KR100507665B1 - Vector for Expression of Ferritin Hetropolymer within Yeast and Recombinant Yeast Transformed with The Expression Vector - Google Patents

Abstract

The present invention provides a recombinant ferritin H / L subunit isoform by functionally binding a ferritin H subunit gene and an L subunit gene derived from a eukaryotic cell with a combination of a predetermined single promoter or / and promoter that operates in the yeast strain. An expression vector having a yeast designed to form as a host and a recombinant yeast transformed therefrom are provided. Recombinant yeast according to the above configuration can express ferritin heterozygotes and accumulate iron ions in a large amount of cells, and thus, may be provided as a source of iron for food use and food industry as well as for medical use.

Description

Vector for expression of ferritin heterozygotes in yeast and recombinant yeast transformed with the expression vector {Vector for Expression of Ferritin Hetropolymer within Yeast and Recombinant Yeast Transformed with The Expression Vector}

The present invention relates to an expression vector designed to increase the intracellular iron ion content of yeast and recombinant yeast transformed thereby. More specifically, to prepare a vector expressing the ferritin heterozygotes in yeast, and transformed using the expression vector to provide a recombinant yeast that can contain a large amount of ferritin heterozygotes.

Ferritin is a representative iron storage protein in cells. The protein consists of a protein shell and a core, which is a globular form of a combination of 24 subunits. The resulting pores (about 8 nm in diameter) have up to 4,500 iron atoms per molecule. Can accumulate. Ferritin protects the cells from intracellular toxicity caused by free iron by retaining iron in the Fe (III) state. When ferritin is required, ferritin is reduced and supplied to Fe (II) state.

Ferritin is present in almost all species and has been isolated from many organisms, including humans, including animals, plants and microorganisms (Harrison, PM and Arosio, P. (1996) The ferritins: molecular properties, iron storage function and cellular regulation , Biochim. Biophys.Acta 1275, 161-203. And Theil, EC (1987) Ferritin; structure, gene, regulation and cellular function in animals, plants and microorganism, Ann. Rev. Biochem . 56, 289-315.). In particular, in the case of human ferritin, ferritin is composed of two types of subunits, namely, H (heart; heavy) and L (liver; light) types. Thus, H-rich ferritin present in the heart and brain has a high compositional ratio of H subunits and a relatively low iron content. On the other hand, L-rich ferritin in the liver or spleen has a high content of L subunits and relatively high iron content (Harrison, PM and Arosio, P. (1996) The ferritins: molecular properties, iron storage function and cellular regulation, Biochim. Biophys. Acta 1275, 161-203.). The difference in function of isoperitin present in each tissue does not appear to be independent of the difference in composition ratio of the subunits. In particular, H-rich ferritin regulates bone marrow cell formation in addition to the function of iron storage (Broxmeyer, HE, Bognacki, J., Dorner, MH, deSousa, M. and Lu, L. (1981) Acidic isoferritins as feedback regulators in normal and leukemic myelopoiesis, Hamatol.Bluttransfus. 26, 243-245) or intracellular iron transport (Harrison, PM, Andrews, SC, Artymiuk, PJ, Ford, GC, Lawson, DM, Smith, JMA, Treffry, A. and White) , JL (1990) Ferritin; in Iron Transport and Storage, Ponka, P., Schulman, HM, Woodworth, RC (eds.), 82-101, CRC Press, Boca Raton, Florida.) It became. In tissues, ferritin homopolymers consisting solely of H or L subunits have not been isolated.

Previously, human ferritin H- and L-subunit (or chain) genes were separated to produce recombinant H-subunit ferritin (H-ferritin) and L-subunit ferritin (L-ferritin) in Escherichia coli (Levi, S). ., Luzzageo, A., Cesareni, G., Cozzi, A., Franceschinelli, F., Albertini, A. and Arosio, P. (1988) Mechanisom of ferritin iron uptake: Activity of the H-chain and deletion mapping of the ferroxidase site, J. Biol. Chem . 263, 18086-18092 .; Levi, S., Yewdall, SJ, Harrison, PM, Santambrogio, P., Cozzi, A., Rovida, E., Albertini, A. and Arosio, P. (1992) Evidence that H- and L-chains have cooperative roles in the iron-uptake mechanism of human ferritin, Biochem. J. 288, 591-596 .; Lee, J., Seo, H.-Y ., Jeon, E.-S., Park, OS, Lee, K.-M., Park, C.-U. and Kim, K.-S. (2001) Cooperative activity of subunits of human ferritin heteropolymers in Escherichia coli, J. Biochem. Mol. Biol . 34, 365-370.). Purified recombinant H-ferritin has peroxidase activity, resulting in faster iron uptake rate than L-ferritin, and L-ferritin is more stable than H-ferritin and superior in pore formation. Experimental production of ferritin heteropolymer mixed with H and L subunits in E. coli showed that L-ferritin was not capable of absorbing iron in cells, and the amount of iron bound to recombinant ferritin was determined by the H subunit in ferritin. It was proportional to the content.

Unlike previous publications on iron storage proteins in yeast (Paguzzi, F., E. Lesuisse, and R. Crichton (1988) Iron storage in Saccharomyces cerevisiae, FEBS Lett . 231, 253-258.) (Eide, DJ (1998) The molecular biology of metal ion transport in Saccharomyces cerevisiae, Annu. Rev. Nutr . 18, 441-69.). Therefore, expression of human ferritin heterozygotes in yeast and development of yeast with increased iron content are utilized not only for pharmaceutical raw materials such as dry yeast preparations that can be used for iron-deficiency anemia, but also for use of yeast with increased iron ion content. By confectionery. By using it in the food industry, such as baking and liquor, it will be possible to produce foods with enhanced iron ions, which will create enormous added value.

The present invention has been made to overcome the limitations of the prior art, and an object of the present invention is to provide an expression vector capable of accumulating ferritin heterozygotes in yeast to accumulate iron ions in cells.

Another object of the present invention is to provide a recombinant yeast capable of containing a large amount of ferritin heterologous complex by transforming using the expression vector.

The present invention provides a functional combination of a ferritin H subunit gene and an L subunit gene derived from a eukaryotic cell with a combination of a predetermined single promoter or / and promoters that operate in a yeast strain to form a recombinant ferritin H / L subunit heterozygote. An expression vector having a yeast designed to form as a host is provided.

The expression vector preferably provides an expression vector using a GAL1, GAL10 or GAL10 / GAL1 promoter that works effectively in yeast.

The expression vector of the present invention according to the first aspect provides an expression vector in which the L subunit gene, the GAL10 / GAL1 promoter, and the H subunit gene are sequentially inserted in a 5 'to 3' direction in a known vector expressible in yeast. do.

The expression vector of the present invention according to the second aspect provides an expression vector in which the H subunit gene, the GAL10 / GAL1 promoter, and the L subunit gene are sequentially inserted in a 5 'to 3' direction in a known vector expressible in yeast. do.

The present invention also provides a recombinant yeast transformed with the expression vector according to the first side or the second side.

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

The present invention is to provide a recombinant vector for producing yeast as a feed material for medical, food industry or livestock, etc. with enhanced iron ion content, and a recombinant yeast prepared therefrom. Therefore, it contains any ferritin gene that can be expressed in edible yeast that accumulates iron ions available to animals, including humans. The ferritin gene is derived from and isolated from eukaryotic cells, that is, animal cells such as humans and livestock (including cattle, horses, etc.), as well as plant cells such as soybean, and includes H subunit and L subunit DNA. .

The expression vector of the present invention can be effectively expressed in yeast, and is expressed under the influence of a predetermined promoter operating in yeast as a structure in which H subunit and L subunit DNA are inserted together in the same vector.

The yeast used in the present invention is Saccharomyces cerevisiae, a true yeast strain.

The expression vector usable in the present invention does not require any special limitation as long as it is expressible in Saccharomyces cerevisiae and genetic recombination is possible. Examples of such expression vectors are 2 micron, pBM272, pBR322-6, pBR322-8, pCS19, pDW227, pDW229, pDW232, pEMBLYe23, pEMBLYe24, pEMBLYi21, pEMBLYi22, pEMBLYi32, pEMBLYr25, pFL2, pFL26, pFL34, pFL34, , pFL39, pFL40, pFL44L, pFL44S, pFL45L, pFL45S, pFL46L, pFL46S, pFL59, pFL59 +, pFL64-, pFL64 +, pG6, pG63, pGAD10, pGAD424, pGBT9, pGKl2, pJRD171, pKDN p3 p163 pON3, pPM668, pRAJ275, pRS200, pRS303, pRS304, pRS305, pRS306, pRS313, pRS314, pRS315, pRS316, pRS403, pRS404, pRS405, pRS406, pRS413, pRS414, pRS415, pRS416, pRS423, pRS424, pRS425, pRS426 pSG424, pSKS104, pSKS105, pSKS106, pSZ62, pSZ62, pUC-URA3, pUT332, pYAC2, pYAC3, pYAC4, pYAC5, pYAC55, pYACneo, pYAC-RC, pYES2, pYESHisA, pYESHisB, pYESHC3, pYESHC3 YCpGAL1, YCplac111, YCplac22, YCplac33, YDp-H, YDp-K, YDp-L, YDp-U, YDp-W, YEp13, YEp213, YEp24, YEp351, YEp352, YEp353, YEp354, YEp355, YEp357R, YEp358, YEp358R, YEplac112, YEplac181 , YEplac195, YIp30, YIp31, YIp351, YIp352, YIp353, YIp354, YIp355, YIp356, YIp356R, YIp357, YIp357R, YIp358, YIp358R, YIp5, YIplac128, YIplac204, YRp2204 Yp2211 , pDB248X, pEA500, pFL20, pIRT2, pIRT2U, pIRT2-CAN1, pJK148, pJK210, pON163, pNPT / ADE1-3, pSP1, pSP2, pSP3, pSP4, pUR18, pUR19, pZA57, pWH5, pART1, pPHY21 , REP3, REP4, REP41, REP42, REP81, REP82, RIP, REP3X, REP4X, REP41X, REP81X, REP42X, REP82X, RIP3X / s, RIP4X / s, pYZ1N, pYZ41N, pYZ81N, pSLFSM, pSLF104, pSLF104 , p2UG, pART1 / N795, pYGT and the like.

(See: http://genome-www2.stanford.edu/vectordb/vector.html, http://pingu.salk.edu/~forsburg/vectors.html)

Hereinafter, a vector production process for the expression of ferritin heterozygotes will be described as a preferred preparation of the present invention using the human ferritin gene.

E. coli JM 109 was used as a host for plasmid propagation and mass extraction, and Saccharomyces cerevisiae 2805 ( MATα pep4 :: HIS3 prb1 Δcan1 GAL2 his3 ura3-52 ) was used as a yeast host cell for human ferritin gene expression. Was used. As a recombinant plasmid, a yeast shuttle vector YEp352 was used.

Genes encoding human ferritin H and L subunits ( hfH and hfL ) were obtained and cloned into E. coli to prepare expression vectors. In order to prepare a vector expressing high human hfH and hfL in yeast, expression vectors pYGL1 and pY1H10L were prepared by modifying the N-terminal codons of the hfH and hfL or the upper genes using GAL1, an inducible promoter. Polymerization chain reaction was carried out using these primers, and the resultant was blunted and inserted into pUC18 to confirm the gene sequence. Then, hfH fragments obtained by cutting BamH I and Sal I were obtained from this vector, and pYGH2 was prepared by inserting into pYGT, a yeast expression vector including a GAL1 promoter and a GAL7 terminator. For the L subunit has obtained the hfL fragments cut by EcoR I and Sma I from the vector confirm the gene sequence was prepared by inserting the pYGL1 pYGT.

To prepare a vector expressed by the GAL1-GAL10 promoter, first, a pYGL1, a ferritin L western unit expression vector in yeast, is cleaved with Xba I and Shp I, and then treated with Klenow fragment to treat GAL7. A hfL DNA fragment with terminator was obtained. The rear of the GAL1-GAL10 promoter of pYGH2 plasmid was then cut with EcoR I and bloated before hfL -tGAL7 was inserted. The results establish a pY1H10L having a gene sequence expressing ferritin release aggregate tGAL7- hfL -GAL10-GAL1- hfH -tGAL7. In addition, pYGH2 plasmid was digested with Xba I and Shp I and blunted to obtain a hfH DNA fragment with a GAL7 terminator in order to construct a pY1L10H plasmid with the gene positions of the ferritin subunit interchanged. Then, the back of the GAL1-GAL10 promoter of pYGL1 plasmid was cut with EcoR I and bloated, and then hfH- tGAL7 was inserted. The results establish pY1L10H having a gene sequence of tGAL7- hfH -GAL10-GAL1- hfL- tGAL7 (Fig. 1).

Transformation of the expression vector into yeast is conventional method (Ito, H., Y. Fukuoka, K. Murata, A. Kimura (1983) Transformation of intact yeast cells treated with alkali cations, J. Bacteriol . 153, 163-168.). Accordingly, each of the plasmids for expression of human ferritin H subunit and L subunit is Saccharomyces cerevisiae. After transformation at 2805, transformants were first screened in uracil deficient minimal media. The selected recombinant yeast was extracted with DNA using glass beads, which was reverse transformed into E. coli, followed by enzyme cleavage to confirm DNA and finally selected. The yeast cells transformed with the selected pY1H10L and pY1L10H were named as Y1H10L and Y1L10H, respectively.They contained higher Y1L10H than the ferritin genes. KACC93005) was selected and deposited.

The transformed yeast prepared by the above process can express ferritin heterozygotes in yeast and accumulate a large amount of iron ions in the cells, and thus can be provided as a source of iron for food use as well as for food industry and animal feed compositions. .

The human ferritin produced according to the present invention may be used as a pure separation from the yeast culture, but more preferably may be added in the form of the cultured yeast itself or dry powder thereof, in this case immediately without a special purification process There is an advantage that can be used. The iron supply preparation may be used for the purpose of functional food additives for iron supply or for the treatment of anemia for the treatment of iron deficiency symptoms, and may be formulated according to the preparation of general pharmaceutical preparations for oral or parenteral administration. .

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and liquid preparations for oral use include suspensions, solvents, emulsions, and syrups. Water is a simple diluent commonly used. In addition to liquid paraffin, various excipients may be included such as wetting agents, sweetening agents, fragrances, preservatives and the like. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.

In the iron supply formulation containing the human ferritin of the present invention as an active ingredient, the iron content of the human ferritin or the yeast producing the human ferritin as an active ingredient is 2000 ppm. In view of this, in order to meet the general iron supply amount of 10 to 50 mg / day can be administered by formulating the appropriate amount according to a known method. At this time, the specific dosage is determined based on the known amount of iron formulation, it can be specifically modified and adjusted according to the condition of the patient.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the content of the present invention, and the scope of the present invention should not be construed as being limited to these embodiments.

<Example>

Strains and Plasmids Used

Escherichia coli JM 109 was used as a host for propagation and mass extraction of plasmids. S. cerevisiae 2805 ( MATα pep4 :: HIS3 prb1 Δcan1 GAL2 his3 ura3-52 ) was used as a yeast host cell for human ferritin gene expression. The recombinant plasmid was used as a yeast shuttle vector YEp352.

Medium and culture method

As a general medium for the cultivation of yeast host cells and recombinant yeast, a complex nutrient medium (YEPD (1% yeast extract, 2% bacto-peptone, 2% dextrose)) was used as a selection medium for selecting the transformants of yeast Uracil deficient minimal medium (0.67% amino acid deficient yeast nitrogen base (YNB), 0.5% casamino acid, 2% glucose, 0.03 g / L adenine and tryptophan) was used and 2% of the ferritin gene was expressed in the expression inducing medium. Galactose was added. Recombinant yeast strain was incubated for 3 days at 30 ℃, 200 rpm in a 300 mL flask (working volume; 40 mL). Cell growth of yeast cells was transferred to a 300 mL flask, and the culture solution collected every 24 hours was diluted at an appropriate ratio (10-100 times) at 600 nm using a UV / Vis spectrophotometer (Ultrospec 3000, Pharmacia Biotech.). Absorbance was measured. The number of cells was measured by using a counter (hemocytometer) by diluting the culture solution collected at regular intervals at an appropriate ratio. Pre-culture and flask culture conditions for basic experiments are described by Lee et al. (Lee, DH, Seo, JH, Sohn, JH, Choi, ES and Rhee, SK (1994) Optimization of environmental conditions for hirudin production form recombinant Saccharomyces cerevisiae, Korean J. Biotechnol. Bioeng. 9 , 8-15.). The amount of protein in yeast is Bradford, MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindin, Anal. Biochem. 72 , 248-254.). Plasmid stability was determined by properly diluting (100-200 times) the cultures taken at daily intervals, then smearing 100 colonies grown on complex nutrient plates to toothpicking with uracil-deficient plates, followed by the ratio of the number of colonies formed (percentage). Was measured.

Example 1 Ferritin Expression Recombinant Plasmid Construction

In order to obtain genes encoding human ferritin H and L subunits ( hfH and hfL ), amplified by a polymerase chain reaction method in human liver cDNA library, and then cloned into Escherichia coli, an expression vector was prepared. In order to prepare a vector expressing high human hfH and hfL in yeast, expression vectors pYGH2 and pYGL1 were prepared by modifying the N-terminal codons of the hfH and hfL or the upper genes using GAL1 as an inducible promoter. . To this end, the upper primer of the ferritin H subunit was substituted with TTTAAA in the GAATTC before the start codon to prepare 5′-AGCTTTG TTTAAA ATG ACGACCGCGTCC-3 ′ of SEQ ID NO: 1. In addition, the lower primer was prepared by 5'-CGCAAGCTTTTAGCTTTCATTATCACTGTCTC-3 'of SEQ ID NO. For the L subunit, the upper primer was prepared by substituting the codon AGC (Ser) after the start codon with TCT (Ser) to prepare 5′-CGAATTC ATG TCT TCCCAGATTCGTCAG-3 ′ of SEQ ID NO: 3, and the lower primer was SEQ ID NO: 4 'of 5'-TCCCCCGGGTTAGTCGTGCTTGAGAGT-3'. Polymerization chain reaction was performed using these upper and lower primers as follows. In the case of the H subunit, the reaction was repeated 35 times at 94 ° C. for 1 minute, 43 ° C. for 30 seconds, and 72 ° C. for 1 minute to amplify DNA, followed by treatment at 72 ° C. for 7 minutes to complete the reaction. In the case of the L subunit, it is the same as the polymerization chain reaction method except that the annealing temperature is set to 52 ° C. The resulting reaction was blunted and then inserted into pUC18 to confirm the gene sequence. Then, hfH fragments obtained by BamH I and Sal I were obtained from this vector, and pYGH2 was prepared by cutting and inserting pYGT, a yeast expression vector including a GAL1 promoter and a GAL7 terminator, using the same restriction enzyme. In the case of the L subunit, a hfL fragment cut with EcoR I and Sma I was obtained from a vector confirming the gene sequence, and inserted into pYGT cut with SalI to prepare pYGL1.

GAL1-GAL10 first to produce a vector to be expressed by the promoter, by cutting the ferritin L subunit expression vector pYGL1 in yeast with Xba I and Shp I and then, by treatment with Klee Smirnoff fragment hfL having GAL7 terminator DNA fragments were obtained. The rear of the GAL1-GAL10 promoter of pYGH2 plasmid was then cut with EcoR I and bloated before hfL -tGAL7 was inserted. The results establish a pY1H10L having a gene sequence of tGAL7- hfL -GAL10-GAL1- hfH -tGAL7 capable of expressing the two kinds of ferritin complex. In addition, pYGH2 plasmid was cleaved with Xba I and Shp I and blunted to obtain a hfH DNA fragment with a GAL7 terminator in order to construct pY1L10H plasmid in which the genes of ferritin subunits were interchanged. Then, the back of the GAL1-GAL10 promoter of pYGL1 plasmid was cut with EcoR I and bloated, and then hfH- tGAL7 was inserted. As a result tGAL7- hfH -GAL10-GAL1- hfL- tGAL7 the pY1L10H having the gene sequence was constructed of.

Example 2 Preparation, Selection, and Intracellular Expression Efficiency of Yeast Transformant

Preparation and Screening of Yeast Transformants

Each of the plasmids constructed in FIG. 1 was analyzed by Ito et al. (Ito, H., Y. Fukuoka, K. Murata, A. Kimura (1983) Transformation of intact yeast cells treated with alkali cations, J. Bacteriol . 153, 163- 168.) was transformed into S. cerevisiae 2805, and then transformants were first screened in uracil deficient minimal media. Selected recombinant yeast extracted DNA using glass beads, reverse transformation of E. coli and enzyme digestion to confirm DNA, and finally Y1H10L and Y1L10H transformants were finally selected.

The transformants were used to examine cell growth, ferritin expression and plasmid stability in uracil-deficient minimal media and complex nutrient media, and to determine the intracellular expression efficiency of ferritin after SDS-polyacrylamide gel electrophoresis. The content of ferritin in the total protein expressed by Tomica (Molecular Dynamics PC-120, USA) was analyzed.

Cell growth and plasmid stability

Productivity of gene products depends on several factors including plasmid stability, culture conditions of recombinant yeast (Romanos, MA, Scorer, CA and Clare, JJ (1992), Foreign gene expression in yeasts, Yeast 8 , 423- 488) Using the recombinant plasmids prepared above, the plasmid stability and growth rate according to the culture conditions were investigated. Both strains gradually increased the cell growth until the end of the culture, the final cell concentration in the complex nutrient medium reached 36-38 OD ₆₀₀ for Y1H10L, 35-36 OD ₆₀₀ for Y1L10H. Most of the cells grown in the uracil deficient medium showed lower cell proliferation than those in the combined nutrient medium, reaching peak levels around 3-4 days, and decreasing thereafter. In particular, when 1% glucose / 3% galactose was added to the YEP medium, the cell growth curve was most increased. The strain Y1L10H also appeared similarly to the case of Y1H10L. In plasmid stability, the plasmids remained relatively stable at about 80% or more until the 3rd day of culture for both Y1H10L and Y1L10H strains.

Effect of Medium on Ferritin Expression

The GAL promoter is a promoter that is strongly inhibited by glucose and induced by galactose. Galactose-induced methods include non-repressing and non-inducing sugar, followed by rapid expression induction by galactose, growth in glucose-added medium, and centrifugation. By removing glucose and growing in galactose medium (this method leads to a lag phase in 3-5 hours), and in a medium containing glucose and galactose, in which case glucose is consumed completely before the use of galactose. do. The first two methods are used at small-scales and are not available at large-scales. In this experiment, the third method was used to investigate the effects of galactose and glucose on ferritin expression. The cultures pre-cultured in uracil deficient minimal media were diluted to 1.0 × 10 ⁷ / mL to add YEP with 2% galactose, 1% glucose / 1% galactose, 1% glucose / 2% galactose and 1% glucose / 3% galactose. Incubated in medium (1% yeast extract, 2% bacto-peptone). As a control, the whole culture cultured in uracil deficient minimal medium was diluted to 1.0 × 10 ⁷ / mL and cultured in uracil deficient medium supplemented with 2% galactose instead of 2% glucose. And while culturing them, the amount of total protein and the amount of ferritin expressed in the total protein was measured. As a result of analyzing the total protein amount in the cells, the total protein amount gradually increased up to 4 days in cultivation in proportion to the growth of the cells, and decreased after 4 days in culture. As in cell growth, growth in YEP media resulted in higher total protein levels than in uracil deficient media.

In order to measure the expression level of ferritin in all the water-soluble proteins, the recombinant yeast extract was mixed with SDS sample loading buffer at 1: 1 (v / v) and heat-treated at 100 ° C. for 10 minutes, followed by 12% SDS-polyacrylamide gel. The results are shown in FIG. 2, and analyzed using densitomitri. As a result of investigating the effect of the medium on the expression of ferritin, the total expression amount of the H and L subunits was the highest in the medium containing 2% galactose for strain Y1H10L. Expression levels were highest during two days of culture in all media tested. Thus, the total expression of H and L subunits corresponding to about 20% of the total water-soluble protein was obtained when cultured in YEP medium containing 2% galactose for 2 days in consideration of cell growth. At this time, the relative expression amount of H subunit was analyzed as 22%.

In the case of strain Y1L10H, the expression of the two subunits of ferritin was the highest at 18% on the first day of culture in uracil deficient medium, and the expression was decreased as the culture day passed. The total expression of H and L subunits according to the medium was the highest when cultured in YEP medium containing 2% galactose as in the case of Y1H10L. In this case, the total amount of expression was highest in two days of culture in all the tested media. Thus, even for this strain, the total amount of expression of H and L subunits corresponding to about 15% of the total water-soluble protein was obtained when cultured in YEP medium containing 2% galactose in consideration of cell growth. At this time, the relative expression amount of H subunit was analyzed to 38%.

Example 3 Formation of Ferritin Heterogeneous Assembly and Measurement of Iron Ion Absorption Capacity

Electrophoresis

SDS-polyacrylamide gels were analyzed by Laemml's method (Lammli, UK (1970), Cleavage of structural protein during the assembly of the head of bacteriophage T4, Nature 227, 680-685.) To analyze expression by recombinant plasmids. Followed. In order to confirm that the expressed ferritin H and L subunits are combined with each other to form a ferritin heterozygote and the combined protein reacts with iron, the cell extract is reacted with 1 mM ferrous ammonium sulfate and then used with an unmodified gel. Electrophoresis was performed. For protein staining, 0.25% Coomassie Brilliant Blue R-250 was used, and for staining Fe (III), 2% K ₄ Fe (CN) ₆ and 2% HCl solution mixed 1: 1 in use immediately. Kim, YT and Kim, KS (1994), Synthesis of active tadpole H-chain ferritin in Escherichia coli, Mol .. Cells 4, 125-129.

In order to confirm the composition ratio of the subunits of human ferritin produced in the Y1H10L and Y1L10H cells, electrophoresis was performed using an unmodified gel, and then the band corresponding to the monomer of the ferritin protein was extracted and denatured again. Was performed, and the relative content of the subunits was analyzed by densitomitri.

As a result of electrophoresis using a non-denatured gel on the recombinant protein, each of the ferritin H and L subunits expressed in the yeast was combined with each other to identify a ferritin protein band composed of heterozygous. The expressed protein is recombinant H-ferritin or L-ferritin expressed in E. coli (Lee, J., Seo, H.-Y., Jeon, E.-S., Park, OS, Lee, K.-M., As in Park, C.-U. and Kim, K.-S. (2001) Cooperative activity of subunits of human ferritin heteropolymers in Escherichia coli, J. Biochem. Mol. Biol . 34, 365-370. Positive staining was observed (FIG. 3). The results indicate that the recombinant ferritin heterozygotes produced in yeast accumulated iron in the pores by oxidizing iron in vitro .

In order to determine the composition ratio of the subunits of human ferritin produced in Y1H10L and Y1L10H cells, electrophoresis was performed using non-denatured gel, and the protein was extracted from the band corresponding to the monomer of ferritin protein and denatured again. As a result of PAGE, the composition ratio of the H: L subunit was confirmed to be about 3: 7 in both strains (FIG. 4). Therefore, the present study confirmed that the recombinant human ferritin produced by the two cells was a ferritin heterozygous assembly combined with H and L subunits.

Compared with the relative content of the subunits obtained above, the content of the H subunit was slightly increased for the Y1H10L strain, and slightly decreased for the Y1L10H. This is inferred because it was extracted from the band of ferritin monomers, not the whole ferritin protein produced in yeast. Previous reports have shown that there is a close relationship between the relative amounts of expressed subunits and the proportion of subunits in the recombinant protein produced, and a relatively limited range of subunit compositions (Rucker, P., Torti, FM and Torti, SV (1997) Recombinant ferritin: modulation of subunit stoichiometry in bacterial expression systems, Protein Eng . 10, 967-973.).

Absorption and atomic absorption photometer of iron ions

Yeasts pre-cultured in uracil deficient medium to induce iron uptake in recombinant microorganisms were incubated in YEP (containing 2% galactose) medium at 30 ° C. for 2 days or 3 days, followed by centrifugation (5,000 × g, 5 Cells were harvested and washed twice with 20 mM MOPS (3- [ N -morpholino] propane sulfonic acid) buffer (pH 6.5). Yeast cells (100 mg / mL) were reacted for 30 minutes in the above buffer containing 5% (w / v) glucose and then reacted for 2 hours in air without light by adding 14.3 mM iron sulfate. . In addition, 0.2 mM ascorbic acid was added to react with iron ions, and the absorption of iron ions was also investigated. Thereafter, the cells were harvested by centrifugation and washed twice with distilled water. In order to analyze the iron content, the reaction was carried out at 250 ° C. for 8 hours in nitric acid and hydrochloric acid (5: 2, vol / vol) solution, and then the iron concentration was analyzed using an atomic absorption spectrophotometer (SpectrAA-880, Varian).

The ferritin heterozygotes were isolated from the recombinant yeast, and the proteins were identified using a transmission electron microscope (Jeol JEM 2010). For staining samples, 5 μL of 2% phosphorous tungstic acid (pH 6.0) and 2 μL of the sample were mixed and reacted for 10 minutes, and then dried and observed under an electron microscope.

The results of analyzing the iron uptake in the recombinant yeast strain producing ferritin heterozygotes are shown in Table 1 and FIG.

TABLE 1

Cell type Fe ²⁺ (mM) Ascorbic Acid (mM) yield(%) Fe ²⁺ absorption ^a Culture (days) YGT  0     0.1 3  14.3 0 8.4 ± 2.0    12.0 ± 2.9 3 Y1H10L 0     0.1 3 14.3 0 12.5 ± 0.4    17.8 ± 0.5 3 0 17.4 ± 0.3    24.9 ± 0.4 2 0.2 19.3 ± 0.4    27.5 ± 0.5 2 Y1L10H 0     0.1 3 14.3 0 14.9 ± 0.8    21.3 ± 0.8 3 0 19.7 ± 0.5    28.2 ± 0.3 2 0.2 20.4 ± 0.2    29.1 ± 0.2 2

^a unit is μmol / g (wet cell weight).

As a result, it was confirmed that the iron content in the cells increased as a result of reacting the yeast strain with Fe ²⁺ . When the cultured yeast host cells were reacted with 14.3 mM Fe ²⁺ , the iron concentration increased about 89 times. The concentration of iron in the cells when the yeast strains Y1H10L and Y1L10H were reacted with 14.3 mM Fe ²⁺ was 17.8 μmol / g (wet weight) and 21.3 μmol / g (wet weight), respectively. Was 12.0 μmol / g (wet weight). This concentration is about 1.5 and 1.8 times higher iron content than the host cell, respectively. When yeast strains Y1H10L and Y1L10H were incubated for two days, these values were 24.9 μmol / g (wet weight) and 28.2 μmol / g (wet weight), respectively. The concentration of iron increased 2.1 times and 2.4 times, respectively, compared to the host cell, indicating that iron uptake increased when the relative expression of ferritin was high. In addition, iron uptake in yeast Y1L10H increased 13-20% than in Y1H10L, which is inferred to be related to the relative amount of expression of H subunits (FIG. 3). In case of addition of 0.2 mM ascorbic acid in the reaction with Fe ²⁺ , these values were 27.5 μmol / g (wet weight) and 29.1 μmol / g (wet weight), respectively, and the iron absorption was slightly increased. Ascorbic acid is an important coenzyme in many metabolic reactions in cells, Hoffman et al. (Hoffman, KE, Yanelli, K. and Bridges, KR (1991) Ascorbic acid and iron metabolism: alterations in lysosomal function, Am. J. Clin. Nutr. 54, 1188-1192.) Reported that ascorbic acid is closely related to the metabolic mechanism of iron ions in cells, in particular inhibiting ferritin degradation and keeping iron ions in a biologically useful state.

Fig. 6 shows the results obtained by separating the ferritin heterozygotes from the recombinant yeast under an electron microscope. From the images of ferritin heterozygotes isolated from the two strains, the ferrite cores with high electron density were identified. As a result of observation from these staining samples, it was confirmed that the ferritin pores were surrounded by a protein band having a low electron density. This demonstrates that the recombinant ferritin heterozygotes produced in yeast maintain the ability to store iron.

In summary, through the present example, a system for successfully producing a human ferritin heterozygotes in yeast was constructed, and the recombinant ferritin heterozygotes produced were found to have iron binding activity. And it can be inferred that the iron content of the yeast is enhanced by expressing the human ferritin gene in the yeast strain. Ferritin is a source of iron for both animals and plants, as well as for humans. Therefore, the recombinant yeast with improved iron content produced through the present invention can be utilized in a wide variety of industries, including medicine, food and feed industry.

According to the present invention, the ferritin isomer can be expressed in yeast to produce yeast having an enhanced iron ion content. The iron ion content-enhanced yeast obtained as described above is a raw material for pharmaceuticals, such as dry yeast preparations that can be utilized for iron deficiency anemia, confectionery. It can be used as a raw material for the food industry such as baking or liquor, or for feed to livestock.

1 is a cleavage map of pY1L10H and pY10L1H prepared according to the present invention

Figure 2 is a mixture of the recombinant yeast produced according to the present invention with SDS sample loading buffer 1: 1 (v / v) mixed with 100 ℃, heat treatment for 10 minutes, the result of performing 12% SDS-polyacrylamide gel

Figure 3 is the result of electrophoresis performed using an unmodified gel for the identification of ferritin heterozygotes expressed in recombinant yeast according to the present invention

Figure 4 is an SDS-PAGE results carried out to determine the composition ratio of the subunit of human ferritin produced in recombinant yeast cells according to the present invention

5 is a result of analyzing the degree of iron absorption in the recombinant yeast strain according to the present invention

6 is a result of observing on a microscope to isolate the ferritin heterozygotes from recombinant yeast according to the present invention

<110> KIM, Kyung Suk JEONG, Yong Seob <120> Vector for Expression of Ferritin Hetropolymer within Yeast and Recombinant Yeast Transformed with The Expression Vector <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer for ferritin H subunit <400> 1 agctttgttt aaaatgacga ccgcgtcc 28 <210> 2 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer for ferritin H subunit <400> 2 cgcaagcttt tagctttcat tatcactgtc tc 32 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer for ferritin L subunit <400> 3 cgaattcatg tcttcccaga ttcgtcag 28 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer for ferritin L subunit <400> 4 tcccccgggt tagtcgtgct tgagagt 27

Claims (6)

delete delete An expression vector obtained by sequentially inserting ferritin L subunit genes derived from eukaryotic cells, GAL10 / GAL1 promoters, and ferritin H subunit genes derived from eukaryotic cells in a 5 'to 3' direction to a known expression vector expressible in yeast. Recombinant Saccharomyces cerevisiae expressing the transformed recombinant ferritin H / L subunit isoforms An expression vector obtained by sequentially inserting ferritin H subunit genes derived from eukaryotic cells, GAL10 / GAL1 promoters, and ferritin L subunit genes derived from eukaryotic cells in a 5 'to 3' direction to a known expression vector expressible in yeast. Recombinant Saccharomyces cerevisiae expressing the transformed recombinant ferritin H / L subunit isoforms delete delete