JPS62248488A - Recombinant dna, transformed yeast containing same and secretion and production of human lysozyme using same - Google Patents

Abstract

PURPOSE:To prepare a human lysozyme by a yeast, by using a recombinant DNA containing a hybrid prelysozyme gene composed of a region coding chick lysozyme signal peptide and a region containing human lysozyme structural gene. CONSTITUTION:A DNA coding a signal peptide of chick lysozyme and having the nucleotide sequence of formula is linked with a human lysozyme structural gene treated with a proper restriction enzyme to obtain a hybrid prelysozyme gene, which is integrated in a proper manifestation vector. The obtained recombinant plasmid is used for the transformation of host yeast cell. The transformed yeast is cultured to obtain large amount of pure human lysozyme at a low cost.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to a novel NA and its uses.

More specifically, the present invention provides a recombinant DNA containing a hybrid prelysozyme gene consisting of a DNA encoding the signal peptide of chicken lysozyme and a human lysozyme structural gene, a yeast transformant harboring the recombinant DNA, and a transformant thereof. This invention relates to a method for secretory production of human lysozyme by expressing it.

[Prior art and problems]

Human lysozyme is found in human tears, saliva, nasal mucus, and milk.

Lymph glands, white blood cells, plasma, cartilage, lungs, kidneys, B'l/,
Muramidase is an enzyme protein found in the viscera, liver, intestinal tract, parotid gland, skin, semen, and urine of leukemia patients, and hydrolyzes the β-1,4 bond of N-acetylglucosamine and N-acetylmuramin. (muramidasc
) is known to have enzymatic activity.

In addition, lysozyme derived from human milk has the sequence 1 shown below.
It is known that it consists of 30 amino acids (for example,
(See Funatsu et al., "Lytic Enzymes," pp. 55-58, Kodansha Scientific (1977)).

Lys-Val-Phc-Glu-Arg-Cys-g
lu-Leu-Ala-Arg-Thr-Leu-Ly
s-Arg-Leu-Gly-Met-Asp-Gly
-Tyr-Arg-Gly-11e-5er-Leu-
Ala-Asn-Trp-Met-Cys-Lcu-A
la-Lys-Trp-Glu-5cr-Gly-Ty
r-Asn-Thr-Arg-Ala-Thrψ-Asn-Dyr-Asn-Ala-Gly-A
sp-Arg-5er-Thr-A, 5p-Tyr
-Gly-11e-Phc-Gln-11e-A? n-
5cr-Arg-Tyr-Trp-Cys-Asn-A
sp-Gly-Lys-Thr-Pro-Gly-Al
a-Val-Asn-Ala-Cys-Ilis-Lc
u-5er-Cys-3cr-Ala-n-5cr-A
rGln-Asp-Asn-11e-Ala-Asp-
Ala-Val-Ala-Cys-Ala-Lys-A
rg-Val-Val-Arg-Asp-Pro-Gl
n-Gly-11a-Arg-Ala-Trp-Val
-Ala-Trp-Arg-Asn-Arg-Cys-
Gin-Asn-Gln-Asp- Asn-11e-Ala-Asp-Ala-Val-A
la-Cys-Ala-Lys-Ar lysozyme has the effect of lysing various bacteria, so
Lysozyme can be used as a preservative for foods, medicines, etc., and as an antibacterial agent by itself or in combination with antibiotics. It also has blood coagulation and hemostasis effects, anti-inflammatory effects,
Since it also has a sputum expulsion effect on people with respiratory diseases, it can also be used as a drug for those patients.

Human lysozyme can be isolated from human milk, urine, etc., but it is inefficient and impractical to meet the demands of industrial production for medical purposes.

The present invention makes it possible to secrete and produce pure human lysozyme in large quantities at low cost using recombinant DNA technology.

Recombinant DNA using a synthetic hybrid prelysozyme gene
The method for producing human lysozyme using this technology will be described in more detail later, but basically consists of the following steps.

(1) Gene design (2) Chemical synthesis of the gene (3) Integration of the gene into an appropriate expression vector (4) Transformation of an appropriate host using the obtained recombinant plasmid (5) Cultivation of the transformant Production and recovery of human lysozyme In the above process, the details of the production method using Escherichia coli as a host have already been described by some of the present inventors in Japanese Patent Application Nos. 59-201366 and 59-201366.
It is disclosed in the specification of No. 201367.

Yeast (Saccharomyces cer
Compared to the case of using Escherichia coli, (a) culture is safer and easier when using E. evisiae), and it is possible to culture at a higher density.

(b) The safety of the product is increased because the contamination of toxic substances such as pyrogens can be avoided.

(c) Gene products derived from higher animals tend to have the correct structure.

(d) Since yeast cells are insensitive to lysozyme, they can produce it more stably.

It has the following advantages.

However, with the method already proposed by the present inventors (Japanese Patent Application No. 6O-139710), human lysozyme expressed in yeast cells cannot be secreted into the medium. Therefore, the yeast cells are collected, the cells are crushed,
Extract the target protein from this.

It needs to be separated and purified. However, since there are a large amount of contaminant proteins inside the yeast cells, separation and purification are quite difficult tasks. Furthermore, in proteins directly expressed in yeast cells, methionine derived from the translation initiation codon AUG may be produced as an undesired adduct added to the N-terminus of the target protein as an N-terminal amino acid. Therefore, if the first expression product can be produced as a fusion protein precursor containing an appropriate signal peptide, then processed within yeast cells, and secreted outside the cells as a mature protein, the above-mentioned problems can be solved at once. It becomes possible to solve the problem.

Incidentally, although the amino acid sequence of human lysozyme is known, neither its precursor protein nor gene have been isolated yet. However, the gene for chicken lysozyme, which has biological activity extremely similar to that of human lysozyme, has been isolated, and from this sequence it is possible to know the precursor protein (watli plelysizyme) and the DNA sequence that encodes it. can. (For example, A. Jung et al.
Proc.

Natl, Acod, Sci, USA 77, 5
759 (1980)). Since lysozyme is a protein that is secreted from the inside of cells to the outside of cells in both humans and chickens, yeast can also utilize the yeast secretion system if an appropriate signal peptide can be added to the N-terminus of the human lysozyme mature protein. It is thought that it is possible to secrete human lysozyme into the culture medium using yeast cells. [Structure of the Invention] Under these circumstances, the present inventors have developed various methods for the extracellular secretion production of human lysozyme using yeast cells. As a result of repeated studies, we discovered that it is possible to secrete and produce correctly processed human lysozyme into a medium at high levels by using DNA encoding the signal peptide of chicken lysozyme, and completed the present invention. reached. The invention will now be described in detail.

(1) Gene design 1-1 DNA corresponding to structural genes Select a codon that satisfies the following conditions from among several codons that specify the amino acids that make up human lysozyme and create a DNA.
Synthesize A.

(A) Choose codons that are used as frequently in yeast as possible.

(B) Ensure that each of the synthetic fragments described below does not have any undesired complementary sequences within or between molecules.

The structural gene used in the present invention is the gene shown in Figure 6, and the details of the method for designing and obtaining the gene have already been disclosed by the present inventors in Japanese Patent Application No. 59-201367. Please refer to it.

1-2 DNA corresponding to the signal peptide In order to enable secretion in yeast, DNA corresponding to the signal peptide sequence necessary for secretion is added to the 5' end of the above structural gene with matching translation reading frames. There is a need. Normally, as a signal peptide used to secrete and produce a foreign protein in yeast, a signal peptide of a yeast protein that yeast originally secretes into the medium is used. This is hereinafter referred to as a "same type signal." For example, the invertase signal peptide, which is an amino acid presequence normally bound to invertase, or the signal peptide contained in the yeast α-factor precursor protein may be used. On the other hand, as described above, the signal peptide of chicken lysozyme was found to have the following structure from the analysis of the isolated chicken lysozyme gene. (For example, A. Jung et al., Pro.
Nat, 1. Acod.

Sci, USA, 775759 (1980))
-set, -arg -sar -1ue -1eu -
Lie −1au = val −1eu−cys −
phc -1eu -pro -1cu -ala -
ala -1eu --I hand-gly-1ys-val-pha-gly-arg・-
However, 10r indicates the N-terminal amino acid of mature chicken lysozyme, and -1 to -18 indicate the signal peptide contained in the precursor chicken lysozyme. Arrows indicate the positions of cleavage when the precursor is converted to the mature form in chicken cells.

Such a chicken-derived signal peptide is hereinafter referred to as a "heterologous signal" in the sense that it is a signal other than yeast's original signal. It is not clear whether such heterologous signals function in exactly the same way as the homologous signals described above, ie, whether they achieve expression, processing, and secretion in yeast cells.

This is because the secretion process of yeast has not yet been fully elucidated, and it is not clear whether it is the same as the secretion process of higher mammalian cells such as chickens and humans.

An example of using a "heterologous signal" for secretory production of a heterologous protein in yeast is the use of a human interferon signal peptide (DNA corresponding to 23 amino acid residues). (R,A, lliLzcman et al.
5science, 219, 620 (1983)) In this case, the interferon secreted into the medium accounts for approximately 1 of the total interferon expressed by the yeast.
It was only 0%. Moreover, the secreted interferon is processed at the desired cleavage site and is about 6
5%, and approximately 35% of undesired interferons of longer length also co-occurred. This means that
This suggests that yeast cells incompletely recognize human-derived signal peptides. In general, secreted proteins are usually formed by translation in the rough endoplasmic reticulum (RER), during which signal peptides interact with putative particles called signal recognition particles (SRPs), which in turn interact with SRP receptors or binding proteins. (docking p
proteins are then transported across the RER membrane (e.g., G, Blobal and Blobal protein).
Dobbarst, cin, J. Ce11. B
iol,.

67, 835 (1975) or P, 1ilalt,
er and G,Blobal.

J, Ca11. Biol,, 91.545 (19
81)).

After this protein is isolated in the RER lumen, it is transported to the Golgi apparatus and then packaged into vesicles.

When the vesicle fuses with the plasma membrane, the vesicle contents (proteins)
is released into the periplasmic space (pcriplasmic space) confined between the plasma membrane and the cell wall.

Although the above secretion process is thought to be valid in yeast as well, the estimated SRP in yeast may not be equivalent to the SRP in mammalian cells. Furthermore, it is not clear whether in vivo modifications of proteins (eg, phosphorylation, glycosylation, etc.) occur during the secretion process, and to what extent they are involved in the localization of secreted proteins. Nevertheless, the reason for assuming that the signal peptide of chicken lysozyme may function correctly in yeast is based on the following reasons.

(A) The signal peptide of chive 1-lyrisozyme is 18
It consists of 3 amino acid residues and has a length that is extremely similar to yeast's original invertase signal (18 amino acids), α-galactosidase signal (18 amino acids), acid phosphatase signal (17 amino acids), etc.

(B) Near the N-terminus of the signal peptide, 11rg is present as a basic amino acid residue often found in homologous signals.

(C) The central part of the signal peptide contains leu, il
There is a region consisting of hydrophobic amino acids such as e, val, and phe, which is also very similar to the homologous signal.

The cleavage sites when chicken lysozyme precursor is converted (processed) into the mature form are gly (-1) and 1ys (+
Regarding 1), in the case of yeast, there is no clear basis for predicting whether or not it will be cleaved at the same site; however, for the reasons mentioned above, it is unlikely that it will be cleaved at the same site. estimated to be high.

Based on the above considerations, we decided to use a heterologous signal as a signal for lysozyme secretion in yeast.

The basic idea behind its design is as follows.

(A) As the nucleotide sequence corresponding to the signal peptide, chicken-derived codons are used as they are in order to prevent secondary structure problems at the mRNA level.

(B) The DNA corresponding to the signal peptide is chemically synthesized as a region covering up to the restriction enzyme 1'a41 site located on the 5' side of the structural gene in order to facilitate connection with the structural gene.

(C) A restriction enzyme recognition sequence is provided on the 5' side to facilitate integration into the plasmid and excision from the plasmid.

A specific example of the signal sequence DNA designed in this way is the DNA represented by the nucleotide sequence formula shown in Figure 7, which can be linked to the human lysozyme structural gene treated with an appropriate restriction enzyme to create a hybrid. After making the prelysozyme gene, it can be expressed and secreted in yeast by incorporating it into an appropriate vector.

(2) Gene synthesis To synthesize the gene designed as described above, each of the + and - chains is divided into several fragments, which are chemically synthesized, and each fragment is linked. It depends on how you do it.

The structural gene used in the present invention is the gene shown in Figure 6, and the details of the method for obtaining the gene have already been disclosed by the present inventors in Japanese Patent Application No. 59-201367. On the other hand, the signal peptide DNA is divided into the DNA fragments shown in Figure 7, and the fragments are synthesized, purified, 5'-hydroxyl group phosphorylated, and the fragments are ligated. , can be carried out according to methods known per se.

(3) Preparation of recombinant plasmid 3-1 Introduction of the prelysozyme gene into a plasmid vector The hybrid prelysozyme gene created as described above is transferred to a plasmid vector that can be propagated in the host or a vector that can be propagated and expressed in the host. into the appropriate insertion site of a plasmid vector constructed as follows.

The incorporation operation itself can be performed according to conventional methods known in the field of molecular biology. For specific methods, please refer to Examples below.

According to the present invention, the hybrid prelysozyme gene can be propagated by p[l
R322, pAT153 (e.g. Twigg et al., N
This can be carried out using various known plasmid vectors such as A Sure, 283.216-218 (1980)). Further, isolation and purification of plasmid DNA can also be performed according to conventional methods known in the field of molecular biology.

The structural gene shown in Figure 6 was converted to BamH of pBR322.
Regarding the method for obtaining pHLY-1 obtained by insertion into the I cleavage site, please refer to Japanese Patent Application No. 59-201367.

Expression of human lysozyme in yeast by the hybrid prelysozyme gene of the present invention can be performed using the ADI11 promoter (e.g. H
ijzcrman et al., Nat, urc 293717~
722 (1981)), the PGK promoter (e.g. t (itzeman et al., 5science 219)
620 (1983)), the GAPDII promoter (e.g. Urdca et al., Proc. Najl.
Acad, Sci, USA class 7461-7465 (
(1983)), PH05 promoter (e.g. Kr
amcr et al., Proc. Nat, 1. Acad,
Sci, USA Dan 367-370 (1984)), the ENO promoter (e.g. Holland et al., J. Biol. Chew, 2561385-1)
395 (1981)) and the GALIO promoter (e.g., Broach et al., Experimenta
l Manipulation of Gene [E
xprcssion 83-117 (1983)) and the like).

One specific example of these expression vectors is YEp51 (
For example, Broach et al., Expcrimcenal
Manipulation of Gene Expr.
ession, 83-117 (1983)).

3-2 Determination of directionality The directionality of the hybrid prelysozyme gene integrated into the plasmid can be determined using restriction enzymes that recognize specific base sequences contained within the gene (e.g., Tag I, Dst, E).
, This can be carried out by analysis using commonly used methods such as gel electrophoresis.

(4) Transformation The recombinant plasmid incorporating the hybrid prelysozyme gene is prepared and propagated using Escherichia coli, and one specific example of using Escherichia coli as a host cell is E. coli C600 (
For example, Na1son et al., Virology 10
8338-350 (1981)).

Expression of human lysozyme by a recombinant plasmid incorporating a hybrid prelysozyme gene is carried out using yeast, and one specific example of using yeast as a host cell is S. cera
visiae KK4 (e.g., Nogi et al., Mo1.
Gen.

(See Genej, 1952'J-34 (1984))
It is. Transformation with a recombinant plasmid incorporating the human lysozyme gene is not limited to the above-mentioned hosts, and various known S, cerevi, and 5iae derivatives (e.g., YeastGenetic 5tock Cent
er) (see catalog) can be used. Many of these S, cercvisiae vI conductors are located in the East Genetic Stock Center (Veal
Genetic St., ock Center) and recognized microbiological institutions, such as the American Type Culture Collection.
, ura Co11ection) and can be distributed there.

4-2 Transformation The transformation itself can be performed according to conventional methods known in the field of molecular biology. (For transformation of E. coli, see, eg, Cohen et al., Proc.

Nat, 1. Acad, Sci, USA 69
2110-2114 (1972) and Maniatis
LaMo1ecular Cloning 249~
253 (19g2), 1 for yeast transformation.
For example 1 (innen et al., Proc. Natl.
Acad, Sci, USA 751929 (19
78) and Ijo et al., J.
．． 153163-168 (1983)).

4-3 Transformants Specific examples of transformants in yeast include S, cercv
isiaeKK4 was transformed with YEp-11LYsIG.
, cereviSiacKK4 (YEp-HL'
/5IG).

5 Secretory production of human lysozyme Human lysozyme is secreted and produced by culturing the transformant thus obtained according to conventional methods known in the fields of molecular biology and fermentation science.

Human lysozyme can be detected by known immunoprecipitation reactions, radioimmunoassays (e.g. Yuzuriha et al. CheIIl, P.
harm.

Bull, 27 2802-2806 (1979) and Yuzuriha et al. Chew, Pharm, Bu
ll, 2690.8-914 (1978)), enzyme activity assays (e.g. 5idhan et al., Agric
, Biol.

Chem, 451817-1823 (1981) and Morsky Anal.

Biocham, 12877-85 (1983)).

In addition, the produced human lysozyme can be recovered and purified by appropriately combining conventional methods known in the fields of biochemistry and fermentation science.

Example Chemical Synthesis of DNA Fragment Corresponding to Signal Peptide The DNA corresponding to the chicken lysozyme signal peptide designed as described above was divided into nine fragments shown in FIG. 7 and synthesized. These oligonucleotide fragments can be prepared using known methods (e.g.

P, L, dcllascth et al., Nuclcic
Ac1ds Res,, 11. 773 (1
983), Aρρ1
DNA synthesizer (Mo
del 38OA). All synthetic reagents such as phosphoramidite reagents are Applied Bi
osyst, manufactured by CMS, was used according to the attached operator's manual (available from Nikkaki Co., Ltd.). The synthesized NA was separated from the silica gel carrier and recovered with the nucleobase and 5' hydroxyl group protected and the phosphate protecting group deprotected. These protected DNA oligomers were treated with 28% aqueous ammonia 3taQ at 60°C for 4 hours to obtain oligomers in which all but the dimethoxytrityl group at the 5' end were deprotected. The same oligomer was analyzed using high-performance liquid chromatography on a reversed phase support (Cosm).
osi15C18 (manufactured by Gyudon Kagaku Co., Ltd.) and purified by elution with a linear concentration gradient of acetonitrile in 0.1 M acetic acid-1 to ethylamine buffer (pH 6,5). After the purified solution was concentrated to about 500 μQ by distillation under reduced pressure, glacial acetic acid was added to give a final concentration of 80%, and the dimethoxytrityl group was removed by treatment at room temperature for 15 minutes.

This was purified by high performance liquid chromatography according to the method described above to obtain a completely deprotected oligomer of 10 to 2000 units.

The nine fragments chemically synthesized above were converted into IIL'/5
It was divided into two blocks 8 and n, IG1-5 and IIL'/SIG 6-9, and each block was linked, and the two blocks were further linked to synthesize the entire DNA corresponding to the signal peptide. Among the nine fragments 1-, M at both ends
The seven fragments excluding L'/5IG-1 and HLYSIG-8 were combined into the two blocks A and B, and the hydroxyl group at the 5' end was phosphorylated, followed by a ligation reaction.

The details of the phosphorylation and ligation reaction are as follows in the case of block A containing 5IG-1 to 5.

Four fragments that perform phosphorylation (llLYsIG2
~5), 2 μg each of the mixture was injected with 15 units of Dye, polynucleotide kinase, '14 parole (Y'P
) ATP<2900 Ci/mmol), - and phosphorylation buffer (1mM spermidine, 50mM dithiothreitol (abbreviated as DTT), 100mM MgCQ2
．． 3 in a 45 pQ reaction solution containing 500 mM Tris-IICQ (pH 7,5) and (LmN EDTA).
After incubation at 7°C for 30 minutes, 80nsolc
of ATP, 24 units of T4-polynucleotide kinase were added and further incubated for 60 minutes at 37°C. This was incubated at 65°C for 10 minutes and then cooled with water. The DNA was recovered by phenol extraction and ethanol precipitation, and then dissolved in 60 μQ of sterilized water. Add 14 μg of fragment IILYSIG to this,
0mM Tris-HCQ (pH 7,5) and 10mM
IM Tris-IIc Q to become MagCQ2
(pH 7,5) and LM MgCQ 2 and 6
After incubating at 5°C for 10 minutes, the mixture was left standing at 37°C for 20 minutes and then at room temperature (approximately 256C) for 20 minutes. Next, ATI' and D1'T were adjusted to 0.4mM and 10mM, respectively.
Add 16 units of T-*-DNA ligase so that the reaction volume is 100μQ, and store in a refrigerator (about 10℃).
) for 12 hours for ligation. Block B was synthesized from HLYSIG6 to 9 in the same manner as when the reaction was completed. However, for block B, after synthesizing block B, the total amount of this ligation reaction product was added to 20 nmoles of ATP and 15 units of T.
4 polynucleotide kinase and the phosphorylation buffer described above for 60 minutes at 37°C to remove the 5' of IIL'/SIG-8 that was added later.
The hydroxyl group was phosphorylated. This was incubated at 65°C for 10 minutes, then at 37°C for 20 minutes, and then at room temperature (approx.
5°C) for 20 minutes. Next, half the amount (10 μQ) of each of the ligation reaction products of block A and block B (phosphorylated IILVsIG-8) were mixed, and the mixture was placed in a reaction solution with the same composition as that used for the ligation reaction of block A, and then refrigerated. (approximately 10° C.) for 12 hours. 8.
The product was separated by electrophoresis on a 10% polyacrylamide gel containing 3M urea to obtain the desired (A+8) block, i.e.
DNA fragment corresponding to the signal peptide (68
mer and 70mar) were prepared.

This was ligated with synthetic gene DNA and cloned into pBR322 vector. The details are described below.

Plasmid pHLV- containing human lysozyme structural gene
1507zg was mixed with 10mM Tris-tlcQ (pH
7,5), 50mM NaCQ, 10mM MgC
2 containing Q z and 190 units of restriction enzyme 5au3AI
After incubation at 37° C. for 4 hours in a 00 μΩ reaction solution, phenol extraction and ethanol precipitation were performed. The reaction product was subjected to 5% polyacrylamide gel electrophoresis to isolate a 418 bp fragment containing the human lysozyme gene.

Next, this DNA fragment was treated with 10mM Tris-tlcQ.
(pi-!7.5)7mM MgCQ2.10mM N
aCQ, 7mM β-mercap 1-ethanol,
100 μg/aQ BSA and 84 units restriction enzyme T
It was incubated at 65° C. for 12 hours in 82 μQ reaction solution containing ag I. This reaction solution was mixed with phenol lJ1
DNA was recovered by ethanol precipitation, and 385 bp (385 bp) (as shown in Figure 6) was isolated by 5% polyacrylamide gel electrophoresis.
0mM ditris-HCQ (pH 7,5),
100a+M NaCQ, 10mM MgCQ2
．． At 37°C in a 100 μQ reaction solution containing 80 units of restriction enzyme 5a121 and 24 units of restriction enzyme Baa+ HI.
After incubation for 12 hours, phenol extraction and ethanol precipitation were performed. 4. This reaction product was subjected to 5% polyacrylamide gel electrophoresis. A fragment of IKb was isolated.

Next, the total amount of the previously prepared signal peptide corresponding DNA fragment 1-38 containing the human lysozyme grooving gene
1/4i of the 5 bp restriction enzyme Tag I/5au3A fragment, and 4. IKb plasmid pBR32
Restriction of 2 # JBamHI/SaΩ Summer Mix half of the cleavage fragments and add buffer 1 (50mM Tri
s-11(jl (pH 7,5), 10mM Mg(12
, 10+sM DTT, 1mM spermidine, 1mM
The reaction was carried out in a refrigerator (approximately 10'C) for 12 hours in a 95 μQ reaction solution containing ATP, 100 μg/sQ BSA) and 10 units of T4 DNA ligase. Extract this reaction product with phenol: 1. -) DNA was recovered by ethanol precipitation. Next, this DNA was used to transform E. Co11 C600 strain to obtain an ampicillin-resistant (60 μg/a+1) transformant.

Identification of recombinant plasmid The above transformant was grown on LB agar medium containing 60 μg/lsQ of ampicillin (Bactodryptone log IQ).

Bakto Yeast Extract 5g7Q, NaCl2
Grown on nitrocellulose filters mounted on 10 g/(IDifco agar L 5 g agar 1 tree prepared). 37℃, left for 4 hours, chloramphenicol 170μg/mQ and ampicillin 50μg
The filters were transferred onto LB agar medium containing /vaQ. After 15 hours of amplification, in 5 situ colony screening methods (e.g., Grunsjein et al., pr.
oc. Natl. Acad. Sci. USA,
Colonies containing the target plasmid were searched using μC3961 (1975)). As a hybridization probe, a 210 bp DNA fragment containing approximately half of the 5' side of the human lysozyme structural gene labeled with Ra was used.

ㅅ、This converts PHLY 1 to restriction enzyme BamHI/Xb.
aI cut and 210
The isolated bρ fragments were subjected to nick translation techniques (e.g., P, W, J, Rigby et al., J.
Mo1. Biol.

μ! □, 237 (1977)) by P-JfA#
! T, ta. Hybridization is 50DIM Tr
is-tlc Q (pH 7,5), 4XSSC (IX
SSC=0.15M NaCQ, 0.015M (sodium citrate)), I X Dcnhardt'
S solution (0.02% BSA, 0.02% Ficoll 40
0.0.02% polyvinylpyrrolidone), 50% formamide and 50 Mg/mQ Salmon-t,e
In the stas DNA, lXl0' cps uP-
Probes were added and incubated overnight at 42°C. Filters were washed three times for 30 minutes at 42°C in (2XSSC+0.1% 5DS).

Dry the filter at -80°C, Dupont,
Approximately 1200 colonies containing 0 recombinant plasmids were exposed to Kodak XAR-5 X-ray film using a Lightning-Plug intensifying screen.''
in situ using P-labeled DNA fragments, u
As a result of hybridization, about one-third of the colonies were found to be hybridized. A small amount of plastic and persmid DNA was collected from 12 of these colonies using Birnb.
Oim and Doly's method (Nucleic and EcoRI/Sal I cleavage). As a result, 12 colonies were analyzed by 5% polyacrylamide gel electrophoresis, each with 244 bp and 829 bp, respectively.
It was found that the target plasmid provided a fragment of p. Transfer this plasmid to PIIL'/SI
It was named G (see Figure 1).

Plasmid -l Plasmid YEp5120Mg was added to buffer ■ (10mM
Tris-HCQ (pH 7,5), 100+sM
NacQ, 10mM MgCQ 2.1mM
DTT), 50 units of restriction enzyme 5afll and 40
After reacting at 37°C for 5 hours in a 70 μΩ reaction solution containing 11 ind Ill of restriction enzyme, 0.7% agarose gel electrophoresis was performed, and a fragment of about 6.9 b containing the GALIO promoter was recovered. . On the other hand, plasmid P
HLYSIG 30Mg in the above buffer ■, 70 units of 5
After reacting for 5 hours at 37°C in a 200 μa reaction solution containing aQl and 84 units of Hind m, 5% polyacrylamide gel electrophoresis was performed.
The reaction mixture was reacted for 12 hours in a refrigerator (approximately 10° C.) in a 0 μm reaction solution for ligation.

Next, E. coli strain C600 was transformed using this reaction solution to obtain a transformant resistant to ampicillin (60 Mg/■Q). Plasmid DNA from these transformants
A, restriction enzyme Sa Q I /Hindnl,
The recombinant plasmid YEp as shown in Fig. 1 was obtained by analyzing the cutting pattern of EcoRI/XbaI etc.
- Transformants containing HLVSIG were selected.

Mieno Using 5 μg of plasmid YEp-HLYSIG.

S. cerevisiae strain KH2 (a. ur
a3. hisl or his3,shirpl,1eu
2, ga180) using the known Q lithium acetate method (e.g.
Ito et al., J. 0actcrio1. , 153.1
63 (1983)) to obtain a leucine non-auxotrophic transformant. This transformant was transformed into 4 (YE
p −+1LVSIG).

The recombinant S, cerevisiae KH2 of human 1zozyme (
YEp-HLYLSIG) and the recombinant S, ceravisiae KH2 (YEp
IIIJ-1) was grown in a medium containing galactose or glucose as a carbon source, and the presence or absence of secretion of human lysozyme into the medium was examined.

Culture was carried out using various amino acids except leucine (final concentration 20-3
75 g/Ω), adenine sulfate (final concentration 2 axis g
/n).

Minimal medium (D) containing uracil (final concentration 20 mg/7 m)
ifc.

manufactured by Sherm, amino acid-free Bacto yeast nitrogen base)
an et al., Methods in Yeast Gene
tics, 62 pages, Cold SpringHarb
our (1982)) 200w+1 and 2% galactose or 2% glucose as a carbon source
``I did it in C.

Recombinant S, cerevisiaa KH2 (VE
p-HLYSIG), using a medium containing galactose (SGal) or glucose (SO) as a carbon source, when the Klet value of the culture medium reached 360 to 380, the human lysozyme activity in the medium was 1 unit
/Peng Q, 22 units/ma (see Figure 2). However, lysozyme activity can be measured using Morsky's method (Anal, oral iochcm, 128.77).
(1983)), the following method was used. Mi
crococcus, 1cus bacterial cells) suspension (freeze-dried bacterial cells 15 + a g / Room 0
50mM sodium phosphate buffer (pH 6,4)) 8
00 μfl was added with 200 μρ of the sample, and after rapid mixing, the decrease in turbidity of the bacterial cell suspension was monitored by measuring the absorbance at /+50 nm in a quartz cell with an optical path length of 1 cm. Enzyme activity is determined by measuring the decrease in absorbance over a 10-minute period from 1 minute to 11 minutes after reaction at room temperature (25°C) using this measurement system, and the ■unit indicates a decrease in absorbance (at 450 nm) of 0.001 per minute. defined as a value. When glucose was used as a carbon source, the secretion of human lysozyme activity into the medium was delayed compared to when galactose was used, but this was due to the fact that the Ga Q 1 used for 1 expression was delayed.
This seems to be because the induction of the 0 promoter was suppressed by glucose in the minimal medium, and the suppression was released as the glucose was consumed as the bacterial cells multiplied. On the other hand 1
Recombinant S, ceravigiaa KK4 (YEp
IILY-1) was grown in a medium containing galactose (SGaQ) as a carbon source, no human lysozyme activity was secreted into the medium (Figure 2).
. This indicates that the signal peptide portion is essential for secretion of human lysozyme activity into the medium.

Various amino acids except leucine (final concentration 20-375t) of human lysozyme
rrg/Q) Adenine sulfate (final concentration 20m
g/12), uracil (final concentration 20-g/12) and the above minimal medium containing 2% galactose as carbon source (
SGa Q ) the recombinant S in 20 Ovan,
ceravisiac KK4 (YEp-HLYSIG
) was grown at 30°C. 18.5 hours after the start of culture,
23.3 hours, 38.8 hours, 61.8 hours, 85
．． 8 hours and 134.3 hours culture solution 10m Q 1
The cells were separated from the supernatant by centrifugation at 500 rpm for 2 minutes. The above-mentioned supernatant was added to the extracellular fraction (Medium).
I made it J. The above bacterial cells were treated with 1.2M sorbitol and 10
After washing twice with buffer 1rsQ consisting of 2 ml of 1.2 M sorbitol, 10 mM KH2PO4 (pH 6,8) and 0
．． After incubation at 30°C for 30 minutes in m buffer containing 6% Zy + Wolyasa 100T (Kirin Brewery tS), the cells were separated from the supernatant by centrifugation at 3000 rpm + m2.
pragmic 5pace) J. 1 of the above bacterial cells
IIQ 1.2M Sorbitol and 10a+M KH
After washing twice with a buffer consisting of 2POa (pH 6,8), it was suspended in a buffer consisting of the same composition as 2+IQ.

The cells in the suspension were disrupted by ultrasonication for 3 minutes, and then centrifuged at 15 Krpm for 2 minutes to separate into a precipitate and a supernatant. The above supernatant was collected as an “intracellular fraction”.
cclular) J. The lysozyme activity contained in each of the "extracellular fraction,""periplasmicfraction," and "intracellular fraction" was determined by N.

It was measured by the bacteriolytic method described above using Iysodeikt, 1 cu. The localization of his-1-lysozyme activity in each fraction changed with cell growth, but after the stationary cell growth phase, it was almost ``extracellular fraction (Madium) J 60%.
, “Periplasmic fraction (Periplasmic 5p
ace) J 10%, “intracellular fraction (Int, race
(Figure 3).

In addition, as a result of quantitative analysis using a commercially available purified human lysozyme preparation (manufactured by Sigma), 1 unit was determined using the above measurement method.
, was found to have a production amount of 0.02% g/mQ in terms of the medium.

S, ccrcvisiac KK4 (Yl!p-HL
YSIG) in the above minimal medium containing 2% galactose (
SGal medium) Cultured in 400taQ at 30°C for 1 day and night with aeration. The culture grown to Klett 400-500 was centrifuged at 10,000 rpm for 10 minutes to remove yeast cells and obtain a culture fluid fraction. After lyophilization, the supernatant fraction was dissolved in a small amount (10 to 20 m m ) of 50-N phosphate buffer (pH 8,0) and thoroughly dialyzed against the same buffer. After dialysis, the concentrated supernatant fraction was subjected to FPLC (Pha
rmacia) using a cation exchange column Mono
The 6 active fractions separated using S11R515 (Phars+acia) were passed through the column at a flow rate of 1 mfi/min for 50 m
0-I for 20 minutes in the presence of M phosphate buffer (pH 8,0)
NK (#) was eluted by applying a linear concentration gradient. At this stage, the elution pattern of lysozyme as seen by bacteriolytic activity completely matched the only major peak eluting around 0.25 MKCfi (Figure 4). Similarly, commercially available purified human lysozyme (Sigma) was added to Mono
When applied to the S force column, the retention time on the column completely matched this main peak, and a single peak eluted around 0.25MKCQ. At this stage, it is possible to obtain a single lysozyme sample, but for the purpose of confirming purity and desalting the eluted fraction, high-performance liquid chromatography ( Further purification was performed by HPLC). Both fractions with lysozyme activity were pooled together using a Mono S column, and directly loaded onto an ODS reverse phase column (Cos
It was purified using n+osil 5C1g (Hawai Chemical).

The active fraction was eluted from the column with a linear gradient of 5-70% acetonitrile over 45 minutes in the presence of 0.1% trifluoroacetic acid (TFA) at a flow rate of IIIQ 1 min. Approximately 50
The peak eluted as a single major peak at the position of % acetonitrile (column retention time of approximately 30 minutes) completely coincided with the elution position of commercially available purified human lysozyme, which was separately run on an ODS column as a control. Also lysozyme Mic
It also completely matched the elution pattern of rococcu+ bacteriolytic activity. Through the above operations, a single human lysozyme preparation could be obtained from the culture supernatant at a yield of about 70% (Table 1). The isolated human lysozyme preparation is available from Microc.
occus showed approximately the same turnover rate as commercially available purified human lysozyme. This indicates that the zozyme protein expressed in yeast by the hybrid prelysozyme artificial gene has no difference in catalytic activity from natural human lysozyme.

Table-1 Mono SO, 24123000954008
1 The amino acid sequence of human lysozyme protein purified from yeast culture supernatant is published by Applied Biogyst, cm
The analysis was performed using a gas phase amino acid sequencer-47OA manufactured by S Company.

The amino terminus was subsequently recovered as PTH-amino acids by Edman degradation. PTII-amino acids were then separated by high performance liquid chromatography using a reversed-phase column and identified by comparison with the retention times of standard PTI+-amino acids. As a result, only lysine was detected as the N-terminal amino acid of this refined M41 product.

Furthermore, as a result of determining the sequence from the N-terminus to the 20th amino acid, H2N-Lys-Val-Pha-Glu-
Arg-Cys-Glu-Leu-Ala-A
rg -Thr -Lou -Lys -Arg ・L
cu ・Gly ・MeheAsp Gly・Tyr
...and completely matched the amino acid sequence already reported for natural human lysozyme. This means that the chemically synthesized genes (31 types of prelysozyme genes) encoding the human lysozyme protein with the secretory signal peptide of Niwatori egg white lysozyme added to the N-terminus are accurately translated, processed, and secreted in yeast. It shows that

[Brief explanation of drawings]

FIG. 1 is a diagram showing the method for creating the expression plasmid YEp-HLYSIG. FIG. 2 is a diagram showing secretory expression of the hybrid prelysozyme gene in yeast. Figure 3 shows the localization of human lysozyme expressed in yeast. Figure 4 shows Mon of human lysozyme secreted and expressed in yeast.
o Figure 5 shows the purification by S column, Figure 5 shows the purification of human lysozyme secreted and expressed in yeast by reverse phase column, Figure 6 shows the nucleotide sequence of the human lysozyme structural gene, and FIG. 7 shows the nucleotide sequence of DNA encoding chicken lysozyme signal peptide. In Figure 2, l is 4('/
[ρ-HLYSIG] (7) Bacterial growth, 2 is the bacterial growth of KK4 (YEp-HLYSIG) cultured in sDte, 3 is the bacterial growth of KK4 (YEp-HLYSIG) cultured in 5Gal.
1) (7) Cell proliferation, 4 is KK4 cultured in 5Gal
(YEp-HLYSIG) no enzyme activity, 5 is sD
Enzyme activity of crab 4 (YEp-11LYsIG) cultured in Te and KK4 (YEp)I cultured in 5Gal
LY-1) is a plot showing the enzyme activity of LY-1). In addition, in Fig. 3, 7 indicates KK4 (
8. Localization of lysozyme activity in the intracellular fraction, 9. Localization of lysozyme activity in the extracellular fraction, and 1. Localization of lysozyme activity in the Haberi plasm fraction. The bar graph shows total lysozyme activity. Patent applicant: Director of the Agency of Industrial Science and Technology Tatsudai Todoroki 2nd
Figure Tunitlmll
(klett 144n Gyuran (h") 3rd Illusion
sn Tyr Asn Ala Gly Asp Ar
g←-HLY-19(LY-21-m-→'1'AC'
AACACr AGA GCT ACT AA
CTACAACGCCl)・1・・・・10Gfff' GACCGr '1'Cr hCr
GAG'r8 (J:;r ATC'ITC
B...+ +--HLY-27HLY-29HLY-3+ -1÷
QAA A'17r AAC'I℃T A(jA
'rAC'rGG Tσr AACGACGGT
AAG ACT CCA GGCBs +N
l EC0 Rulo

Claims (13)

[Claims] (1) A recombinant DNA containing a hybrid prelysozyme gene consisting of a region encoding a chicken lysozyme signal peptide and a human lysozyme structural gene region, and a promoter. (2) The recombinant DNA according to claim 1, wherein the region encoding the signal peptide has the following nucleotide sequence. [There is a gene sequence] (3) The promoter is a yeast promoter, and the region encoding the signal peptide is located downstream of the yeast promoter and upstream of the structural gene, both of which are in the same reading frame. The recombinant DNA according to claim 1 or 2. (4) The recombinant DNA according to any one of claims 1 to 3, wherein the hybrid prelysozyme gene has the following nucleotide sequence. [There is a gene sequence] (5) The hybrid prelysozyme gene has a restriction enzyme cleavage site (Sol I site) at the 5' end, and two translation termination signals (TAA TGA) and a restriction enzyme cleavage site (Sol I site) at the 3' end.
The recombinant DNA according to any one of claims 1 to 4, which has a Sau3AI site). (6) The recombinant DNA of claim 1, which is the plasmid pHLYSIG. (7) The recombinant DNA according to any one of claims 1 to 5, wherein the promoter is a yeast GAL 10 promoter. (8) The recombinant DNA of claim 7, wherein the promoter is from plasmid YEp51. (9) The recombinant DNA of claim 8, which is plasmid YEp-HLYSIG. (10) Human lysozyme, which is characterized by culturing a yeast transformant carrying a hybrid prelysozyme gene consisting of a region encoding a chicken lysozyme signal peptide and a human lysozyme structural gene region, and a recombinant DNA containing a promoter. Production method. (11) The transformant is S. cerevisiae
The manufacturing method according to claim 10, which is KK4 (YEp-HLVSIG). (12) A yeast transformant carrying a hybrid prelysozyme gene consisting of a region encoding a chicken lysozyme signal peptide and a human lysozyme structural gene region, and a recombinant DNA containing a promoter. (13) S. cerevisiae KK4 (YEp-
13. The yeast transformant according to claim 12, which is HLVSIG).