatment can be performed at 65 ° C. for about 24 hours, and the recombinant parine is stable to the heat treatment. This is an example of the iron binding ability using the purely isolated recombinant partin in one embodiment of the present invention to see the iron absorption rate at 30 ℃ and 58 ℃ after the iron was added to the recombinant parritin between the two temperatures Not only is there a difference, but it is supported by the result that the absorption rate does not drop even after 24 hours after reaching the highest absorption rate after the reaction, indicating that the recombinant parine is very stable to heat.

The composition comprising the parine-producing strain of the present invention or the iron-containing parritin produced from the strain may include the strain or the parine alone, or may further include an excipient which can be fed to other animals. Excipients are preferably used according to the use of the composition, wherein the content of the strain or parritin may be included in the total composition of 0.1 to 70% by weight. In addition, the composition may further comprise a wild-type strain in the same ratio, when the parine-producing strain is heat-treated to include in the form of dead bacteria.

The formulation of the paritin-producing strain of the present invention or the composition comprising the iron-containing paritin produced from the strain may be a liquid, powder, emulsion, granules, suspensions, injections or tablets, it is appropriate to adjust according to the method of use desirable.

 Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example

1. Experimental Materials and Methods

1-1. Strains and Strain Cultures

Plasmids were maintained and propagated in E. coli HB101 or DH5α (Sambrook, J., EF Fritsch, and T. Maniatis. 1989. Molecular Cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.). Saccharomyces cerevisiae 2805 ( MATαpep4 :: HIS3 prb1-δCan1 GAL2 his3 ura3-52, Park, EH, YM Shin, YY Lim, TH Kwon, DH Kim, and MS Yang. 2000. J. Biotechnol . 81: 35-44) and Saccharomyces cerevisiae 15Dau ( MAT ade1 his2 leu2-3,112 trp1-1 ura3Δns, Valdivieso, MH, K. Sugimoto, KY Jahng, PM Fernandes, and C. Wittenberg. 1993. Mol. Cell. Biol 13:. 1013-1022) from 2 has a different selectable marker (ura- and leu-), was used as for the purpose of breeding ascus spores later cured with a heterologous expression involves Parry tin body pep4- for. Crossing, sporulation induction and tetrad analysis were performed according to the publication (Mortimer, RK, and DC Hawthorne. 1969. The Yeasts, Vol. 1, Academic Press, New York.).

That is, two strains of opposite breeding types were crossed on YEPD plates, and diploids were sporulated. The resulting sanders were excised on YEPD plates and selected progeny requiring uracil and leucine. Uracil-deficient (ura ^-) selective medium (0.67% amino acid-deficient yeast nitrogen base (Sigma), 30 mg / L adenine and tryptophan, and 0.5% casamino acids, 2% dextrose and 2% agar) and leucine-deficient ( leu ^-) selective medium (0.67% amino acid-deficient yeast nitrogen base (Sigma), 0.14% yeast synthetic drop-out medium (Sigma), 30 mg / L tryptophan, 2% dextrose and 2% agar) is prepared by each mating One later yeast and transformant were used as a medium for screening.

Saccharomyces cerevisiae was maintained at 30 ° C. in YEPD medium. The primary inoculum was prepared with 5 ml of uracil selection medium and incubated for 24 hours, and then 10 ⁷ cells were inoculated in a 300-ml Erlenmeyer flask in 40 ml YEPD medium. Cultures for expression were grown by continuous stirring culture at 30 ℃, after which the cells were harvested and confirmed the parine expression.

In order to verify the iron resistance, the transformants were cultured in uracil selection medium containing citric acid to measure colony forming units (CFU).

1-2. Vector building

To express different levels of the heavy chain and the light chain of parine, the low copy integration vector YIplac128 (Gietz, RD and Sugino, A. (1988) Gene 30: 527-534) and the high copy episomal vector YEp352 ( Park, EH, YM Shin, YY Lim, TH Kwon, DH Kim, and MS Yang. 2000. J. Biotechnol . 81: 35-44) were constructed to express heavy chains and light chains. In addition, the same promoter was used for both vectors to minimize errors due to differences in promoter strength.

That is, between the ADH2 -GPD hybrid promoter and the galactose-1-P uridil transferase ( GAL7 ) end, a parine heavy chain gene ( HuFH) containing the lower 142 bp from the translation stop codon from the upper 41 bp from the translation start codon. : Accession no. BC016009) was inserted to prepare an expression cassette, which was inserted into the Eco RI / Hin dIII site of the YIplac128 vector. In addition, the nucleotide sequence including the human paritin ride chain gene ( HuFL : accession no. BC004245), the upper 41 bp from the translation start codon, and the lower 45 bp in the translation stop codon, the ADH2 -GPD hybrid promoter of the YEp352 vector and the GAL7 termination site Inserted in between.

In addition, a high copy episomal vector YEp352 was used to produce paritin light chain homopolymers and heavy chain homopolymers.

1 shows an expression cassette containing a human paritin heavy chain and a light chain, respectively, and a restriction enzyme map thereof. FIG. 1A is an expression cassette containing a human paritin heavy chain ( HUFH ), and FIG. An expression cassette comprising a light chain ( HuFL ). Each box shown in FIG. 1 is a respective gene or functional domains corresponding thereto, p AG is an ADH2-GPD hybrid promoter and t GAL7 is a galactose-1-P uridil transferase termination site. At the bottom of each box, the nucleotide sequence constituting each linking site at the promoter-paritin-terminating site is indicated, the transcription start site is indicated by "**", and the translation start and stop codons are shown in bold.

Transformation of Saccharomyces cerevisiae strains was carried out by known methods (Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. J. Bacteriol . 153: 163-168). Transformed strains were selected on leucine selection medium or uracil selection medium, and the presence of plasmid in the strain was confirmed by reverse-transformation into Sudden blot or Escherichia coli.

The stability of the plasmids introduced in yeast was screened according to known methods (Shin, YM, TH Kwon, KS Kim, KS Chae, DH Kim, and MS Yang. 2001. Appl. Env. Microbiol . 67: 1280-1283). It was confirmed using.

1-3. Northern blot analysis

Transformants were grown in YEPD medium and total RNA was isolated (Mitchell, DA, L. Farh, TK Marshall, and RJ Deschenes. 1994. J. Biol. Chem . 269: 21540-21546). RNA was then electrophoresed with 1.2% agarose gel on formaldehyde, transferred to nylon membrane and reacted with HuFH or HuFL labeled with ³² P (Sambrook, J., EF Fritsch, and T. Maniatis. 1989. Molecular Cloning Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

1-4. Preparation of Crude Extract with Cells Removed from Yeast

Strains were incubated for 3 days, harvested and washed twice with extract buffer (50 mM Tris-HCl, 2 mM EDTA) and ground three times for 1 minute with Beadspec Products Inc. The lysate was centrifuged (10 min at 10000 x g ) to develop the supernatant by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Since parritin is heat stable (Kim, YT, and KS Kim. 1994. Mol. Cells . 4: 125-129) and the host cells are prophase deficient, all steps were performed at room temperature (25 ° C.).

1-5. Polyacrylamide Gel Electrophoresis

SDS-polyacrylamide gel electrophoresis was performed by Lammmli (UK 1970. Nature , 227: 680-685), using 12% acrylamide separation gel and 5% stacking gel each containing 1% SDS. It was. Samples were heat treated at 100 ° C. for 10 min on 125 mM Tris / Glycine buffer (pH 6.8) containing 1% SDS and 1% β-mercaptoethanol. Electrophoresis was carried out with a development buffer (25 mM Tris / glycine, pH 8.8, containing 0.1% SDS) at a constant current of 10 mA for 3 hours, and the gel was stained with 0.025% Coomassie Brilliant Blue R-250.

In addition, the expressed parine heavy chain subunits were stained with Prussian blue to confirm whether they were bound with a large polymer capable of accumulating iron. The cell extracts were heat-treated at 75 ° C. for 15 minutes, subjected to 6.0% native PAGE under non-denatured conditions, and then 2% K ₄ Fe (CN) ₆ and 2% of 11.6 M HCl (1: 1, v prepared immediately before use). / v). Electrophoresis was performed at 25 mA for 5 hours on 25 mM Tris / glycine buffer, and parine was purchased and used as a control.

1-6. Protein purification

The expressed protein products were purified by gel filtration with native PAGE prepared at 6.0% under undenatured composition and then stained with copper chloride (Lee, C., A. Levin, and D. Branton. 1987. Anal. Biochem . 166 : 308-312). Protein bands were separated by an electro-elutor (Model 422 Electro-Eluter; BIO-RAD) and concentrated in an Amicon concentrator with a 30 kDa molecular cut-off. The purity of the isolated parine was confirmed by native PAGE, and each subunit of parine was confirmed by SDS-PAGE. Biochemical properties of the purified parine product were confirmed in subsequent experiments.

1-7. Phytase expression

Since several studies have confirmed that Saccharomyces cerevisiae 2805 is suitable for external gene expression, later heterologous expression efficiency was compared with 2805 strain using phytase, a reporter enzyme. The phytase expression plasmid obtained in the previous study was introduced into a cross-seeded posterior follicle spore strain, and the transformants were analyzed for phytase activity in comparison with the 2805 strain. Phytase activity was measured by known methods (Lim, YY, et al. 2001. J. Microbiol. Biotechnol . 11: 915-921), and 1 unit of yeast releasing 1 umole of phosphoric acid per minute at 58 ° C. The enzyme activity measurement was repeated three times, and similar results were obtained.

1-8. Atomic absorption photometer

Iron-absorption analysis was performed on samples incubated in uracil sorting medium provided with iron citrate.

Strains were incubated at 30 ° C. for 4 days on a uracil-selective medium containing iron citrate, followed by centrifugation (10 min, 3500 × g ), and then washed three times with distilled water and dried at 60 ° C. for 48 hours. The dried strains were placed in a glass tube, 6 ml of a mixture of 14 M nitric acid: 10 M perchloric acid in a 2: 1 volume ratio was injected into the glass tube, and left at 250 ° C. for 8 hours to decompose the strains. Iron concentrations were measured by atomic absorption photometer (AAS 208; Hitachi Inc., Japan) (Mateos, F., et al. 1999. J. Infect . 38: 18-21).

1-9. Statistical processing

Statistical processing was performed by t -test of SAS (version 6.0 for PC; SAS / STAT Institute Inc.) (SAS Institute, Inc. 1985. SAS / STAT: Language Guide for Personal Computers.Vesion 6 Ed. SAS Institute, Inc.). , Cary, NC. 378 pp.)

2. Experimental Results

2-1. Preparation of host cell

To protect the expressed foreign protein, Saccharomyces cerevisiae 2805 strain induced protease deficient ( pep4 :: HIS3 ) mutation. PEP4 wild-type allele removal was performed by PCR using primers of SEQ ID NO: 2 corresponding to the primer and the HIS3 gene of SEQ ID NO: 1 corresponding to the PEP4 gene.

In addition, co-using the YIplac128 and YEp352 - to produce the expression system, a host cell is at least two selectable markers are needed ^{(leu 2 - -, ura 3} ). Thus, in order to introduce additional screening marker leu 2- to the Saccharomyces cerevisiae 2805 strain, it was crossbred with Saccharomyces cerevisiae 15Dau having an opposite hybridization type including the selection marker leu 2 ⁻ . The formed follicle spores were screened with two screening markers ( leu 2 ⁻ , ura 3 ⁻ ) to select strain 58 (25.8%) strains with a leucine and auraxotrophic phenotype from 224 later.

In the later 58 weeks, the strain containing the mutant PEP4 allele was confirmed by PCR, and 19 of them were found to contain the mutant. Later 19 weeks were designated Saccharomyces cerevisiae 2805-a1 to Saccharomyces cerevisiae 2805-a19.

FIG. 2 is an electrophoretic photograph PCR-amplified pep4 :: HIS3 allele of the posterior 19 follicle spores, λ / H is a λDNA size cut by Hin dIII, α and a are parent strains 2805 and 15Dau, Lanes 1 to 19 are later 19 weeks. In Figure 2 it can be seen that the later 19 weeks contain a DNA fragment of about 2.0 kb, ie pep4 mutant.

Phytase expression was confirmed at the later 19 weeks. Ten weeks showed almost the same phytase activity as the parental strain 2805 (FIG. 3). It was confirmed that the phytase expression rate in the parent strain and later strains of Figure 3, it was shown by measuring the phytase activity. Α and a in the graph are parental strains 2805 and 15Dau, and 1 to 19 are later 19 strains (2805-a1 to a19). Table 1 shows the characteristics of each strain.

Strain Allele ^a leu2 ura3 pep4 MAT phytase 2805 + - - α ** 15Dau - + + a ND ^b 2805-a1 - - - a ** 2805-a2 - - - a ** 2805-a3 - - - α ** 2805-a4 - - - α ** 2805-a5 - - - α ** 2805-a6 - - - α ** 2805-a7 - - - α ** 2805-a8 - - - α ** 2805-a9 - - - a ** 2805-a10 - - - α ** 2805-a11 - - - α * 2805-a12 - - - a * 2805-a13 - - - α * 2805-a14 - - - a * 2805-a15 - - - α * 2805-a16 - - - α * 2805-a17 - - - α * 2805-a18 - - - a * 2805-a19 - - - a * ^a : leu2 (leucine demand), ura3 (uracil demand). MAT ( crossed position), pep4 ( pep4 deficient), phytase (phytase expression) **: > 2805 *: <2805 ^b : not detected

Saccharomyces cerevisiae 2805-a7, which shows the highest phytase activity among the latter 19, was selected as a host cell.

2-2. Heteroparatin Expression in Recombinant Yeast

In order to produce a high proportion of the parine light chain, the parine light chain gene expression cassette was cloned into the YEp352 vector, which is a high-copy episomal vector, and the transformants produced by Northern blot confirmed that the parine light chain was strongly expressed. It was. As a result, the most efficient transformant TYEFLAG-1 was selected among the 10 transformants, and YIplac128, a low-copy integration vector containing a parine heavy chain, was introduced to successively introduce the parine heavy chain gene. 10 candidate strains (TYFHLAG-1) were selected by transformation.

In addition, transformants TYEFHAG-1 and TYEFLAG-1, which produce a single polymerized paritin, were prepared, respectively.

The episomal vector showed high stability, and more than 80% of the strains cultured in liquid phase contained the vector. Also no loss of integration vector was observed. When cultured in YEPD medium, all the transformants showed almost the same growth curve as the host cell, so that the expression of the parine gene did not interfere with the growth of the strain.

In previous studies, iron-tolerance has been identified as a feature in recombinant yeast expressing parine subunit genes. Iron-tolerance was measured in ura -selective medium with 25 mM iron citrate, and when all 46 transformants were cultured, distinct colonies were formed, whereas non-transformants did not form colonies. This is due to the presence of homologous or heterologous parine.

2-3. Heteropolymer polymerization parine production verification

Iron-binding ability of the expressed parine was confirmed by Prussian blue staining.

FIG. 4 is a Prussian blue staining photograph for each transformant cell extract, A is a transformant cell extract pretreated with 1 mM ferrous ammonium sulfate, and then stained with Prussian blue by non-denatured PAGE, and B is sulfuric acid Unmodified PAGE without ferrous ammonium treatment and stained with Coomassie Blue, C is Coomassie blue stained SDS-PAGE of heteropolymerized parine separated by gel purification. In FIG. 4, "1" is a heavy polymer homopolymer, "2" heteropolymer, "3" light chain homopolymer, and "SM" is size marker.

In FIG. 4A, blue bands were observed in the cell extracts of all transformants. This means that the expressed parine subunits were combined into a functional protein capable of iron binding. Cell extracts of the paritin light chain transformants required a reaction time of more than 4 hours in ferrous ammonium sulfate reaction, which is a step before electrophoresis on an unmodified gel. The converter required a reaction time of less than 20 minutes.

In Figure 4B, ferrous ammonium sulfate reaction showed no difference in the electrophoretic mobility of the expressed parine. In FIG. 4B, parine was isolated by gel lysis, followed by SDS-PAGE. The molecular size measured in SDS-PAGE is 27 kDa for the parine heavy chain and 24 kDa for the parine light chain. Heteropolymer type parine showed a marked difference in the band concentration between the light chain and the heavy chain, and the expression ratio was different. As a result of checking the ratio of each subunit, the ratio of the light chain to the heavy chain was 1: 6.8. Was measured.

In addition, in Figures 4A and B, the transformant of the present invention can be seen that only forms a heteropolymeric parine, but does not form other homopolymers.

In addition, each transformant was determined to produce heavy chain polymerized parine, light chain polymerized parine and heteropolymer paritine at 42, 36 and 37 ug per mg of total water soluble protein, repeated three times. In the experiment, there was only a difference of less than about 5%. That is, heavy chain polymerized parine was produced more than light chain polymerized parine, and heteropolymer paritine was produced in the middle, but it was almost similar to heavy chain polymerized parine production strain, and light chain polymerized parine. Production strains and heteropolymeric paritin-producing strains were found to have almost the same expression efficiency.

The iron uptake of parritin was determined by measuring absorbance at 310 nm.

Figure 5 is a graph showing the iron absorption rate of mono- and a hetero-polymer apoperytin heteropolymer, "●" is a hetero-polymer apopery when using a ratio of iron: heteropolymer apoperytin 2000: 1 It shows the iron absorption rate of tin, "○" shows the iron absorption rate of heteropolymer apopatini when iron: heteropolymer type apoparitin is used in 1000: 1 ratio, "▲" is iron: heavy chain The ratio of iron absorption of the polymer-type apoperytin, which is a heavy chain, is shown when the polymerized apopatin is used in a 2000: 1 ratio, and "Δ" is used when the polymer-type apopatin is iron: heavy, in a 1000: 1 ratio. It shows the iron absorption of the heavy chain polymerized apoptotin.

The iron: parity reaction ratio of FIG. 5 is 1000: 1, and the iron absorption rate of the initial polymerization-type paritin of the initial reaction is slightly later than that of the heavy chain polymerized parine, but it shows a similar absorption rate within three minutes, and reaches its peak. The iron absorption rate of the light chain polymerized parine was so low that it took 8 hours to show a sharp rise and then 12 hours to reach its peak.

At 2000: 1 iron-paritine reaction ratio, the double-polymer parine and heavy chain polymerized parine took a little slower reaction than the maximum absorption rate of 9 and 6 minutes, respectively. The iron absorption rate of the polymerized parine was significantly different from each other, and the iron absorption rate of the polymerized paritine, which is heavy, was measured to be about 30% lower than that of the heteropolymerized parine. The light chain polymerized parine also showed a very slow reaction, and it took 12 hours to reach the maximum iron absorption rate, and the iron absorption rate was 90% lower than that of the heteropolymerized parine.

2-3. Iron accumulation in recombinant yeast

In host cells, light chain polymerized parine-producing transformants (TYEFLAG-1), heavy chain polymerized parine-producing transformants (TYEFHAG-1) and heteropolymeric paritine-producing transformants (TYFHLAG-1) Iron accumulation rates were measured, respectively, and are shown in Table 2 below. In addition, the known strain TYETFAG-1 was used as a positive control group, which is a strain expressing the heavy chain of tadpole parine.

Strains Iron citrate concentration Cells / ml ^a Iron content ^b (ppm) ^c Host cell (2805-a7) 0 3.0 x 10 ⁸ ± 0.8 x 10 ⁷ 6.3 ± 1.2 20 1.0 x 10 ⁸ ± 1.3 x 10 ⁷ 210 ± 12 TYETFAG-1 0 3.2 x 10 ⁸ ± 1.2 x 10 ⁷ 5.3 ± 0.9 20 1.5 x 10 ⁸ ± 2.1 x 10 ^{7 *} 500 ± 51 ^* TYEFHAG-1 0 3.3 x 10 ⁸ ± 1.3 x 10 ⁷ 5.9 ± 0.9 20 1.5 x 10 ⁸ ± 2.2 x 10 ^{7 *} 505 ± 62 ^* TYEFLAG-1 0 3.2 x 10 ⁸ ± 1.3 x 10 ⁷ 6.5 ± 0.9 20 1.4 x 10 ⁸ ± 2.0 x 10 ^{7 *} 760 ± 81 ^* TYFHLAG-1 0 3.2 x 10 ⁸ ± 1.1 x 10 ⁷ 5.5 ± 0.9 20 1.4 x 10 ⁸ ± 2.1 x 10 ^{7 *} 1004 ± 61 ^{* a} : Result of 2 replicates ^b : Mean ± standard deviation ^c : Parts per million of dry cell mass. ^* : Significant difference compared to host cell ( p = 0.05)

In Table 2, the iron accumulation rate was found in the order of heteropolymerized parine-producing transformant >> light chain polymerized parine-producing transformant> heavy chain polymerized parine-producing transformant, in particular heteropolymer parry The iron accumulation rate of the chitin-producing transformant was significantly higher than that of the heavy-chain polymerized parine-producing transformant. The light chain polymerized parine-producing transformant also exhibited a high iron accumulation rate, but in reality, the iron absorption time is very long, which is inefficient.

The heteropolymerized paritin-producing transformant (TYFHLAG-1) was produced by Saccharomyces cerebrum on March 27, 2003 at the Korea Agricultural Microbiological Conservation Center (KACC) of the National Institute of Agricultural Biotechnology (NIAB), Suwon, Gyeonggi-do, Korea. TYFHAG1 was deposited in Biji and received accession number KACC93006.

Heteropolymerized parine-producing transformants of the present invention not only have a high paritine production efficiency but also can absorb a large amount of iron in a short time and accumulate the highest level of iron.

As mentioned above, the method for producing heteropolymeric paritin according to the present invention produces a heteropolymeric paritin in which a light chain and a heavy chain are combined in a 5: 1 to 10: 1 molecular ratio, thereby bioavailable a large amount of iron. It can be stored in this high form, and can be used as a food additive, feed additive or medicament to prevent and treat diseases caused by iron deficiency.

<110> KIM, DAE HYUK          YANG, MOON SIK          JANG, YONG SUK <120> METHOD OF PRODUCING HETEROPOLYMERIC FERRITIN <130> dpp20024295 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tgggcagcag catagaac 18 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tcactacata agaacacctt 20

Claims (15)

In the method for producing heteropolymeric paritin in yeast, the method Introducing parity light chain expression vector I and parity heavy chain expression vector II, which are designed to express the paritin light chain in a high molecular ratio compared to the parine heavy chain in a single host cell, and both vectors are introduced. Preparing transformants; And As a method comprising culturing the transformant to express a heteropolymeric paritin, The parine light chain expression vector I is an episomal vector comprising a polynucleotide comprising (a) an ADH2-GPD hybrid promoter and (b) a nucleotide sequence encoding a parine light chain, The parity heavy chain expression vector II is an integrative vector comprising a polynucleotide consisting of a base sequence encoding the (a) ADH2-GPD hybrid promoter and (b) the parine heavy chain. The method of claim 1, wherein said host cell is a protease deficient ( pep4 :: HIS3 ) mutant strain and is a yeast having at least two auxotrophic phenotypes. The method of claim 1, wherein the heteropolymeric parritin comprises a heavy chain: light chain in a 1: 5 to 1:10 molecular ratio. delete delete delete The method of claim 1, wherein the method further comprises culturing the transformant in an iron-containing medium to produce heteropolymerized paritin with stored iron. delete Introducing parity light chain expression vector I and parity heavy chain expression vector II, which are designed to express the paritin light chain in a high molecular ratio compared to the parine heavy chain in a single host cell, and both vectors are introduced. Preparing a transformed transformant, The parine light chain expression vector I is an episomal vector comprising a polynucleotide comprising (a) an ADH2-GPD hybrid promoter and (b) a nucleotide sequence encoding a parine light chain, The parritin heavy chain expression vector II is an integrative vector comprising a polynucleotide comprising (a) an ADH2-GPD hybrid promoter and (b) a nucleotide sequence encoding a partin heavy chain. Method for producing heteropolymeric paritin production strain Heteropolymerized parine-producing strain prepared by the method of claim 9. (a) By introducing the parity light chain expression vector I and parity heavy chain expression vector II designed to express the paritin light chain in a high molecular ratio compared to the parine heavy chain in a single host cell, the two vectors To prepare a transformant with all introduced; And (b) culturing the transformant in a medium containing iron It includes, The parine light chain expression vector I is an episomal vector comprising a polynucleotide comprising (a) an ADH2-GPD hybrid promoter and (b) a nucleotide sequence encoding a parine light chain, The parritin heavy chain expression vector II is an integrative vector comprising a polynucleotide comprising (a) an ADH2-GPD hybrid promoter and (b) a nucleotide sequence encoding a partin heavy chain. Method for producing a strain stored iron. The iron stored strain prepared by the method of claim 11. Between EcoRI and HindIII Restriction Enzymes of YEp352 Vector (a) an ADH2-GPD hybrid promoter; (b) a polynucleotide consisting of a nucleotide sequence encoding a parritin light chain; And (c) Galactose-1-P uridil transferase termination site Parity light chain expression vector comprising a. Between EcoRI and HindIII Restriction Sites of YIplac128 Vector (a) an ADH2-GPD hybrid promoter; (b) a polynucleotide consisting of a nucleotide sequence encoding a paritin heavy chain; And (c) Galactose-1-P uridil transferase termination site Parity heavy chain expression vector comprising a. The feed additive comprising a heteropolymer-type paritin prepared by the method of claim 7 or a strain containing iron of claim 12 as an active ingredient.

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