JP2002218980A - New ferritin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new ferritin gene. SOLUTION: This ferritin gene originating from a plant or a gene recombination coding for a subunit having molecular weight of 28 kDa having sequence Ala-Ser-Asn-Ala-Pro-Ala for the 6 residues from the amino end after breakage of TP(Transit Peptide). This gene is capable of sustaining an iron content caused to resistance to breakage compared to conventional 28 kDa subunit.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel ferritin.

[0002]

2. Description of the Related Art Ferritin is an iron storage protein widely present in the living world from higher animals including humans to microorganisms such as plants and bacteria, and supplies stored iron to other iron-binding enzymes and the like. Its primary physiological role is to take in excess inorganic iron that can damage the living body and protect cells by detoxifying it. This protein is very large (molecular weight 540 kDa), has an outer diameter of about 13 nm, and has 24 subunits stacked symmetrically, as if in a bag-like structure. It is estimated that up to 4500 atoms of iron are stored in this bag-like structure. The present inventors have found that by introducing a ferritin gene capable of storing such a large amount of iron into a plant as a foreign gene, the iron content of the plant can be increased,
A patent application was filed earlier (see JP-A-9-201190). Conventional plant ferritin was thought to be formed from a subunit (28 kDa) derived from one gene. The 28 kDa subunit was said to be easily converted to a 26.5 kDa subunit by cleavage of an extension peptide (EP) upon release of iron. Although the 28 kDa subunit has been reported to be in an iron-containing form, the 26.5 kDa
The iron content of the Da subunit is not known.
Therefore, it is considered that without this conversion by releasing iron, the plant can store iron more efficiently.

[0003]

DISCLOSURE OF THE INVENTION The present invention has been studied variously for ferritin capable of storing more iron in a plant, and as a result, has found ferritin having a subunit different from the conventional one. Was completed.

[0004]

That is, the present invention relates to ferritin and a ferritin gene containing a subunit having a molecular weight of 28 kDa which is not easily cleaved. More specifically, ferritin derived from plants, particularly soybean. In the present invention, "not easily cleaved" is used in the sense that it is not cleaved under the condition that a 28 kDa subunit of ferritin derived from a plant, which is conventionally known, is converted into a 26.5 kDa subunit. The ferritin gene of the present invention comprises a transit peptide (TP: Transit Peptid).
e) The sequence of 6 residues from the amino terminus excluding Ala-Ser-As
It encodes a subunit having a molecular weight of 28 kDa, which is n-Ala-Pro-Ala. Also, in the conventional 28 kDa subunit of soybean ferritin, the 234th position of the carboxyl (C-) terminal residue is arginine, whereas in the ferritin gene of the present invention, it is characterized by being substituted with leucine. Due to this difference, the 28 kDa subunit of the invention is not easily cleaved. These specific sequences of the present invention can be obtained by genetic recombination techniques. Accordingly, the present invention encompasses ferritin and ferritin genes derived from plants and by genetic modification.

[0005]

DETAILED DESCRIPTION OF THE INVENTION "Not easily cut" of the present invention
The following experiments were performed to confirm the 28 kDa subunit of ferritin. A. Experimental method Purification of Natural Soybean Ferritin 500 g of dried soybean seeds (Kitano-shiki variety) were pulverized with a grinder. The soybean seed flour was suspended in 50 mM Tris-HCl buffer containing 1 mM EDTA (ethylenediaminetetraacetic acid) and 10 mM 2-ME (2-mercaptoethanol), homogenized, and centrifuged at 10,000 × g for 10 minutes. The supernatant was fractionated with 20% saturated ammonium sulfate. The amber precipitate was collected by centrifugation and 50 mM Tris-HCl (pH
7.5) Dialysis in buffer. The dialyzed sample was prepared using DEAE-Toyopearl (TOS) previously equilibrated with 50 mM Tris-HCl buffer containing 1 mM EDTA.
OH) added to the column. The column was washed with a buffer containing 0.15 M sodium chloride and eluted amber ferritin. The solution containing soy ferritin was again fractionated with 20% saturated ammonium sulfate.
The supernatant collected by centrifugation was applied to a butyl toyopearl (TOSOH) column and eluted with a 20-0% saturated density gradient ammonium sulfate. Fractions containing soy ferritin were pooled, dialyzed in 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA, and purified by Q-Sepharose column (Amersham-Pharmacia).
Was added to Protein was eluted with a 0 to 0.7 M density gradient of sodium chloride. The fractions containing soy ferritin were pooled and concentrated, and finally 20 mM Tris-HC
l Attached to a Superdex 75 pg gel filtration column (Amersham-Pharmacia) equilibrated with (pH 7.5) buffer.

[0006] Apoferritin was obtained by a conventionally known method. The purified ferritin was dialyzed in 50 mM Hepes / NaOH buffer (pH 7.0) containing 1% thioglycolic acid, followed by 0.1
The dialysate was dialyzed against Heaps buffer containing 1% and 0% thioglycolate. Then the protein is 13g / l Chilex-100
(Bio-Rad) and Heaps buffer containing 0.2 M sodium chloride, and finally dialysed in deionized water. Purified protein concentration is determined using the protein assay kit (Bi
o-Rad) and densitometer (Pharmacia).

[0007] 2. Amino acid sequence analysis Soy ferritin subunits were separated by SDS-PAGE using a 12.5% polyacrylamide gel and transferred to a PVDF membrane. The membrane was stained with 0.1% Ponceau-S (Sigma) dissolved in 2% (V / V) acetic acid. Two characteristic bands on the membrane were each cut from the membrane. The amino terminal amino acid sequences of both proteins are
Applied Biosystems Model 477A Pulse-
Automated Edman degradation with a liquid amino acid sequence analyzer (sequencer). The carboxyl (C-) terminal amino acid sequence of the 26.5 kDa subunit was digested with lysyl endopeptidase, and the C-terminal containing peptide fragment was digested with DITC-glass (SIG).
MA). Thereafter, the sequence of the C-terminal fragment was determined by automated Edman degradation.

[0008] 3. Novel ferritin gene cD from soybean
Cloning of NA (complementary DNA) Soybean EST registered with Gene Bank
(Expressed Sequence Tag) Sequence (AW185
525, AI966037, AW397605, AI443722, AI900240) were used for primer design. 5'race ( r apid a mplificat
ion of c DNA e nds) and 3'race has a total RNA extracted from 1 week seedling after germination was carried out in a manufacturing method of a template (template). Ten candidate sequences for the target sequence encoding the L subunit were determined.

4. Preparation of Recombinant Soy Ferritin The DNA sequence encoding the mature part of soy ferritin uses (a) primer-TP (5'-GCGCATATGTCAACGGTGCCTCTCAC-3 ') and (b) C (5'-GCGGGATCCTAATCASAGAAGTCTTTG-3'). PCR (polymerase chain reaction). -TP and C contained NdeI site (position) and BamHI site, respectively. The obtained fragment containing no TP sequence was digested with NdeI and BamHI, and NdeI on the expression vector pET3a (Novagen) was digested.
And BamHI to obtain plasmid pESF. The expression plasmid pESF was transformed into Escherichia coli (E. coli) BL21 (DE3) pLys strain. E. coli containing expression plasmids The E. coli strain was isolated at 37 ° C. from ampicillin (50 μg / ml) supplemented LB (Luria-Bertani).
The cells were cultured in a medium. When grown to A ₆₀₀ (absorbance) = 0.6, isopropyl-
β-D-thiogalactopyranoside (IPTG) was added. Bacteria were harvested by centrifugation, and proteins were extracted with a BugBuster protein extraction reagent (Novagen). The purification process of recombinant ferritin containing the complete mature region is similar to that of native ferritin.

[0010] 5. Soybean ferritin degradation (degradation) experiment 200 mg of soybean leaves were extracted with extraction buffer [50 mM K ₂ PO
₄ , 1 mL of 10 mM 2-ME, 0.1% Triton X-100, 0.1% sarcosine) and sea sand were homogenized, and centrifuged at 12000 rpm to extract soluble proteins. The supernatant was used for a soybean ferritin degradation experiment. 150 ng each of recombinant ferritin and natural ferritin are added to 20 μl of leaf extract,
Incubated at room temperature. SDS-PAGE and PV
After electroblotting on the DF membrane, immunodetection of the protein was performed. The antiserum was raised against the soy ferritin (%) subunit. Blot detection with soy ferritin antibody was performed using anti-rabbit IgG sheep immunoglobulin conjugated with biotinylated horseradish peroxidase. For visualization of the signal, Immunostain HRP-1000 (Konica) was used.

6. Measurement of iron uptake and release Iron uptake reaction by natural soybean ferritin and recombinant ferritin was performed at room temperature with Fe2 ⁺ / ferritin molar ratio of 1,0
100 (1 mM ferrous sulfate and 0.1 μM ferritin) in 100 mM Hepes / NaOH buffer (pH 7.0)
It was carried out in. Iron incorporation into ferritin has an absorbance of 310.
Measured in nm. In the iron release experiment, native soybean and recombinant apoferritin (2 μM each) add fresh ferrous sulfate (1 mM) dissolved in 0.1 M MOPS containing 0.2 M sodium chloride. Mineralization by
Subsequently, the cells were cultured at room temperature for 2 hours, and then left overnight at 4 ° C. Iron release was initiated by adding sodium ascorbate and ferrozine to final concentrations of 1 mM and 4 mM, respectively. Ascorbate has been reported to promote reduced release of iron from the ferritin shell. Exogenous Fe ²⁺ is a Hitachi Ultraviolet Spectrophotometer
(Hitachi, Tokyo, Japan) using the absorbance of the Fe ²⁺ / ferrozine complex at a wavelength of 560 nm.

The following results were obtained from the above experiments. B. Results 1. Isolation of soy ferritin from dried seeds Soy ferritin was purified from dried seeds by two-step anion exchange chromatography, hydrophobic chromatography, and gel filtration. Soy ferritin eluted as a single peak at each chromatographic step. However, purified ferritin includes SDS-PAGE.
(Sodium dodecyl sulfate / polyacrylamide gel electrophoresis) analysis showed that the molecular weight was 28 kDa and 26.
Two bands estimated to be 5 kDa were observed (FIG. 1).
A). According to the density analysis of these bands, the purified ferritin contained almost equal amounts of the 28 kDa and 26.5 kDa subunits (28 kDa / 26.5 kDa ratio was 1 /
(Estimated as 1.09). From purified soy ferritin, the molecular weight is about 550-56.
Only a single band estimated to be 0 kDa (FIG. 1B) was detected.

[0013] In Figure 1A, purified soybean ferritin is analyzed by SDS-PAGE, CBB (Coomassie Brilliant Blue: C oomassie B rilliant
Were stained by the B lue) (lane 1). FIG. 1B shows the results of non-denaturing PAGE analysis of soybean ferritin. Lane 1 shows natural soybean ferritin purified from soybean seeds.
Lane 2 shows the recombinant soybean ferritin expressed in Escherichia coli, and the protein marker (M) indicates the molecular mass.

2. Amino acid sequence analysis of both subunits Six residues from the amino (N-) terminus of both subunits were determined. The sequence of the 28 kDa subunit is Ala-Ser-
Asn-Ala-Pro-Ala and the 26.5 kDa subunit was Ala-Ser-Thr-Val-Pro-Leu. The determined N-terminal sequences of both subunits are different from each other, and the 28 kDa subunit is a novel sequence that has not been reported so far. Furthermore, the N-terminal sequence after the sixth residue also suggested that the 28 kDa subunit was a novel subunit (FIG. 2). 28 kDa and 26.5
The kDa band was completely separable and no contamination peak was detected in the sequence profiles of both subunits. On the other hand, the N-terminal sequence of the 26.5 kDa subunit was identical to the sequence of the EP region of the known sequence. 26.
From the N-terminal sequence analysis of the 5 kDa subunit, it was found that the transit peptide was cleaved by the cysteine at the 48th residue on the carboxyl side, instead of the alanine at the 49th residue on the carboxyl side as reported (see FIG. 2). here,
28 kDa and 26.5 kDa are defined as L (large) subunit and S (small) subunit, respectively.

In the figure showing the deduced amino acid sequence of the subunit of soy ferritin in FIG.
Showing the amino acid sequence of the S subunit which is the same as the sequence already known; The sequence below shows the amino acid sequence of the L subunit according to the invention. The arrow indicates the cleavage site (Proteolytic cleavage) of the S subunit. Also,
The portion surrounded by a solid line indicates the N-terminal 32 residues of the L subunit determined by N-terminal sequence analysis, and the portion surrounded by a dashed line indicates the 17th fragment formed by digestion with lysyl endopeptidase. Show. This fragment is located at the carboxyl terminus of the S subunit.

Next, in order to detect the cleavage site, S
The carboxyl (C-) terminal amino acid sequence of the subunit was analyzed. The sequence of the peptide fragment containing the C-terminal residue was Ser-Glu-Tyr-Val-Ala-Gln-Leu-Arg-Arg (FIG. 2). Although this sequence is found in the known soybean ferritin sequence, the C-terminal residue was determined to be arginine at position 234, which is 18 residues from existing reports on the C-terminus of the soybean ferritin sequence. The base was located upstream. These data indicated that the S subunit in soybean seed ferritin involves a proteolytic cleavage of the C-terminal 17 residues.

3. Amino acid sequence of novel ferritin subunit The cDNA for the newly identified L subunit of soy ferritin was cloned by a PCR-based strategy. The amino acid sequence deduced from the cDNA sequence is shown in FIG. 2 together with the existing reported examples. From precursor to TP
The mature region of the L subunit from which was dissociated was composed of 209 residues, which was 7 residues longer than the S subunit (consisting of 202 residues). The amino acid sequence of the mature region of the L subunit is 82% that of the S subunit.
Showed homology. Contrary to the high homology of the mature region, T
The P sequence showed only low sequence homology (41%) between L and S. In the sequence of the mature region, in the deduced helical region (A to E helix), B and C
The helix-binding loop region was relatively mutated, but high homology was maintained between the L and S subunits; that is, the homology of the amino acid sequences of the helical, loop, and EP regions was 90, respectively. %, 81% and 63%. Table 1 shows the results.

[0018] Note) The amino acid sequences of both subunits were compared with each other in eight portions. TP: Transfer peptide EP: Extension peptide

The L subunit had an extension at the C-terminus consisting of 5 residues, 4 of which were charged residues that were basic or acidic amino acids. All residues believed to be ferrooxidase sites were conserved in both subunits. Iron (III) -tyrosinate
Tyrosine residues believed to form a complex were also conserved. However, in the sequence of the S subunit, the 231rd arginine residue at which the carboxyl side is cleaved was replaced by leucine in the L type subunit. It is believed that this substitution renders the L subunit no longer subject to carboxyl terminal cleavage. As described above, the arginine residue that is cleaved at the carboxyl side in the case of soybean S subunit is CP3
Sequences were also found.

4. Degradation of soy ferritin S subunit E. col
i. Non-denaturing PAGE (Polyacrylamide ・
Gel electrophoresis) (see FIG. 1B) and gel filtration analysis indicated that recombinant soybean ferritin could assemble into a 24-mer as well as the native protein. FIG. 3 shows the results obtained by culturing the homopolymer of the recombinant S subunit with the soybean leaf extract. About half of the S subunit was cleaved to the 26.5 kDa form within 10 minutes. However 1
After incubation for 2 hours, the 28 kDa form disappears and a small amount of 2 kDa
Only the 6.5 kDa type could be detected. After 2 hours, 26.
The 5 kDa form was completely degraded and could not be detected. On the other hand, in natural soybean ferritin containing almost equal amounts of S and L subunits, the 28 kDa form was not present,
26.5 kDa type could be detected even after 2 hours of culture. Since the anti-S subunit antibody was used in this experiment, the L subunit of natural ferritin could not be completely detected.

In the figure showing the degradation of the recombinant and natural soybean ferritin in FIG. 3, the recombinant and natural soybean ferritin were cultured in a soybean leaf extract as described above, Subunit is anti-S
Detected by subunit antibody. In the figure, lane 1 is soybean leaf extract, lane 2 is 150 ng recombinant S subunit, and lanes 2 to 8 are 150 ng recombinant S subunit and leaf extract are 0, 1, 1 respectively.
S subunit after culturing for 0, 30, 60 and 120 minutes, lane 9 shows 150 ng of natural soy ferritin,
Lanes 10 and 11 show the results of 150 ng of natural ferritin cultured with leaf extract for 60 and 120 minutes, respectively.

5. Iron Uptake and Release To study the effect of C-terminal deletion on iron uptake and release, iron uptake and release of native soybean ferritin (50% L) and recombinant ferritin were tested. Natural soy ferritin had equal amounts of both 28 kDa and 26.5 kDa subunits, whereas the recombinant consisted of the entire subunit mature region including the C-terminal sequence. The 26.5 kDa form of the S-subunit lacking the C-terminus did not assemble into the 24-mer. Both recombinant and native soy ferritin showed iron uptake activity (Figure 4A). According to the continuous curve (progression plot), the uptake rate of natural soybean ferritin was lower than that of the recombinant, even though all the constituent subunits had ferrooxidase sites. Both incorporation rates were higher than the Fe (II) autoxidation rate (control).

The rate of reductive release from ferritin was also evaluated (FIG. 4B). The iron nucleation reaction of native and recombinant ferritin is performed at 4 ° C. overnight at a ferritin / iron molar ratio of 1/500,
The culture was performed using ferrous sulfate as a Fe (II) source. Since the ferrous (Fe (II)) atom outside the ferritin shell is bound to ferrosin, the absorbance of this complex was measured. Fe (I
The amount of I) / ferrozine complex was greater for native soy ferritin than for recombinant at any time course.
The difference in the rate of iron release between both subunits is statistically significant with a t-test, p = 0.05, where cleavage of the C-terminal 16 residues promotes iron release from the protein shell. It was shown that.

FIG. 4A is a graph showing a continuous curve plot of the iron uptake reaction of soybean ferritin in 0.1 M Heaps-Na, pH 7.0, containing 0.1 M ferritin subunit and 0.1 mM ferrous sulfate. 2 shows the results of each ferritin performed in the above. The control indicates the autoxidation rate. FIG. 4B is a graph showing the effect of subunit co-existence on assembled ferritin. Native (Native) and recombinant (Recombinant) ferritin (2 μM)
Mineralized in test tubes by mixing 0.1 M MOPS (pH 7.0) with 1 mM ferrous sulfate dissolved in 0.2 M sodium chloride. Iron release from mineralized ferritin was initiated by the addition of 1 mM ferrosin and 4 mM sodium ascorbate to promote reductive release from internal iron. The released iron is Fe ²⁺
The absorbance of the / ferrozine complex was detected by monitoring at 560 nm. The results were obtained from three experiments.

In the present invention, the present inventors detected two different polypeptide chains of soy ferritin and isolated a cDNA encoding a novel subunit. According to the present invention, it has been proved that two different types of ferritin coexist in soybean seeds in almost equal amounts. One subunit had a known amino acid sequence with a molecular weight of 28 kDa.
-Easily converted to 26.5 kDa (S subunit) by cleavage of the terminal 17 residues. Other subunits had a novel amino acid sequence and remained as a 28 kDa type (L subunit). According to the amino acid sequence profile, the S subunit was completely converted to the 26.5 kDa form. These data indicate that the 26.5 kDa subunit was generated by proteolytic cleavage of the amino-terminal extended peptide (EP) of the 28 kDa subunit;
The kDa and 26.5 kDa subunits were contrary to the existing hypothesis, which in principle was thought to be identical.

In a gradation experiment using a soybean leaf extract, the homo submer of the S subunit was unstable as compared to natural soybean ferritin (FIG. 3). S
The subunit instability may be due to cleavage of the C-terminal 17 residues, as mutants lacking the C-terminal 17 residues of the S subunit cannot assemble into oligomers. On the other hand, the L subunit was unaffected by cleavage of the C-terminal region and was stable in leaf extracts.
These data indicate that the L subunit stabilizes the ferritin 24-mer in soybean seeds.

[0027] The present invention relates to the method of
Conversion to form was shown to result from cleavage of the C-terminal 17 amino acid residues. Based on the amino acid sequence matching data and the deduced three-dimensional structure of plant ferritin, the cleaved 17 residues are considered to correspond to an “E-helix”. This "E-helix" is a ferritin 2 such as an animal.
In the tetramer, it is involved in the interaction between subunits near the 4-fold symmetry axis, and forms a narrow channel. The large holes created by the cleavage of the E-helix are expected to have a sufficient diameter to allow the iron to move around freely. The iron release rate from natural soy ferritin is higher than the recombinant release rate (FIG. 4B). These data suggested that iron release was enhanced by cleavage of the E-helix. The large pores formed as a result of cleavage of the E-helix provide a new mechanism of iron release from ferritin.

In plant ferritin, several functional genes have been discovered. The present inventors have found a novel subunit of soybean ferritin whose maturation process differs from existing ferritin, and have shown that this new subunit is one of the major subunits of soybean seed ferritin. The primary structure of the new ferritin subunit is
It was close to the already reported subunit (82
% Homology). However, differences in the maturation process are recognized as justification for the new function of "iron release". Soybean ferritin is thought to function as a buffer for iron in cells by actively taking up and releasing iron molecules. The 28 kDa and partially cleaved 26.5 kDa subunits are also detectable in pea and other legume ferritins.
Amino acid substitutions based on the findings of the present invention render the L subunit of soybean unaffected by proteolytic cleavage of the C-terminal residue, but this phenomenon can be applied to ferritin from other legumes.

To summarize the invention, ferritin is composed of at least two types of subunits in soybeans, S and L. The L subunit, present as a 28 kDa molecule, is involved in the stability of the protein shell, while the S subunit is mediated by cleavage of the E-helix.
Converted to 6.5 kDa form. Cleavage may be related to the mechanism of action of iron release from ferritin. The existence of various subunits having different functions is presumed to be expressed in legumes other than soybean.

[0030]

The ferritin formed from the subunit derived from the gene of the present invention can be easily estimated that iron release is reduced and more iron can be accumulated in the ferritin molecule. Therefore, if the ferritin gene of the present invention is introduced into a plant, it can be expected to have a great effect on the production of a plant having a high iron content proposed by the present inventors.

[Brief description of the drawings]

FIG. 1 shows the results of SDS-PAGE and non-denaturing PAG analysis of soybean ferritin. (A) The purified soy ferritin was obtained by SDS-PAGE.
Analyzed by CBB (Coomassie Brilliant
Blue: stained with C oomassie B rilliant B lue). (Lane 1) (B) Non-denaturing PAGE analysis of soy ferritin. Lane 1: natural soybean ferritin purified from soybean seeds, Lane 2: E. coli. recombinant soybean ferritin expressed in E. coli. The protein marker (M) indicates the molecular mass.

FIG. 2 shows the deduced amino acid sequence of the soybean ferritin subunit. Above: the amino acid sequence of the S subunit, which is the same as the sequence already known. Bottom: amino acid sequence of the L subunit according to the invention. The cleavage site of the S subunit is indicated by an arrow. Solid line: 32 residues at the N-terminus of the L subunit determined by N-terminal sequence analysis. A portion surrounded by a broken line: a fragment No. 17 formed by digestion with lysyl endopeptidase. This fragment is located at the carboxyl terminus of the S subunit.

FIG. 3 shows the degradation of recombinant and natural soy ferritin. Recombinant and native soy ferritin were cultured in soy leaf extract. Ferritin subunit was detected by anti-S subunit antibody. Lane 1: soybean leaf extract, lane 2: 150 ng of recombinant S subunit, lanes 2 to 8: 150 ng of recombinant S subunit with leaf extract, 0, 1,
S subunit after culturing for 10, 30, 60 and 120 minutes. Lane 9: 150 ng of natural soy ferritin, lanes 10 and 11: 150 ng of natural ferritin cultured with leaf extract for 60 and 120 minutes, respectively.

FIG. 4 (A) is a graph showing a continuous curve plot of the iron uptake reaction of soy ferritin. (B) Graph showing the effect of subunit co-existence on assembled ferritin.

Continued on the front page (72) Inventor Toshikazu Yoshihara 1646 Abiko, Abiko-shi, Chiba F-term within the Abiko Research Institute, Electric Power Research Institute 4B024 AA08 BA80 DA01 EA04 GA11 4H045 AA10 BA10 CA33 EA05 FA72 FA74

Claims (6)

[Claims] 1. Ferritin comprising a 28 kDa subunit that is not easily cleaved. 2. A plant-derived non-cleavable 28 kD
The ferritin according to claim 1, comprising the subunit of a. 3. The ferritin according to claim 2, wherein the plant is soy. 4. The sequence of 6 residues from the amino terminus after cleavage of TP (Transit Peptide) is Ala-Ser-Asn-Ala-Pro-Ala.
Ferritin gene encoding a subunit having a molecular weight of 28 kDa. 5. The ferritin gene according to claim 4, which is derived from a plant. 6. The ferritin gene according to claim 4, which is obtained by genetic recombination.