JPH062062B2 - Novel DNA and its use - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to a novel D containing a promoter obtained from yeast.
NA and its use in genetic engineering.

2. Description of the Related Art With the widespread use of recombinant DNA technology, it has become possible to produce useful polypeptides using prokaryotes and eukaryotes. Up to now, Escherichia coli has been mainly used for the large-scale production of these substances, but the use of eukaryotes is particularly desired for the production of pharmacologically important polypeptides. Yeast has many similarities with other eukaryotes, especially mammalian cells, and thus is advantageous for expressing mammalian-derived protein genes. Furthermore, yeast cells do not contain endotoxin, yeast is a microorganism and therefore easy to culture, mass production has been industrially performed for a long time, and its safety has been confirmed. Attention has been focused on the use of yeast as a host because of the large number of statistical analyzes.

Currently, some yeast vectors for gene cloning are known, but strong yeast promoters for efficiently expressing heterologous genes are not well known.

It is known that two types of alkaline phosphatases are present in the cell lysate of the yeast Saccharomyces cerevisiae. One of them is an inhibitory alkaline phosphatase, whose production is suppressed by inorganic phosphate and has wide substrate specificity, and the other is constitutively produced by using only p-nitrophenyl phosphate as a substrate. It is a specific p-nitrophenyl phosphatase. The pho8 and pho9 mutants have been isolated as mutants lacking inhibitory alkaline phosphatase activity.The pho8 gene is the structural gene for inhibitory alkaline phosphatase and the pho9 gene is pho8.
It is considered that it is essential to activate precursors of the vacuolar hydrolase group after transcription. On the other hand, specific p-nitrophenylphosphatase mutants have not been isolated.

Problems to be Solved by the Invention At present, various yeast vectors for gene cloning are known and available. However, in order to efficiently express the homologous or heterologous gene, it is necessary to select a strong yeast promoter suitable for the host used.

Means for Solving Problems The present inventors have found that the yeast p-nitrophenylphosphatase promoter (referred to as PHO13 promoter) is used.
Focusing on the above, the region encoding p-nitrophenylphosphatase was cloned, and its nucleotide sequence was determined. As a result, the promoter was a novel DNA, and the same or heterologous gene was efficiently expressed using this promoter. And that the p-nitrophenylphosphatase obtained by expressing a structural gene encoding p-nitrophenylphosphatase, which is one of the cognate genes, is a novel yeast. As a result of further research based on these, the present invention has been completed.

The present invention provides (1) PHO13 promoter, (2) PHO
Vector incorporating 13 promoters, and (3)
It is a yeast transformed with a vector having a PHO13 promoter and a structural gene downstream thereof.

Examples of the structural gene in the present invention (4) include, for example, a structural gene encoding p-nitrophenylphosphatase, and examples of the gene product include p-nitrophenylphosphatase.
-Nitrophenyl phosphatase.

PHO13 promoter and p-NPPas of the present invention
The DNA containing the structural gene encoding e (referred to as PHO13) can be isolated and collected from yeast cells.

The yeast may be any yeast, but Saccharomyces spp. Is particularly preferable, and examples thereof include S. cerevisiae, and specific examples thereof include commercially available baker's yeast.

For extracting DNA from yeast, for example, Methods in C
ell Biology, vol.12, p. 13-44 (1975) or a method similar thereto.

Next, the obtained DNA is treated with an appropriate restriction yeast, and then a plasmid treated with the same restriction yeast or a restriction yeast that produces the same sticky ends, or a DNA bank obtained by incorporating the above-obtained DNA fragment into a gene bank is prepared. Create. The plasmid is, for example, pBR322 or Escherichia coli-yeast shuttle vector YEp13 [see Gene 8 , 121 (1979)], and the charge is charon type [J.Vi].
rol. 29 , 555 (1979)] etc. are used. If necessary, for example, E. coli-yeast shuttle vector YEp16.
[See Gene 8 , 17 (1979)] and the like for further subcloning.

The vector carrying the DNA thus cloned is transformed into a host microorganism.

As the host microorganism, yeast is preferable, and examples thereof include Saccharomyces spp. More specifically,
Satsucaromyces cerevisiae NA75-2A strain, NA
74-3A strain and the like.

The host microorganism is preferably yeast of pho9.

The above-mentioned NA75-2A strain and NA74-3A strain are obtained by the usual crossing method (Handbook Cf Genetics p.366 Plenum press, Ne.
w York1974). That is NA
The 75-2A strain is the AL211-2B strain (α, pho3-
1, pho8, arg6) [Mol. Cell Biol. 2 , 127 (1982)],
AH22 strain (a, leu2, his4, can1) [Proc.Natl.Aca
d.Sci.USA 80, 1 (1983)], D13-1A strain (a, trp1,
his3, gal2, SUC2) [Proc.Natl.Acad.Sci.USA, 76, 103
5 (1979)] and YAT228 strain (a, leu2, lys10, cy)
h, karl-1) [J. Bacteriol., 145 , 1421 (1981)] and the NA74-3A strain was AL203-.
9A strain (a, pho3-1, pho9, trp5) [Mol.Cell.Biol.
2 , 127 (1982)], AH22 strain, D13-1A strain, respectively.

The gene bank or subclone thus obtained was used to transform the yeast of pho9 into P
A transformant carrying a vector in which a structural gene encoding p-NPPase is linked to the HO13 promoter and the downstream thereof can be obtained.

For transformation, known methods such as Proc.Natl.Aca
d.Sci USA 75 , 1929 (1978), Nature 275 , 104 (1978), Col
d Spring Harbor Symp., Quant Biol. 43 , 1305 (1979), Pr
oc.Natl.Acad.Sci.USA 76 , 1035 (1979) or a method similar thereto.

Saccharomyces cerevisiae NA75-2A / pAL2, which is a transformant produced in Example 1 described below, was deposited at the Fermentation Research Institute (IFO) on July 5, 1984 under accession number IFO 10133, and This microorganism was accepted by the Ministry of International Trade and Industry, Institute of Industrial Technology (FRI), on July 13, 1984, under the contract number FERM P-
It has been deposited as 7712.

Whether or not the transformant obtained by the above method retains the PHO13 promoter and the structural gene encoding p-NPPase can be confirmed by the method described below. That is, the transformant is allowed to form colonies on, for example, Bark holder-modified medium [J. Bac-teriol. 113 , 727 (1973)]. These colonies are then treated with chloroform vapor and then overlaid with agar containing p-nitrophenyl phosphate. If the DNA containing the promoter and p-nitrophenylphosphatase gene has been cloned, the colonies show a yellow color, which confirms the presence of the PHO13 gene. Next, a plasmid can be extracted from the transformant containing the PHO13 gene, which can be digested with, for example, restriction yeast, and the DNA containing the inserted gene can be isolated by, for example, agarose gel electrophoresis or polyacrylamide gel electrophoresis. .
This series of basic operations is already known, for example, Mole.
cular Cloning (1982) Cold Spring Harbor Laboratory.

DNA containing p-nitrophenylphosphatase gene
The nucleotide sequence of, for example, the dideoxynucleotide synthetic chain termination method [Proc. Natl. Acad. Sci. USA 74 , 5463 (1977)], Ma
xam-Gilbert method (Proc. Natl. Acad. Sci. USA, 74 , 560 (197
7)] and the like. The position of the p-nitrophenylphosphatase gene in DNA is inferred from the presence of an open reading frame that is decoded based on the determined nucleotide sequence of DNA. The promoter is expected to be located upstream of its open reading frame, and the existence of the promoter can be known by reducing or deleting p-nitrophenylphosphatase activity by constructing a plasmid lacking this upstream portion. You can Thus, a desired plasmid can be obtained by incorporating a DNA fragment containing the known promoter into a plasmid.

Examples of the medium used for culturing the transformant thus obtained include Burkholder's minimum medium known per se [Proc. Natl. Acad. Sci. USA 77 , 4505 (1980)].

The conditions such as culture temperature and time are determined so as to maximize the production of the target gene product, but the temperature is generally about 15 ° C to 40 ° C, preferably about 24 ° C to 37 ° C.
C. or time is about 10 hours to 96 hours, preferably about 24 hours to 72 hours, and aeration and stirring can be added if necessary.

The gene product accumulated in the culture is crushed into cells by a conventional method in the art, for example, a method using a lytic enzyme such as Zymolyase (manufactured by Kirin Brewery Co., Ltd.) or a mechanical crushing method using glass beads. To extract the target product. If necessary, a surfactant such as Triton-X100 or a protein denaturant such as guanidine hydrochloride may be added to facilitate extraction of the target substance. The target product can be separated and purified from the extract thus obtained by a generally known protein purification method, for example, a precipitation method using a precipitant, a dialysis method, an electrophoresis method, a chromatographic method using an ion exchange resin, or the like. Method, gel filtration method, and method using an antibody column can be combined.

Among the gene products thus obtained, p-nitrophenyl phosphatase hydrolyzes p-nitrophenyl phosphatase, and thus can be used as a reagent.

Meanings of symbols used in the present specification and drawings are as follows, and in the present invention, when amino acids are represented by abbreviations, IUPAC-IUB Commission on Bioche
Based on the abbreviations by mical Nomenclature or abbreviations commonly used in the field. When an amino acid may have optical isomers, L- unless otherwise specified.
It shall indicate the body.

DNA: deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine Met: methionine Thr: threonine Ala: alanine Gln: glutamine Gly e: gluine: para lysine: para lysine: proline: lysine lysine: proline: lysine: proline: proline: lysine: proline: proline: lysine: lysine: : Phenylalanine Leu: Leucine Asp: Aspartic acid Tyr: Tyrosine Cys: Cysteine Trp: Tryptophan Ser: Serine Arg: Arginine His: Histidine H: Hind III E: EcoRI B: P IB: BamH1 MbI: BamH1 M: BglIIS: SaII Examples The present invention will be described in more detail below with reference to Examples.

Example 1 Production of transformant: Yeast (Saccharomyces cerevisiae) gene bank (JBHick, USA) prepared by inserting an MboI partial degradation fragment of yeast chromosomal DNA into the BamH1 cleavage site of yeast-Escherichia coli shuttle vector YEp13 Saccharomyces cerevisiae NA74-3A (MATa pho9-1,
leu2-3, 12 his4-519) and coronal staining using p-nitrophenyl phosphate as a substrate [Biochim Bi
ophys.Acta 428 , 182 (1976)], the one showing an alkaline phosphatase-producing ability (Alp ⁺ ) was screened.
When about 1.6 × 10 ⁴ Leu ⁺ transformants were examined, 5
Alp ⁺ transformants were obtained. Three of them did not show activity against α-naphthylphosphate, which is a substrate for inhibitory alkaline phosphatase, and did not recover the sporulation ability, which is one of the phenotypes of the pho9 mutation. The remaining two Alp ⁺ transformants exhibited activity against α-naphthylphosphate and also recovered the sporulation ability. From these results, it was considered that the p-nitrophenylphosphatase gene was cloned in the former.

From these three Alp ⁺ transformants, the method of Cameron et al. [Nucle
ic Acids Res. 4 , 1429 (1977)] with E. coli JA221 [J. Mol. Biol. 120 , 517 (197).
8)] was transformed. Ampicillin-resistant tetracycline-accepting transformants obtained from each of the three types of DNA were selected one by one and the method of Birnboim & Doly [Nucleic Acids R
es. 7 , 1513 (1979)].
A was separated and analyzed using restricted yeast. As a result, Ec
Since the oRI degradation pattern was the same, they were all considered to be the same plasmid, and this plasmid was named pAL2. Thus, pAL2 isolated as a plasmid that restores the activity against p-nitrophenyl phosphate using the pho9 mutant as a host restores the p-nitrophenylphosphatase activity in the pho8 host as well.
It was strongly suggested that the p-nitrophenylphosphatase structural gene was cloned into AL2.

With the pAL2 described above, the yeast Saccharomyces cerevisiae NA75-2A (MATa pho8, trp1, leu2, his4, cn
1) and the resulting transformant was transformed into Saccharomyces cerevisiae NA75-2A / pAL2 (IFO
10133, FERM P-7712).

Example 2 Production of phosphatase: The yeasts shown in the following Table 1 were each placed in a 500 ml Sakaguchi flask containing 100 ml of Bark holder modified medium at 30 ° C.
After shaking culture for 18 hours, the bacterial cells were collected and crushed using a French press to prepare a crude enzyme solution [Mol. Cell. Bio.
l. 2 , 127 (1982)] and the reference Mol. Cell. Biol. 2 , 127 (1982);
The alkaline phosphatase activity was measured according to the method described in Biochimica et Biophysica Acta 428 , 182 (1976). The results are shown in Table 1.

a) High phosphoric acid and low phosphoric acid have KH ₂ PO ₄ concentrations of 1.5 mg / m ₂ each
l) 0.03 mg / ml b) Only the genotype for phosphatase is noted c) pNPP and αNP are p-nitrophenyl phosphate,
As shown in Table 1 showing α-naphthylphosphate, Saccharomyces cerevisiae NA75-2A / pAL2 and Saccharomyces ·
S. cerevisiae NA74-3A / pAL2 shows activity only against p-nitrophenyl phosphate, the value of which is not affected by the concentration of phosphate, and the production amount thereof is S. saccharomyces cerevisiae NA75-2A, NA74-. Three
It was about 30 times that of A. This high p-nitrophenylphosphatase activity is considered to be due to the gene broadening effect of the PHO13 gene.

Example 3 Construction of restriction enzyme map of DNA fragment containing PHO13 gene: restriction enzyme reaction buffer (100 mM Tris-HCl, pH 7.5, 10 mM MgC) was added to pAL2 DNA (1 μg) obtained in Example 1.
4-6 units of various restriction enzymes (BamH1, Bg1II, EcoRI, HindIII, PstI, SalI, Xho in 1 ₂ , 50 mM NaCl)
I) was acted alone or in combination of two kinds, and reacted at 37 ° C. for 1 hour. The reaction solution was subjected to 1% agarose electrophoresis, the molecular weight of the resulting DNA fragment was estimated, and a restriction enzyme cleavage map was prepared and shown in FIG.

Example 4 Estimation of the position of the PHO13 gene on the cloned DNA fragment: In order to estimate the position of the PHO13 gene on the cloned DNA fragment, a deletion derivative of pAL2 was prepared.
After reacting pAL2 (2.3 μg) in 50 μl of a restriction enzyme reaction buffer containing 4 units of EcoRI at 37 ° C. for 5 minutes, the reaction was stopped by heating at 65 ° C. for 10 minutes to obtain a partially decomposed product. T4 ligase reaction solution containing this reaction solution (40 μm) (5 mM MgCl ₂ 10 mM dithiothreitol, 0.055 mM
100 μ of ATP, T4 ligase 3 units) was reacted at 4 ° C. for 18 hours to re-bond the degradation products. Then 10μ
E. coli JA221 was transformed with the T4 ligase reaction solution of E. coli and the obtained transformant was used to transform Birnboim & Dol described above.
The plasmid DNA was separated according to the method of y to obtain deletion plasmids pAL2-D1, pAL2-D5, pAL2-D6 and pAL2-D7. Meanwhile, pAL2 DNA (2.3 μg) was added to 6 units of Hind II.
After treating with I for 1 hour at 37 ° C., heat treatment, T4 ligase treatment and transformation of E. coli JA221 were carried out in the same manner as above to obtain ampicillin-resistant transformants. Saccharomyces cerevisiae NA74-3A was transformed with the obtained deletion derivative plasmid DNA, the Alp phenotype of 10 Leu ⁺ transformants was examined, and the plasmid showing Alp ⁺ was designated as pAL2-D9. The relationship between the above deletion plasmids and Alp phenotype is shown in FIG. From this result, the PHO13 gene is on the fragment sandwiched between the HindIII and EcoRI cleavage sites (see FIG. 2). (Part) It was expected to exist in the vicinity.

Example 5 Confirmation of PHO13 gene existing position: In order to confirm the position of PHO13 gene, a deletion plasmid was further constructed based on pAL2-D9. pAL2-D9
(5 μg) of 6 units each of HindIII and BglII
The reaction was carried out in a restriction enzyme reaction solution containing 50 μl at 37 ° C. for 1 hour, and the resulting 2.9 kb DNA fragment was separated and purified by 1% low-melting point agarose electrophoresis, and 30 μm TE buffer (10 mM Tris-HCl, pH7 .6, 1 mM EDTA). On the other hand, YE treated with HindIII and BamH1 by the same procedure.
p13 (1 μg) was heated at 65 ° C. for 10 minutes. The obtained reaction mixture (5 μ) and the previously obtained HindIII-BglII DNA fragment (2.9 k
b) 100μ of T4 ligase reaction mixture containing 20μ of solution
Were reacted at 4 ° C for 18 hours, and 20 μ of the mixture was used for E.co.
li JA221 was transformed. A plasmid was isolated from the transformant showing ampicillin resistance and named pAL15.

Similarly, pAL2-D9 (5μ) was treated in 50μ of a restriction enzyme reaction solution containing 6 units of XhoI, and then LEU.
The XhoI fragment (6.6 kb) containing the 2 genes and the 2μ plasmid replicon was separated and purified by agarose electrophoresis, and 3
It was dissolved in 0 μ of TE buffer. On the other hand, pBR322 (3.2 μg)
Was reacted at 37 ° C. for 1 hour in 50 μl of a restriction enzyme reaction solution containing 6 units of SalI, followed by heat treatment at 65 ° C. for 10 minutes.

Then, 100 μl of T4 ligase reaction liquid containing 5 μl of the pBR322 reaction liquid treated with SalI and 20 μl of the XhoI DNA fragment (6.6 kb) solution was reacted at 4 ° C. for 18 hours, and then 2
0 μ was used to transform E. coli JA221. Plasmid DNA was isolated from a transformant showing ampicillin resistance and non-leucine auxotrophy, and named pAL16 (Figs. 2 and 3). As is clear from FIG. 2, pA
Transformants harboring L15 showed p-nitrophenylphosphatase activity. Further, since the p-nitrophenyl promoter activity of the transformant carrying pAL16 was reduced, it was expected that the promoter was located on the right side of the XhoI site. As shown in Figure 3, pBR322
Since a promoter capable of working in yeast in the same direction as the PHO13 gene could be presumed near the SalI cleavage site of pA, pA
The weak p-nitrophenylphosphatase activity of L16-bearing bacteria is believed to be due to this sequence.

Example 6 Determination of nucleotide sequence: The nucleotide sequence of the DNA inserted into pAL2 was determined by Maxam & Gilb.
It was determined according to the ert method (supra) and is shown in FIG. As expected in Examples 4 and 5, It is considered that the region from ATG to TAG, which is surrounded by a circle, encodes p-nitrophenylphosphatase. Therefore, the amino acid sequence of p-nitrophenylphosphatase expressed by the PHO13 gene is considered to be that shown in FIG. The promoter is expected to be present in the nucleotide sequence containing the XhoI cleavage site upstream of the ATG.

Example 7 Construction of expression vector using PHO13 promoter: The outline of construction of an expression vector using PHO13 promoter is as shown in FIG. First, pAL2DNA
The 0.7 kb DNA fragment obtained by treating E.coli with EcoRI was treated with DdeI.
66, and then partially decomposing it with AvaI
A 4 bp DNA fragment is obtained. This sticky end is DNA
After repairing with Polymerase I Large Fragment, Sa
II Link the linker. After treating this with SaII, it was inserted into the SaII cleavage site of pBR322 in the same manner and the plasmid pAL100 was inserted.
To get Next, pAL100 is simultaneously decomposed with Sau3A and SaII to 64
An expression vector pAL200 can be obtained by obtaining a 5 bp DNA fragment and inserting it into pSH19 [Mol. Cell. Biol. 4 , 771 (1984)] which was co-treated with BamHI and SalI. The obtained pA
By inserting a homologous or heterologous gene into the SalI cleavage site of L200 according to the orientation of the promoter, an expression plasmid for the desired gene product can be obtained.

Effects of the Invention The expression vector using the PHO13 promoter of the present invention can be advantageously used for expression of various genes. For example, the p-nitrophenyl phosphatase of the present invention is
Since other phosphatases are repressible, they are constitutively produced, so that the PHO13 promoter of the present invention can be used to advantageously produce various gene products. The p-nitrophenylphosphatase obtained by the present invention can be advantageously used as a reagent.

[Brief description of drawings]

Fig. 1 shows a restriction map of the pAL2 insertion site, Fig. 2 shows the relationship between various deletion plasmids and p-nitrophenylphosphatase activity expression, Fig. 3 shows the structure of pAL16, and Fig. 4 shows Is the nucleotide sequence of the DNA containing the PHO13 promoter and the structural gene encoding PHO13, FIG. 5 is the amino acid sequence of the structural gene encoding PHO13 and its gene product, PHO13, and FIG. 6 is PHO1.
3 shows the construction of expression vectors using 3 promoters.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication (C12N 15/11 C12R 1: 865) (C12N 1/19 C12R 1: 865) (C12N 15/81 C12R 1: 865) (C12N 15/55 C12R 1: 865)

Claims (4)

[Claims] 1. A DNA fragment having promoter activity comprising a nucleotide sequence represented by the following sequence (PHO13 promoter): TTCGTCGTATCAGACGTAAAATCCATATTTGCGTT CTTTGCCGACCTAGTAGTATCAACAAGATCATTGG AAAGTATCGCTAAGAGGTCGTATGGATTCGAATGA GATCAGTTGTGCTAAAAGCATAATTGATGGTGCTC ACGCATTGAAACTTAAAACAAGAATTTGGGGAGCC CCAACTCGGCCAGCTAACCTGGTGGAAACTATTTC TCGCCAAATTATTCATGACTTGGGTTCCGATTTAC TTAACCTGACAATTTATTCATGGCATCGTCATTGA TATAAGTGGCTTGAGCTGTGGATAAGAAAAGCCGT ATATTTATATAAACATTTAGATATGAATAGGAAGT AGATTGTTCGACGCAACTACCCGTTCAAGAAATAT AATGGGGAATGGTCGTCGGTCTTCCCTCACAGGGG ATATAGTTCTCTGAAGAGATACATACGTTTATGTA TACTATGCTTCTTTATCAACTCAAGTTTTGTAGAG GAAGACGTTGAAGATGGTGATGTCACATCTTTACT ATTCTCCAGCACGTTTTCAGCATTCACTCAATCGC ACATTAATGACGTTCCTCATCCATTAACTTTCCGG TTTTTCTTTTTTTCGGTGAATGTTCTTTCCGTTTT AGCGAATTTTTCAATTGTAATTGACGCAATCGGTT TATAACAAGCAGACATAAATGTCAAGCTCGAGCCA AATACAAAAAAAGCCTTATAGCTTGCCCTGACAAA GAATATAACAACTCGGGAAA 2. A vector having a PHO13 promoter incorporated therein. 3. The vector according to claim 2, wherein the PHO13 promoter and a structural gene are integrated downstream thereof. 4. A yeast transformed with a vector having a PHO13 promoter and a structural gene downstream thereof.