JPH01117790A - Recombinant plasmid integrated with gene coding prealbumin and production of prealbumin using said plasmid - Google Patents

Abstract

PURPOSE:To easily and safely obtain a large quantity of prealbumin useful in the field of pharmaceuticals, at a low cost, by culturing a transformed yeast containing a specific plasmid introduced therein. CONSTITUTION:A double-stranded DNA obtained by the genetic engineering of mRNA of human liver is cloned with E.coli to obtain a gene (A) coding a peptide from the 1st to the 147th amino acids translated from a normal human prealbumin gene having the sequence of formula. A shuttle vector PAM82 (B) is prepared by combining a yeast DNA containing ars1, 2muori and leucine- resistant gene with an E.coli plasmid pBR322. The component B is incised with a restriction enzyme and the product is linked with the component B and integrated at the downstream side of a promoter to obtain an integrated recombinant plasmid (C). The component C is introduced into Saccharomyces cerevisiae and the obtained transformed yeast is cultured under a culture condition suitable for the promoter to obtain the objective prealbumin.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a cDNA encoding human prealbumin.
A method for producing prealbumin using a recombinant plasmid that has been integrated with the plasmid, and a transformed yeast obtained by introducing the plasmid into yeast. That is, cDNA fragments encoding normal human prealbumin and, furthermore, atypical prealbumin possessed by FAP patients are propagated in both E. coli and yeast and placed downstream of the expression regulatory region (promoter) carried by the shuttle vector. This invention relates to a method for obtaining prealbumin (normal prealbumin or atypical prealbumin) produced by obtaining a recombinant plasmid, feeding it to yeast to cause transformation to produce a transformed yeast, and culturing it.

Prealbumin is one of the serum proteins present in the blood at approximately 300 μg/ml, and in the blood, it exists as a complex of four molecules with a molecular weight of 55,000. This complex has two binding sites for thyroid hormone.
It is involved in the transport of the same hormone, and
This complex has four binding sites for vitamin A-binding protein and is also involved in the transport of the vitamin. In addition, the detailed functions of prealbumin have not yet been accurately elucidated, and future research results are expected.

Recently, it has been revealed that atypical prealbumin in which the 30th valine from the N-terminus is mutated to methionine is deeply involved in the pathogenesis of the genetic disease familial amyloid neuropathy (FAP). Diagnosis is also possible.

Among these, atypical prealbumin, which is considered to be the cause of FAP, must be obtained from the blood of FAP patients, which is a major constraint in elucidating the relationship between its function and pathogenesis.

Under these circumstances, a promising clue to solving the constraints in obtaining raw materials for prealbumin, particularly atypical prealamines, would be the development of technology that enables mass production by applying genetic recombination. However, to date, there have been no reports of attempts to express prealbumin or atypical prealbumin using techniques such as genetic recombination, and of course there are no reports of successful expression.

Under these circumstances, the present inventors developed a human-derived prealbumin gene, an atypical prealbumin gene (Mi
ta et al, Biochem, Biophys
.

Res, Comn+un, 124.558-564
(1984), we first attempted to express prealbumin using E. coli as a host. However, no favorable results were obtained, and attempts at expression using large MW as a host ended in failure. Subsequently, the present inventors further investigated mass production of prealbumin using yeast as a host, and as a result, a new method was developed that contained yeast genes and Escherichia coli genes and incorporated the prealbumin gene under the control of a yeast promoter. We prepared recombinant DNA, transformed yeast with it, and successfully mass-produced prealbumin having the same molecular weight and immunological properties as human-derived prealbumin using the transformed yeast. It has been completed.

That is, the present invention provides a novel recombinant plasmid incorporating a prealbumin gene, a transformed yeast using the same, and a method for producing prealbumin using the yeast. Furthermore, the present invention makes it possible to supply a large amount of atypical prealbumin without limit, which has hitherto been difficult to separate from human blood and has had problems with manual sampling.

In the method for producing prealbumin of the present invention, a shuttle vector containing genes for both Escherichia coli and yeast and capable of propagating in either is used, and the prealbumin gene is integrated downstream of the yeast promoter carried by the vector. A prealbumin gene expression vector is obtained. A desired transformed yeast can be obtained by allowing the prealbumin gene expression plasmid thus obtained to act on yeast to cause transformation by a conventional method.

The desired prealbumin can be mass-produced by culturing this transformed yeast under conditions where the used expression promoter works efficiently.

The completion of the present invention has solved the problem of obtaining prealbumin in vitro, in which amino acids have been artificially mutated at any site, easily and quantitatively, and this new technology has yielded extremely interesting findings. It provides possibilities for future creation.

This technology also provides a means to easily obtain human prealbumin without using human blood as a raw material.
In future commercialization of this protein, an extremely safe and low-cost prealbumin preparation that does not require consideration of contamination with unknown infectious factors derived from human blood, unlike when purified from human blood using conventional methods. This makes it possible to supply Furthermore, the present invention solves the limitations of atypical prealbumin, which had limitations on the amount of specimens that can be prepared manually, and makes it possible to easily and infinitely provide this, making it possible to use it for future FAP. It is believed that this will greatly contribute to research.

The recombinant plasmid, transformed yeast, and production of prealbumin thereby of the present invention will be explained in more detail below.

(1) Prealbumin gene cDN encoding prealbumin used in the present invention
In A, double-stranded cDNA was synthesized using reverse transcriptase according to a conventional method using mRNA prepared from human liver as a starting material, and this was cloned using Escherichia coli.

The cloned prealbumin gene contains the entire region encoding the prealbumin protein and has the base sequence shown in FIG.

The prealbumin cDNA prepared in the present invention is
The prealbumin cDNA consists of 669 base pairs and contains the complete sequence of the amino acid coding region.
ξContains 26.3'-untranslated region and 161 base pairs in untranslated region.

A DNA fragment having the restriction enzyme diagram in Figure 1 and the nucleotide sequence shown in Figure 2 was added to the E. coli plasmid Okayama-Be.
The oligo dG or dC inserted into the ry vector is usually treated with Pst I-Pyu II to obtain a prealbumin gene fragment, which is used for plasmid construction as described below. If necessary, in order to directly express mature prealbumin in recombinant yeast, the first to second translated
The gene encoding the amino acids up to the 0th amino acid, ie, the signal peptide portion, can also be removed in advance. In this case, since the start codon ATG is also removed at the same time, it is necessary to add or add ATG, which becomes the start codon, when integrating the prealbumin gene into the shuttle vector described later.

Note that the prealbumin gene described in the present invention is not limited to that having the base sequence shown in FIG.

In addition, the gene encoding atypical prealbumin is also FA
cDNA encoding atypical prealbumin can be similarly prepared from wRNA prepared from the liver of patient P. The atypical prealbumin gene obtained in this way has no difference in one base compared to the normal prealbumin gene sequence, and when the translation start codon of the prealbumin gene is set to +1, the 149th (+14
The only difference is that the cytosine in 9) has been mutated to adenine.

Furthermore, the gene for atypical prealbumin can also be prepared by using a normal prealbumin gene and causing point mutations in this gene and converting bases only at necessary locations.

(2) Shuttle vector The shuttle vector used in the present invention is a plasmid vector that contains yeast genes and E. coli genes and carries a yeast expression control region.

This yeast gene generally includes the NA sequence, which is necessary for the plasmid to propagate independently of the chromosome in yeast;
For example, the NA sequence (arsl) is necessary for the replication of yeast chromosomes.
), 2μm NA sequence required for DNA replication (2μor
i) and, if desired, further contains a gene that serves as a selection marker for transformed yeast. The selection marker includes a leucine-producing gene, a histidine-producing gene, a tryptophan-producing gene, a uracil-producing gene, an adenine-producing gene, etc., and one or more of these may be used.

The genes on the E. coli side include NA sequences, such as Co1E, which are necessary for the plasmid to proliferate within the E. coli body.
It has the DNA sequence (ori) of the replication origin of the I-based plasmid, and preferably further contains a gene that serves as a selection marker for transformed E. coli. Examples of the selection marker gene include an ampicillin resistance gene, a kanamycin resistance gene,
Examples include a tetracycline resistance gene and a chloramphenicol resistance gene, and one or more of these genes may be used. Such Escherichia li [pBR322, which has an ampicillin resistance gene and a tetracycline resistance gene as an INA, is generally used.

The expression control region (promoter) necessary for producing prealbumin by the recombinant yeast is derived from yeast. Examples of preferred promoters include promoters that express traits such as enzymes that are originally necessary for yeast, such as acidic futphatase promoter and glutaraldehyde dehydrogenase promoter.

A specific example is the yeast repressible acid phosphatase promoter, which is the promoter of the 60,000 Dalton polypeptide (p60) that normally constitutes phosphatase, and its promoter activity is quite strong. There is a great advantage in being able to control promoter activity by controlling the phosphorus wS level in the medium.

A representative example of such a shuttle vector was prepared by the present inventors. arsl as a yeast gene,
Shuttle vector PAM82 (Japanese Unexamined Patent Application Publication No. 1989-1992
-36699), which is constructed as follows.

A restriction enzyme EcoRI fragment of approximately 8000 base pairs (8 to 7 b) containing the gene for a 60,000 dalton polypeptide (p60) constituting inhibitory acid phosphatase obtained from the yeast 5288G DNA bank [PNAS, 7
Volume 7, pages 6541-6545, (+980) and PN
AS, Vol. 79, pp. 2157-2161, (1982)
[5u
tcliffe, J.G., Cold 5prin8H
arbor Sy+mposium, vol. 43, 77-9
0, (1979)] is used as the starting material.

Note that this 8-bDNA fragment has one recognition site for the restriction enzyme 5alI at a position that divides it into approximately 2.8 Kb and approximately 5.2 Kb, and the 2.8 Kb side is on the ampicillin resistance gene side of pBR322. It has been inserted.

This plasmid was cut with the restriction enzyme Sal I and reannealed with T4 DNA ligase to create pBR32.
A plasmid in which the 5.2 Kb side of the acid phosphatase gene fragment was lost was obtained from the 5a11 site of pAT2.
It is called 5. This ρAT25 consists of a fragment of about 3.7 b from the EcoR1 site containing the ampicillin resistance gene of pBR322 to the 5alI site, and a fragment of about 2.b from the EcoRI site of the yeast acid phosphatase gene to the 5al1 site.
This is a plasmid in which fragments 8 and b are joined at their corresponding ends. Next, EcoR1 of the above pAT25
The site contains an NA sequence (arsl
) and the 1.4b Ec containing the yeast Trpl gene.
oRI fragment [Pro, NAS.

76, pp. 1035-1039, (1979).

The resulting plasmid is called pAT26. Furthermore, this a
The fragment containing rsl-Trpl has one recognition site for the restriction enzyme old ndm in its Trpl gene.

The former ndm site of pAT26 contains the yeast leucine production gene (Leu2) and the NA necessary for the replication of 2 μm DNA.
Hjndm fragment (Tohe,
A., Guerry.

P., Wichener, R.B.; J.Ba.
Chteriol, 141.41:3-416, 19
Insert 80 pj. Is the plasmid thus obtained the shuttle vector pAT? ? (Unexamined Japanese Patent Publication No. 59
-36699).

This pAT77 is pBR322 as an E. coli gene.
ECOR containing the ampicillin resistance gene (Ap')
It has the I site to the 5al1 site, and as a yeast gene, the a
rsl, 2 μori.

It is located in the order of the eu2 gene, and then it has the 5al1 site from upstream of the acid phosphatase gene. It has a structure in which these E. coli genes and yeast genes are linked at the EcoRI and 5alI sites. This pAT77 can proliferate in Escherichia coli by the replication origin DNA sequence (ori) of pBR322, and can proliferate in yeast by arsl and U2μori. Furthermore, this plasmid has an ampicillin resistance gene (Ap') and a leucine production gene (Leu2) as selection markers, and can proliferate in either E. coli or yeast cells, fully satisfying the conditions as a shuttle vector. ing.

Note that this shuttle vector is used to prepare the recombinant plasmid described later using E. coli, and at the stage of transforming yeast with the recombinant plasmid,
There is no problem even if the E. coli gene is removed.

Such shuttle vector pAT77 is digested with restriction enzyme 5a.
This is treated with exonuclease BAL31 to remove part or all of the acid phosphatase structural gene and, if desired, various parts upstream thereof.

This removal occurs upstream of the acid phosphatase structural gene -50.
It is carried out to an appropriate site up to before bpO, and is appropriately adjusted by the exonuclease treatment conditions.

After removing the entire acid phosphatase structural gene or its upstream portion as described above, a synthetic or natural linker 1, such as a 5all linker or a Xho I linker, is incorporated into this site and the acidic A shuttle vector is obtained that is capable of expressing a foreign gene in pure form under the control of a phosphatase promoter. The shuttle vector in which 33 bp upstream of the acid phosphatase structural gene was removed in this way is pAM82.

This shuttle vector is suitable for integrating a desired gene because its insertion site can be easily cleaved by treatment with common restriction enzymes 5alI or XhoI. Such a shuttle vector pAM
In 1982, the present inventors published JP-A-59-3669.
9, and this pAM82 was incorporated into Satucharomyces cerevisiae AH22 (
Satucharomyces cerevisiae AH22/pAM82) has been deposited as FIKEN Article No. 313.

(3) Construction of prealbumin gene expression plasmid The recombinant plasmid of the present invention, that is, the plasmid incorporating the prealbumin gene, is prepared by first adding a restriction enzyme corresponding to the linker using the shuttle vector, e.g.
The DNA is cleaved by treatment with all or Xho I, and the above prealbumin DNA is applied to the cleavage for ligation. This is amplified in Escherichia coli, and only those that have been integrated in the correct coordination are selected by various restriction enzyme analyzes to obtain the desired recombinant plasmid.

(4) Transformation of yeast The yeast to be transformed is a mutant strain having a mutation that is complemented by the selection marker gene of the transformed yeast carried by a plasmid, such as a leucine auxotrophic mutant strain of Satucharomyces cerevisina. x (Saccharo
myces cerevisiae) AH22(a
leu2 his4 Can1 (Cir”)), Saccharomyces cerevisiae (Saccharoa + yce
s cerevisiae) AH22pho80(a
Ieu2 his4 Can1 (Cir"") p
ho130) etc. are used. After propagating the above recombinant plasmid in Escherichia coli, it is allowed to act on the yeast mutant strain by a conventional method to form, for example, a spheroplast, and then the calcium-treated bacterial cells are mixed with the plasmid DNA to cause transformation. Transformed yeast are selected and isolated using the expression of a gene complementary to the mutation in the host yeast carried on the vector, such as a leucine-producing gene, as an indicator.

In addition to leucine auxotrophic mutants, yeasts include histidine auxotrophic mutants, tryptophan auxotrophic mutants,
Examples include uracil auxotrophic mutants and adenine auxotrophic mutants.

(5) Cultivation of transformed yeast and production of prealbumin The transformed yeast obtained by the above method is cultivated to obtain the desired prealbumin. In this case, it is preferable to devise culture conditions depending on the promoter used. For example, when an acid phosphatase promoter is used, the resulting transformed yeast is cultured in a medium containing phosphoric acid under normal culture conditions. After pre-culture, the hedrons in the logarithmic growth phase are transferred to a phosphate-free medium and cultured under conditions in which the acid phosphatase promoter is not inhibited. After culturing, if the prealbumin gene from which the signal peptide region has been removed is used, it will be present in the bacterial cells, and if the entire prealbumin gene containing the signal peptide region is used, it will be present in the medium II solution and on the surface of the body membrane. A large amount of secreted prealbumin accumulates. Depending on the type of yeast used, for example, P
When the ho80 mutant strain is used, there is no need to use conditions that do not suppress the acid phosphatase promoter, and the transformed yeast can be directly cultured to produce a large amount of the desired prealbumin.

Prealbumin obtained by the above method is immunologically difficult to distinguish from that present in human blood, and since prealbumin is secreted and released into the culture medium, it can also be used as a model for protein secretion research in yeast. Useful.

Next, the present invention will be explained in more detail with reference to Examples.

l: Shirumi's (1) pre-albumin gene [1 (1) mRNA
The purified human liver was removed during surgery and immediately frozen in liquid nitrogen, which was used by Chirwin et al.
et al., Biochemistry, 24
mR according to the method of 5294-5299°1979)
NA was prepared.

(U) Synthesis of cDNA and large% 1iliHB101
0kay based on mRNA obtained from transformed human liver.
aa+a-Berg method (Okayan+a, H, an
d Berg, P., Mo1. Ce1l Bio
l, 2°161-170.1982), cDNA
A plasmid containing A was created and introduced into E. coli H8101.
was transformed to prepare a CDNA library.

(m) Identification of prealbumin cDNA Kanda et al.
J, Riot, Che+w,.

249, 6796-6805.1974), synthesized NA of the part corresponding to 1n112 in Asp6L-.
Wallace et al.
ce, R,B, et al, Nucleic A
c1ds Res,, 6.3543-3557.19
The cDNA library was screened using (r-32P) ATP as a probe using the method described in 79), and Escherichia coli containing the prealbumin gene was selected.

(mV) of plasmid DNA! ! Matsubara et al.
et al., J. Virol, 16
．． The plasmid was prepared by the method of 479-485 (1975).

This plasmid was obtained by cloning the CDNA of the entire region encoding prealbumin into the Okayama-Berg vector, and was designated pPA1.

(2) si + ) ruhe') 9-pAM82 (7) rA
i! Approximately 8000 base pairs (p60) containing the gene for the 5oooo dalton polypeptide (p60) constituting the inhibitory acid phosphatase obtained from the yeast 5288G DNA bank.
Using a plasmid obtained by inserting a 8Kb) restriction enzyme EcoRI fragment into the EcoR1 site of E. coli plasmid pBR322 as a starting material, this is cut with restriction enzyme 5all, and then reannealed with T4 DNA ligase to extract acid phosphatase from the 5μ11 site of pBR322. Plasmid pAT25 that has lost the 5.2 Kb side of the gene fragment
(This is approximately 3.7Kb from the EcoR1 site containing the ampicillin resistance gene of p8R322 to the 5all site.
fragment of yeast acid phosphatase gene EcoR
It is a plasmid in which approximately 2.8 Kb fragments from site 1 to site 5μ11 are ligated with their corresponding ends)
get.

Next, a 1.4 Kb EcoRI site containing the arsl and Trpl genes obtained by treating plasmid YRP7 with EcoRI was added to the EcoRI site of pAT25.
Plasmid FAT26 is obtained by inserting the oR1 fragment (this fragment containing arsl-Trpl has a unique recognition site for the endoenzyme old ndm of the Trpl gene).

Plasmid psLE1 was added to the old nduI of pA726 above.
Yeast Leu2 and 2 obtained by treating with old ndm
Shuttle vector pAT77 is obtained by inserting the old ndm fragment containing μ0"1. This pAT77 is integrated into Satucharomyces cerevisiae AH22 (Satsucharomyces cerevisiae AH22/pAT??).
It has been deposited as No. 24.

pAT77 (1Mg) obtained by the above method was added to 5al
After cleavage with l, 20n+M) Lis-IC+(pH
8,2), 12mM CaCl2.12-M MgCl2
．． 0.2M NaCl, 1mM EDTA solution 5 solution 5
Width! Let it act for a minute. After phenol extraction and ethanol precipitation, 1 pmol of Xho I linker and T
The ligation is carried out for 12 hours under the reaction conditions of 4 DNA ligase.
Escherichia coli x 177B was transformed with this reaction solution, and plasmid DNA
Maxam-Gilbert method (Maxam, A, & G11ber) for each θHA
t, W. ; Pro. N. A. S.

, 74, 560-564), the nucleotide sequence is examined to determine the acid phosphatase gene region removed by BAL31 treatment. Plasmid pA from which the phosphatase structural gene region has been completely removed
M82 (Figure 3) is obtained.

÷ A of the codon ATG that encodes methionine, the first amino acid of the product p60 of the phosphatase structural gene.
1, pAM82 has been removed to −33. In addition, this pAM82 was incorporated into Satucharomyces cerevisiae AH22 (Satucharomyces cerevisiae AH22/l) AM82), which has been deposited as FIKEN Article No. 313.

(3) Prealbumin gene expression plasmid (pNP
Preparation of plasmid pPA1 (3) into which a DNA fragment containing the entire region encoding prealbumin (see Figure 1) is inserted.
μg) was cut with restriction enzymes Haem and Xba 1,
A DNA fragment consisting of 46 bp from positions 63 to 108 was purified, and a synthetic NA having an EcoRI cut end was ligated to this, and this was further digested with EcoR1-Xba I of plasmid pυC19, which had been cleaved with EcoRL Xba I.
Inserted on the side. Then, this plasmid was transformed into Xba
I, Hinc■ cleavage treatment, and then pPA 1 was
a I, P, vn ■ 5706 bp D obtained by cutting
The NA fragment was inserted. The thus obtained plasmid has prealbumin cDNA in which the prealbumin signal region has been removed and ATG has been added as a double-station start codon, that is, N-terminal methionine has been added. Next, this plasmid was infected with EcoRI, Hindm
The cDNA of prealbumin was cut out into the 8th part, and an Xho I linker was ligated to this.

In this way, a prealbumin gene fragment whose ends were cut with Xho I was obtained. This DNA fragment and Xho
After the shuttle vector pAM82 cleaved with I was mixed with T4 DNA at a molecular ratio of 5:1 and ligated with the T4 DNA, Escherichia coli H8101 was transformed with this reaction solution. Plasmid DNA was prepared from the obtained ampicillin-resistant transformant, and it was treated with EcoRI, Xba I, Xho
Integration of the prealbumin gene into the vector and its direction were confirmed by analysis with I.

The selected plasmid has the prealbumin gene inserted in the correct direction downstream of the phosphatase promoter of the vector, and is referred to as the prealbumin gene expression plasmid ρNPAI.

FIG. 4 shows the flow of construction of the prealbumin gene expression plasmid.

(4) Preparation of transformed yeast The yeast Satucharomyces φ cerevisiae AlI22[a
Ieu2 his4 Cant (Cir"")] (Feikoken Jokyo No. 312) was used, and this was added to YPD medium (2%
polypeptone, 1z yeast extract, 2x glucose)
After inoculating 100ml and culturing at 30°C overnight,
Centrifuge to collect bacteria. Wash the bacterial cells with 20ml of sterilized water, then add 1.2M sorbitol and 100μgem.
It is suspended in solution 51 of I zymolyase 60,000 (manufactured by Seikagaku Corporation) and kept at 30°C for about 30 minutes to form spheroplasts. The spheroplasts were then washed three times with 1.2M sorbitol solution, followed by 2M sorbitol, 10mM CaCl2 and 10d) Lis-11cI.
(pH 7.5) and suspended in a solution of 0.61 μl.
Dispense 1 m each into small test tubes. Add to this 30μ of the recombinant plasmid pNPA1 solution prepared in (3) above! Add
Mix thoroughly, add 0.1 M CaCl2 (3 μm) to a final concentration of 1011 MCI, and bring to room temperature for 5-1 m
Leave for 0 minutes. This was then added with 20x polyethylene glycol 4000.10mM CaC1p and 10mM
) Add 11 liters of Lis-HCI (flH7,5) solution, mix, and leave at room temperature for about 20 minutes. This mixture 0.
21 of each were added to 101 of regeneration medium (22% sorbitol, 2x glucose, 0.7x yeast nitrogen base amino acids, 2χYPD, 20dg/m I histidine, 3χ agar) kept at 45°C, mixed gently, and prepared in advance. Minimal medium containing 1,2M sorbitol (0,7
After layering on a $ yeast nitrogen-based amino acid, 2x glucose, 20 dg/ml histidine, 2x agar) plate and solidifying, the mixture was cultured at 30°C to obtain a colony of leucine non-auxotrophic yeast. This colony is 20dg/
Bulkholder minimal medium containing s+I histidine (Tohe, A, et al; J, Bac
hterol,.

113, 727-738 (1973)) to transform the yeast Satucharomyces cerevisiae pNPA.
Get 1.

(5) Method for producing prealbumin using transformed yeast (
Each colony of transformed yeast obtained in 4) was further divided into 20
It is spread on an agar plate of bulk holder minimal medium containing dg/ml histidine and cultured at 30°C to form colonies (to reconfirm transformants that have become non-leucine auxotrophic). The bacterial cells are separated, inoculated into 10 ml of bulk holder Minimardium containing 20 μ8/Histidine, and cultured at 30°C. Approximately 24 hours later, the cells in the logarithmic growth phase are collected by centrifugation, and then added to a phosphate-free minimal medium (KH2PO contained in the bulk holder minimal medium).
(replaced with CI and further added 20 dg/m I histidine) 101 with approximately 4 x 10'cells/a
The cells were suspended to a volume of +1 and cultured at 30°C. In this way, prealbumin produced within the yeast cells was obtained.

The values measured by enzyme immunoassay of prealbumin before and after lowering the phosphoric acid concentration and inducing promoter activity are shown in Table 1 of Example 2 below.

2: (1) Preparation of atypical prealbumin gene of Liliumin The liver of a FAP patient was removed during surgery, and a cDNA encoding atypical prealbumin was prepared in the same manner as in Example 1.
was cloned into the Okayama-Berg vector, and this was designated as plasmid pPA3.

(2) Atypical prealbumin gene expression plasmid (pM
Preparation of PAI) containing the entire region encoding this atypical prealbumin
Plasmid pPA3 (3μ
g), shuttle vector p in the same manner as in Example 1.
An expression plasmid pMPA1 was obtained in which an atypical prealbumin gene was integrated downstream of the acid phosphatase promoter of AM82.

(3) Method for producing atypical prealbumin using transformed yeast The plasmid pMPA+ described above was introduced into the yeast Saccharomyces cerevisiae Al22 in the same manner as in Example 1 to obtain transformed yeast, which was then cultured in the same manner. Table 1 shows the results of enzyme immunoassay of atypical prealbumin before and after lowering the phosphate concentration and introducing promoter activity.

Table 1 The immunological properties of prealbumin (normal) and atypical prealbumin obtained in Examples 1 and 2 were investigated by comparing them with those of human blood-derived prealbumin.

As a result, we would like to confirm that the reactivity of the crude extract obtained by disrupting normal prealbumin-producing yeast bacteria in the enzyme immunoassay is the same as that of prealbumin derived from human blood. figure). In general, the Western blot results also confirmed that yeast-produced normal prealbumin has the same molecular weight as human blood-derived prealbumin. (61st!! I, lane l
: human serum-derived prealbumin, lane 2: normal prealbumin-expressing yeast cell disrupted solution, lane 3: atypical prealbumin-producing yeast hedron disrupted solution, lane 4: negative control host yeast hedron disrupted solution) Similarly, atypical prealbumin is F.
It was found to be immunologically identical to atypical prealbumin derived from AP patient blood. (See Figures 5 and 6) 4. Brief explanation of the figures Figure 1 shows the restriction enzyme cleavage map of the prealbumin gene;
Figure 2 shows the nucleotide sequence of the prealbumin gene and the amino acid sequence predicted from this.

FIG. 3 shows a plasmid diagram of shuttle vector pAM82.

FIG. 4 shows a construction diagram of a prealbumin gene expression plasmid.

FIG. 5 shows the reactivity of yeast-produced normal prealbumin, atypical prealbumin, and human blood-derived prealbumin in enzyme immunoassay.

Figure 6 shows Western plot images of human blood-derived prealbumin (lane 1), yeast-produced normal prealbumin (lane 2), yeast-produced atypical prealbumin (lane 3), and host yeast crude extract (lane 4). .

E; Escherichia coli-derived gene i4〇- Figure 4 Absorbance (oD45o)

Claims (14)

[Claims] (1) It is a shuttle vector that contains yeast genes and Escherichia coli genes and carries the yeast gene expression regulatory region, and is characterized by incorporating cDNA encoding human prealbumin downstream of the gene expression regulatory region. recombinant plasmid. (2) The recombinant plasmid according to item (1) above, wherein the cDNA is a gene encoding normal human prealbumin. (3) The recombinant plasmid according to item (2) above, wherein the cDNA contains a gene encoding a peptide from the 1st to the 147th amino acids translated from the normal human prealbumin gene. (4) The recombinant plasmid according to item (3) above, wherein the cDNA is a gene fragment containing the following gene sequence. [There is a gene sequence] (5) The recombinant plasmid according to item (2) above, wherein the cDNA contains a gene encoding a peptide from the 21st to the 147th amino acids translated from the normal human prealbumin gene. (6) The recombinant plasmid according to item (5) above, wherein the cDNA is a gene fragment containing the following gene sequence. [There is a gene sequence] (7) The recombinant plasmid according to item (1) above, wherein the cDNA is a gene encoding atypical prealbumin possessed by FAP patients. (8) The cDNA containing the gene encoding the peptide from the 1st to the 147th amino acids translated from the atypical prealbumin gene possessed by FAP patients.
7) Recombinant plasmid described in section 7). (9) The recombinant plasmid according to item (8) above, wherein the cDNA is a gene fragment containing the following gene sequence. [There is a gene sequence] (10) The recombinant plasmid according to item (7) above, wherein the cDNA contains a gene encoding a peptide from the 21st to the 147th amino acids translated from the atypical prealbumin gene possessed by a FAP patient. (11) The recombinant plasmid according to item (10) above, wherein the cDNA is a gene fragment containing the following gene sequence. [There is a gene sequence] (12) Transfer the recombinant plasmid of item (1) above to yeast Sa
A method for producing human prealbumin, which comprises obtaining a transformed yeast by introducing it into ccharomyces cerevisiae, and culturing the transformed yeast. (13) The method according to item (12) above, wherein the prealbumin is normal human prealbumin. (14) The method according to item (12) above, wherein the prealbumin is human atypical prealbumin.