JPH0811074B2 - Recombinant plasmid incorporating a gene encoding prealbumin and method for producing prealbumin using the same - Google Patents

Description

The present invention relates to a recombinant plasmid incorporating a cDNA encoding human prealbumin, and a method for producing prealbumin by a transformed yeast obtained by introducing the recombinant plasmid into yeast. That is, cDN that encodes normal human prealbumin and also variant prealbumin possessed by FAP patients
A gene expression regulatory region (promoter) carried by a shuttle vector capable of propagating A fragment in both E. coli and yeast
A recombinant plasmid integrated downstream of is obtained, transformed into yeast by feeding it to yeast, and transformed to obtain prealbumin (normal prealbumin or variant prealbumin). Regarding the method.

Prealbumin is one of the serum proteins present in blood at about 300 μg / ml, and 4 molecules of this preassemble in the blood and exist as a complex having a molecular weight of 55,000. This complex has two binding sites for thyroid hormone and is involved in the transport of the hormone. Furthermore, this complex has four binding sites for vitamin A binding protein and is involved in the transport of vitamin A. In addition, the detailed function of prealbumin has not been clarified yet, and future research results are expected.

Recently, it was revealed that a variant prealbumin in which valine at the 30th position from the N-terminus is mutated to methionine is deeply involved in the pathogenesis of familial amyloid neuropathy (FAP), a genetic disease. It is possible.

Among them, in particular, atypical prealbumin, which is considered to be the etiology of FAP, has no choice but to use the blood of FAP patients as a raw material, and there is a major limitation in clarifying the relationship between its function and etiology.

In such a situation, a powerful clue that can solve the restriction on the acquisition of raw materials for prealbumin, particularly variant prealbumin, will be the development of a technology that enables gene production and mass production. However, there have not been any reports that have attempted to express prealbumin or atypical prealbumin using techniques such as gene recombination, and, of course, no reports have succeeded in expression.

Under these circumstances, the present inventors have previously cloned human-derived prealbumin gene, variant prealbumin gene (Mi
ta et al, Biochem.Biophys.Res.Commun.124,558-564 1
984), the expression of prealbumin was first tried using Escherichia coli as a host. However, as a result, favorable results were not obtained, and the expression attempt using E. coli as a host failed. Subsequently, the inventors further studied the mass production of prealbumin using yeast as a host, and as a result, a new prealbumin gene containing a yeast gene and an Escherichia coli gene and incorporating the prealbumin gene under the control of the yeast promoter was developed. The recombinant DNA was prepared, and yeast was thereby transformed, and it was possible to mass-produce prealbumin having the same molecular weight and immunological properties as human-derived prealbumin by using the transformed yeast, and the present invention was obtained. It came to completion.

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 by the yeast. Further, the present invention makes it possible to supply an abundant amount of atypical prealbumin, which has been difficult to obtain from a human blood until now, and which had a problem in obtaining a sample.

In the method for producing prealbumin of the present invention, a shuttle vector equipped with genes for both Escherichia coli and yeast and capable of growing in any of them is used, and the prealbumin gene is incorporated downstream of the yeast promoter carried by the vector. To obtain a prealbumin gene expression vector. A desired transformed yeast can be obtained by allowing the thus obtained prealbumin gene expression plasmid to act on yeast by a conventional method to cause transformation. The desired prealbumin is mass-produced by culturing this transformed yeast under the condition that the used expression promoter works efficiently.

The completion of the present invention has solved a means for easily and quantitatively obtaining prealbumin in which an amino acid at an arbitrary site is mutated in vitro. It offers the possibility of future development.

The present technology does not use human blood as a raw material and also provides a means by which human prealbumin can be easily obtained.In the future commercialization of the protein, as in the case of purifying from human blood by a conventional method, This makes it possible to supply a prealbumin preparation that is extremely safe and at low cost without considering the contamination of unknown infectious factors derived from human blood. Furthermore, the present invention also solves the restriction of atypical prealbumin, which has a restriction on the acquisition of samples, and makes it possible to provide this easily and indefinitely, which will greatly contribute to future research on FAP. It is considered to be something.

Hereinafter, the recombinant plasmid of the present invention, the transformed yeast, and the production of prealbumin by the yeast will be described in more detail.

(1) Prealbumin gene cDNA encoding prealbumin used in the present invention
Is a double-stranded cDNA synthesized by reverse transcriptase according to a conventional method using mRNA prepared from human liver as a starting material and cloned in E. coli. The cloned prealbumin gene contains the entire region encoding the prealbumin protein and has the nucleotide sequence shown in FIG.

The prealbumin cDNA prepared in the present invention has 66
It consists of 9 base pairs and contains the complete sequence of the amino acid coding region. In addition, the prealbumin cDNA contains 26 in the 5'-untranslated region and 161 base pairs in the 3'-untranslated region.

A DNA fragment having the nucleotide sequence shown in Fig. 1 and the nucleotide sequence shown in Fig. 2 was inserted into the Escherichia coli plasmid Okayama-Bery vector by oligo dG or dC method.
-Treat with PyuII to obtain a prealbumin gene fragment, and use it for plasmid construction described below. If necessary, in order to directly express the mature prealbumin in the recombinant yeast, the translated amino acids 1 to 20, that is, the gene encoding the part of the signal peptide can be removed in advance. In this case, since the start codon ATG is also removed at the same time, the AT that becomes the start codon when the prealbumin gene is integrated into the shuttle vector described later.
A device to add G is required.

The prealbumin gene described in the present invention is the second
It is not limited to those having the base sequence shown in the figure.

In addition, the gene encoding atypical prealbumin also contains FA
A cDNA encoding atypical prealbumin can be prepared in the same manner from mRNA prepared from the liver of P patient.
The variant prealbumin gene thus obtained is
Compared to the normal prealbumin gene sequence, there is only one base difference, and when the translation initiation codon of the prealbumin gene is set to +1 the 149th (+149) cytosine is mutated to adenine. Also, for the variant prealbumin gene, the normal prealbumin gene is used,
It can also be prepared by causing a point mutation in this gene and converting the bases only at the required sites.

(2) Shuttle Vector The shuttle vector used in the present invention is a plasmid vector containing a yeast gene and an Escherichia coli gene and carrying a yeast expression control region.

As this yeast gene, generally, a DNA sequence necessary for a plasmid to grow independently of a chromosome in yeast,
For example, a DNA sequence (ars1) required for yeast chromosome replication, 2
There is a DNA sequence (2 μori) required for replication of μm DNA, and if desired, a gene that serves as a selectable marker for transformed yeast is further included. The selectable marker includes leucine-producing gene, histidine-producing gene, tryptophan-producing gene, uracil-producing gene, adenine-producing gene, etc., and one or more of these may be used.

The gene on the Escherichia coli side has a DNA sequence necessary for the growth of the plasmid in Escherichia coli, for example, a DNA sequence (ori) at the origin of replication of a ColEl-based plasmid, and is preferably a gene which serves as a selectable marker for transformed E. coli. including. Examples of the gene for this selection marker include an ampicillin resistance gene, a kanamycin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, and one or more of these genes are used. PBR322 having an ampicillin resistance gene and a tetracycline resistance gene as such E. coli DNA
Is generally used.

Yeast-derived ones are used as the trait expression control regions (promoters) necessary for producing prealbumin by recombinant yeast. Examples of preferable promoters include promoters for expressing traits such as enzymes originally required for yeast such as acid phosphatase promoter and glutaraldehyde dehydrogenase promoter. A specific example is the yeast repressible acid phosphatase promoter. The acid phosphatase promoter normally constitutes phosphatase 60,0
It is a promoter of a 00 dalton polypeptide (p60), its promoter activity is also quite strong, and there is a great merit in that the promoter activity can be controlled by controlling the phosphate concentration in the medium.

Representative examples of such shuttle vectors have been prepared by the present inventors. Ars1 and 2 as yeast genes
Yeast D with μori and leucine resistance gene (Leu2)
A shuttle vector PAM82 (JP-A-59-36699), which is a combination of NA and E. coli plasmid pBR322, is constructed as follows.

A 60,000-dalton polypeptide (p6, which constitutes an inhibitory acid phosphatase from the yeast S288G DNA bank.
8000 base pair (8 Kb) restriction enzyme EcoR containing the gene (0)
I fragment [PNAS, 77, 6541-6545, (1980) and PNA
S, 79, pp. 2157-2161, (1982)] to the known E. coli plasmid pBR322 [Sutcliffe, JG, Cold Spring Har.
bor Symposium, 43, pp. 77-90, (1979)]
The plasmid obtained by inserting it into the oRI site is used as the starting material. This 8 Kb DNA fragment has a recognition site for the restriction enzyme SalI.
We own one in the position to divide into 2.8Kb and approximately 5.2Kb, and 2.8Kb side is p
It is inserted so as to be on the ampicillin resistance gene side of BR322.

This plasmid is cleaved with restriction enzyme SalI, and then T4DN
It was reannealed with A ligase to obtain a plasmid in which the 5.2 Kb side of the acid phosphatase gene fragment was lost from the SalI site of pBR322, and this is called pAT25. This pAT25 is pBR3
SalI from the EcoRI site containing 22 ampicillin resistance genes
It is a plasmid in which a fragment of about 3.7 Kb up to the site and a fragment of about 2.8 Kb from the EcoRI site to the SalI site of the yeast acid phosphatase gene are linked at their corresponding ends. Next, at the EcoRI site of pAT25 described above, a 1.4 Kb EcoRI fragment containing a DNA sequence (ars1) necessary for autonomous growth of yeast and the Trp1 gene of yeast [Pro.NAS, Vol. 76, 1035-1039,
(See (1979)]. The resulting plasmid is pA
Called T26. The fragment containing this ars1-Trp1 is the Tr
It has one recognition site for the restriction enzyme HindIII in the p1 gene.

Yeast leucine-producing gene (Leu2) at the HindIII site of pAT26 and a DNA sequence (2μori) required for replication of 2μm DNA
HindIII fragment containing (Tohe, A., Guerry, P., Wichener, R.
B .; J. Bachteriol, 141, 413-416, 1980). The plasmid thus obtained is shuttle vector pAT77 (see JP-A-59-36699).

This pAT77 has an EcoRI site containing the ampicillin resistance gene (Ap ^r ) of pBR322 as a gene for E. coli to a SalI site, while a yeast gene has ars1, 2μori, and Leu2 genes in that order from the EcoRI site bound to pBR322. It is located further upstream from the acid phosphatase gene to the SalI site. And its EcoRI and Sal
The structure is such that these E. coli genes and yeast genes are bound at the I site. This pAT77 is pBR32 in E. coli.
It can be propagated by the origin of replication 2 DNA sequence (ori) and can be propagated by ars1 and 2 μori in yeast.
In addition, this plasmid contains ampicillin resistance gene (Ap ^r ) and leucine producing gene (Leu) as selectable markers.
It has 2) and can grow in both E. coli and yeast cells, and fully satisfies the conditions as a shuttle vector.

The use of this shuttle vector is for preparing a recombinant plasmid described below 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. Absent.

Such a shuttle vector pAT77 is cleaved by treatment with the restriction enzyme SalI, which is then exonuclease BA.
Treatment with L31 removes some or all of the acid phosphatase structural gene and, optionally, various portions further upstream thereof. This removal is carried out up to an appropriate site upstream of the acid phosphatase structural gene up to -50 bp and is appropriately controlled by the exonuclease treatment conditions.

Control of the acid phosphatase promoter by removing all or even the upstream portion of the acid phosphatase structural gene as described above, and then incorporating a synthetic or natural linker, for example, SalI linker or XhoI linker into this site and returning it to the circular plasmid again. Below, a shuttle vector is obtained which allows the foreign gene to be expressed in pure form. In this way, the shuttle vector removed up to -33 bp upstream of the acid phosphatase structural gene,
It is pAM82.

This shuttle vector uses the usual restriction enzyme SalI or
Since the integration site can be easily cleaved by treating with XhoI, it is suitable for incorporating a desired gene. Regarding such a shuttle vector pAM82, a patent application has been filed by the present inventors as JP-A-59-36699. It should be noted that this pAM82 is incorporated into Saccharomyces cerevisiae AH22 (Saccharomyces cerevisiae AH22).
22 / pAM82) has been deposited as JIKO No. 313.

(3) Construction of Prealbumin Gene Expression Plasmid To prepare the recombinant plasmid of the present invention, that is, the plasmid incorporating the prealbumin gene, first, a restriction enzyme such as SalI or XhoI corresponding to the linker using the shuttle vector is prepared. It is treated and cleaved, and the prealbumin DNA is allowed to act on it to ligate it. This is amplified in Escherichia coli, and only those that have been incorporated into the correct coordination by various restriction enzyme analyzes are selected to obtain the target recombinant plasmid.

(4) Transformation of yeast As the yeast to be transformed, a mutant having a mutation complemented by a selection marker gene of the transformed yeast carried by a plasmid, for example, Saccharomyces cerevisiae which is a leucine-requiring mutant. (Saccharomyces ce
revisiae) AH22 [a leu2 his4 Can1 (Cir ⁺ )], Saccharomyces cerevisiae
AH22 pho80 [a leu2 his4 Can1 (Cir ⁺ ) pho80] is used. After the above recombinant plasmid is grown in Escherichia coli, it is allowed to act on the yeast mutant strain by a conventional method, for example, spheroplast formation and calcium treatment are mixed with the plasmid DNA to cause transformation. The yeast thus treated is selected and isolated by using the expression of a gene, such as a leucine-producing gene, which complements the mutation of the host yeast carried on the vector as an index.

In addition to the leucine-requiring mutant strain,
Examples include histidine-requiring mutants, tryptophan-requiring mutants, uracil-requiring mutants, adenine-requiring mutants, and the like.

(5) Cultivation of transformed yeast and production of prealbumin The transformed yeast obtained by the above method is cultured to obtain the target prealbumin. In this case, it is preferable to devise culture conditions depending on the promoter used. For example, when the acid phosphatase promoter is used, the obtained transformed yeast is pre-cultured in a phosphate-containing medium under normal culture conditions, and cells in the logarithmic growth phase are cultured in a phosphate-free medium. Then, the cells are cultured under the condition that the acid phosphatase promoter is not suppressed. After culturing, when the prealbumin gene from which the signal peptide region has been removed is used, it is secreted into the cells, and when the whole prealbumin gene containing the signal peptide region is used, it is secreted into the culture solution and on the surface of the cell membrane. The prepared prealbumin is accumulated in a large amount. Depending on the type of yeast used, for example, Ph
When the o80 mutant strain is used, there is no particular need to adopt 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 indistinguishable from that present in human blood, and since prealbumin is secreted and released into the medium, it is used as a model for protein secretion research in yeast. Is also useful.

Next, the present invention will be described more specifically with reference to examples.

Example 1: Expression of prealbumin (1) Preparation of prealbumin gene (I) Purification of mRNA Human liver was removed at the time of surgery and immediately frozen in liquid nitrogen, which was then used by Chirwin et al. a
l, Biochemistry, 24 5294-5299, 1979),
mRNA was prepared.

(II) Synthesis of cDNA and transformation of E. coli HB101 Based on mRNA obtained from human liver, the Okayama-Berg method (Okavama, Ha
nd Berg, P., Mol. Cell Biol. 2, 161-170, 1982), c
A plasmid containing DNA was prepared and transformed into Escherichia coli HB101 to prepare a cDNA library.

(III) Identification of prealbumin cDNA Kanda et al. (Kanda, Y. et al, J. Biol. Chem., 249, 6679-680
5, 1974), 16 synthetic DNAs corresponding to Asp ⁵⁸ -Gln ⁶² were synthesized based on the amino acid sequence of prealbumin, and were synthesized by Wallace et al. (Wallace, RBet
al, Nucleic Acids Res., 6,3543-3557,1979) and labeled with (r ^-32 P) ATP.
The DNA library was screened and E. coli containing the prealbumin gene was selected.

(IV) Preparation of plasmid DNA From Escherichia coli containing the prealbumin gene, Matsubara et al.
A plasmid was prepared by the method of ubara et al, J. Virol., 16, 479-485, 1975).

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

(2) Preparation of shuttle vector pAM82 A 60,000 dalton polypeptide (p60) constituting inhibitory acid phosphatase obtained from yeast S288G DNA bank
The plasmid obtained by inserting the restriction enzyme EcoRI fragment of about 8000 base pairs (8 Kb) containing the gene of Escherichia coli into the EcoRI site of Escherichia coli plasmid pBR322 was used as a starting material.
The plasmid pAT25, which had been cleaved with T4 DNA ligase and reannealed with T4 DNA ligase, lost the 5.2 Kb side of the acid phosphatase gene fragment from the SalI site of pBR322 (this is approximately 3.7 Kb from the EcoRI site containing the ampicillin resistance gene of pBR322 to the SalI site). And a fragment of about 2.8 Kb from the EcoRI site to the SalI site of the yeast acid phosphatase gene are linked at their corresponding ends).

Next, plasmid YRP7 was added to the EcoRI site of this pAT25.
A 1.4 Kb EcoRI fragment containing the ars1 and Trp1 genes obtained by EcoRI treatment was inserted into the plasmid PAT2.
6 is obtained (the fragment containing ars1-Trp1 uniquely recognizes the recognition site of the restriction enzyme HindIII in the Trp1 gene).

HindI containing Leu2 and 2 μori of yeast obtained by treating plasmid pSLE1 with HindIII in HindIII of pAT26
Insert the II fragment to obtain shuttle vector pAT77. This p
The AT77 integrated into Saccharomyces cerevisiae AH22 (Saccharomyces cerevisiae AH22 / pAT77) has been deposited as Micro Engineering Research Article No. 324.

After cleaving pAT77 (1 μg) obtained by the above method with SalI, 20 mM Tris-HCl (pH 8.2), 12 mM CaCl ₂ , 12
0.1 U of exonuclease BAL31 is allowed to act for 30 seconds to 1 minute in 50 μl of a solution of mM MgCl ₂ , 0.2 M NaCl and 1 mM EDTA.
Then, after phenol extraction and ethanol precipitation,
The ligation is performed for 12 hours under the reaction conditions of 1 pmol of XhoI linker and T4 DNA ligase. Escherichia coli x1776 was transformed with this reaction solution, and a plasmid DNA was prepared from the obtained ampicillin-resistant transformant. For each DNA, Maxamu Gilbert's method (Maxam, A, & Gilbert, W .; Pro.NAS, 74,560 ~ 5
(See 64), the nucleotide sequence is examined and the acid phosphatase gene region removed by BAL31 treatment is determined.
From these, a plasmid pAM82 (Fig. 3) from which the phosphatase structural gene region has been completely removed is obtained.

+1 for A of codon ATG encoding methionine which is the first amino acid of the product p60 of phosphatase structural gene
As a result, pAM82 has been deleted up to −33. Incorporating this pAM82 into Saccharomyces cerevisiae AH22 (Saccharomyces cerevisiae AH22 / pAM82)
Has been deposited as Mikori Kenjo No. 313.

(3) Prealbumin gene expression plasmid (pNPA1)
Preparation of whole region encoding prealbumin (see Figure 1)
Plasmid pPA1 (3μ containing the DNA fragment containing
g) was digested with restriction enzymes HaeIII and XbaI to purify a DNA fragment consisting of 46 bp at 63-108th position, and synthetic DNA having a cut end of EcoRI was ligated to the fragment, which was further digested with EcoRI and XbaI.
It was inserted into the EcoRI-XbaI side of the plasmid pUC19 that had been cleaved with. Then, this plasmid was digested with XbaI and HincII, and pPA1 was digested with XbaI and PvnII to obtain 5
A 706 bp DNA fragment was inserted. The thus obtained plasmid had the prealbumin cDNA in which the signal region of prealbumin was removed and ATG, that is, the N-terminal methionine was added as the translation initiation codon. Next, this plasmid was digested with EcoRI and HindIII to excise the prealbumin cDNA portion, and XhoI
The linker was attached.

In this way, a prealbumin gene fragment having an XhoI-cut end was obtained. This DNA fragment and the shuttle vector pAM82 cleaved with XhoI were mixed at a molecular ratio of 5: 1 to produce T4DNA.
E. coli HB101 was transformed with this reaction solution after ligation. Plasmid DNA was prepared from the obtained ampicillin-resistant transformants, and EcoRI, Xba
The integration of the prealbumin gene into the vector and its direction were confirmed by analysis with I and XhoI. The selected plasmid has the prealbumin gene inserted in the correct direction downstream of the phosphatase promoter of the vector, and this is called prealbumin gene expression plasmin pNPA1. The flow of construction of the prealbumin gene expression plasmid is shown in FIG.

(4) Preparation of transformed yeast As a yeast, Saccharomyces cerevisiae AH22 [a leu2
his4 Can1 (Cir ⁺ )] (Mikken Kenjoyori No. 312),
This is inoculated into 100 ml of YPD medium (2% polypeptone, 1% yeast extract, 2% glucose), cultured at 30 ° C. overnight, and then centrifuged to collect the cells. The cells were washed with 20 ml of sterilized water, and then suspended in 5 ml of a 1.2 M sorbitol and 100 μg / ml thymolyase 60,000 (Seikagaku Corporation) solution.
Keep at 0 ° C for about 30 minutes to make spheroplasts. Then, the spheroplast was washed three times with a 1.2 M sorbitol solution, and then suspended in 0.6 ml of a solution of ₂ M sorbitol, 10 mM CaCl ₂ and 10 mM Tris-HCl (pH 7.5), and its 60 μm was suspended.
Dispense l into each small test tube. To this, 30 μl of the recombinant plasmid pNPA1 solution prepared in (3) above was added, mixed well, and 0.1 M CaCl ₂ (3 μl) was added to give a final concentration of 10 mMC.
Let it be aCl ₂ and leave at room temperature for 5-10 minutes. Then to this,
20% polyethylene glycol 4000, 10mM CaCl ₂ and 10m
Add 1 ml each of M Tris-HCl (pH 7.5) solution, mix, and leave at room temperature for about 20 minutes. Regeneration medium (22% sorbitol, 2% glucose, 0.7% yeast nitrogen base amino acid, 2% YP
D, 20 μg / ml histidine, 3% agar) 10 ml, mixed lightly, and prepared in advance with 1.2 M sorbitol-containing minimal medium (0.7% yeast nitrogen base amino acid, 2% glucose, 20 μg / ml histidine, 2% agar). ) After overlaying on a plate and solidifying, it is cultured at 30 ° C. to obtain a leucine-non-requiring yeast colony. Bulk colonies containing this colony containing 20 μg / ml histidine in a minimal medium (To
he, A. et al; J. Bachterol., 113, 727-738 (1973)) to obtain transformed yeast Saccharomyces cerevisiae pNPA1.

(5) Method for producing prealbumin by transformed yeast Each colony of the transformed yeast obtained in (4) above is further coated on an agar plate of a bulk holder minimal medium containing 20 µg / ml histidine, and cultured at 30 ° C. To form colonies (for reconfirmation of transformants that have become non-leucine auxotrophic), and then the cells are separated from the colonies and inoculated in 10 ml of a bulk holder minimaldium containing 20 μg / ml histidine, Incubate at ℃. about
After 24 hours, the cells in the logarithmic growth phase were centrifuged to collect the cells, and this was collected in a minimal medium containing no phosphate (KH ₂ PO contained in the bulk holder minimal medium was replaced with KCl, and further 20
μg / ml histidine was added) 10ml to about 4 × 10 ⁶ c
The cells were suspended at ells / ml, and the culture was continued at 30 ° C.
In this way, prealbumin produced in yeast cells was obtained.

The values measured by enzyme immunoassay for prealbumin before and after the reduction of phosphate concentration and the induction of promoter activity are shown in Table 1 of Example 2 below.

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

(2) Atypical prealbumin gene expression plasmid (pMPA
Preparation of 1) DN containing the entire region encoding this variant prealbumin
Using the plasmid pPA3 (3 μg) in which the A fragment was inserted, an expression plasmid pMPA1 in which the variant prealbumin gene was integrated downstream of the acid phosphatase promoter of the shuttle vector pAM82 was obtained in the same manner as in Example 1.

(3) Method for producing atypical prealbumin by transformed yeast The plasmid pMPA1 was introduced into the yeast Saccharomyces cerevisiae AH22 in the same manner as in Example 1 to obtain transformed yeast, which was similarly cultured. Table 1 shows the results of enzyme immunoassay of atypical prealbumin before and after introducing the promoter activity by decreasing the phosphate concentration.

Example 3: Analysis of Prealbumin Produced By comparing the immunological properties of prealbumin (normal) and variant prealbumin obtained in Example 1 and Example 2 with that of human blood-derived prealbumin Examined.

As a result, it was confirmed that the reactivity of the crude extract obtained by crushing yeast producing normal prealbumin in enzyme immunoassay was the same as that of prealbumin derived from human blood (No. 5). Figure). Furthermore, the results of the Western blot also confirmed that the yeast-produced normal prealbumin had the same molecular weight as human blood-derived prealbumin. (FIG. 6, Lane 1: Human serum-derived prealbumin, Lane 2: Normal prealbumin expressing yeast cell disruption solution, Lane 3: Atypical prealbumin producing yeast cell disruption solution, Lane 4: Negative control host yeast cells Crushed liquid) Similarly, the variant prealbumin in Example 2
It was found to be immunologically identical to the variant prealbumin derived from the blood of FAP patients. (See FIGS. 5 and 6)

[Brief description of drawings]

FIG. 1 is a restriction enzyme cleavage map of the prealbumin gene,
FIG. 2 shows the chlorine sequence of the prealbumin gene and the amino acid sequence predicted from it. 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, variant prealbumin and human blood-derived prealbumin in the enzyme immunoassay. FIG. 6 shows Western blot images of human blood-derived prealbumin (lane 1), yeast-produced normal prealbumin (lane 2), yeast-produced variant prealbumin (lane 3) and host yeast crude extract (lane 4). .

Claims (13)

[Claims] 1. A recombinant plasmid containing a yeast gene and an Escherichia coli gene and having a cDNA encoding human prealbumin inserted downstream of the shuttle expression vector of the shuttle expression vector responsible for the yeast expression control region. Saccha
A method for producing human prealbumin, which comprises obtaining a transformed yeast by introducing it into romyces cerevisiae and culturing the transformed yeast. 2. In the shuttle vector, the yeast gene serves as a selectable marker for ars1 and 2 μori and transformed yeast, and the Escherichia coli gene has a DNA sequence and a selectable marker required for growth in E. coli cells. The method according to the above (1), wherein the gene, and the trait expression control region are yeast-derived acid phosphatase promoters. 3. The method according to claim 1, wherein the prealbumin is human variant prealbumin. 4. The method according to claim 3, wherein the cDNA encoding the variant prealbumin contains a gene encoding a peptide from the 1st to 147th amino acids translated from the human variant prealbumin gene. . 5. The method according to item (4), wherein the cDNA is a gene fragment containing the following gene sequence. 6. The method according to claim 3, wherein the cDNA encoding the variant prealbumin contains a gene encoding a peptide from the 21st to the 147th amino acid translated from the human variant prealbumin gene. . 7. The method according to item (6), wherein the cDNA is a gene fragment containing the following gene sequence. 8. The method according to claim 1, wherein the prealbumin is normal human prealbumin. 9. The method according to item (8), wherein the cDNA encoding the normal prealbumin contains a gene encoding a peptide from the 1st to 147th amino acids translated from the human normal prealbumin gene. . 10. The method according to item (9), wherein the cDNA is a gene fragment containing the following gene sequence. 11. A cDNA encoding the normal prealbumin.
No. 21 translated from the normal human prealbumin gene
The process according to (8) above, which comprises a gene encoding a peptide from the 14th to the 147th amino acid. 12. The method according to item (11), wherein the cDNA is a gene fragment containing the following gene sequence. 13. The method according to claim 1, wherein the transformed yeast is a mutant strain having a mutation complemented by a selectable marker gene of the transformed yeast carried by a shuttle vector.