JPS63214195A - Novel protein - Google Patents

Abstract

PURPOSE:To obtain a safe protein, having a specific amino acid sequence and IgG aggregation activity, produced in large amounts and useful in the immunochemical field, etc. CONSTITUTION:A DNA capable of coding a protein having amino acid sequences expressed by formula I, II or III is integrated into a plasmid to provide a plasmid which is a recombinant DNA. The resultant plasmid is then introduced into Escherichia coli K-12c600 to afford a transformant strain, which is subsequently inoculated into a culture medium containing a carbon source, such as glucose, nitrogen source, such as NH4Cl, and nutrient sources, such as vitamin B1, and cultivated at pH5.5-8.5 and 18-40 deg.C for 5-90hr by spinner culture with aeration. The resultant culture is subjected to collection of microbial cells, which are treated with a lysozyme, then crushed by freezing or melting and subsequently centrifuged to provide a supernatant. The obtained supernatant is extracted to collect the aimed protein.

Description

Detailed Description of the Invention: Retired Emperor Doguri ■1 Minute The present invention provides a novel protein that specifically binds to immunoglobulin G (hereinafter abbreviated as 1gG), a DNA encoding the protein, and a recombinant DNA containing the DNA. and regarding their manufacturing methods.

The novel protein of the present invention has TGC binding properties equivalent to protein A produced by Staphylococcus aureus.

Protein A is a cell membrane component produced by Staphylococcus aureus (hereinafter abbreviated as S aureus) that binds to mammalian immunoglobulin and exhibits a specific serum reaction. This reaction is similar to the normal antigen-antibody reaction. Unlike I
It strongly binds to the Pc portion of gG in a non-immunospecific manner. For this reason, protein A does not inhibit the specific binding reaction of the Fab part of IgG with antigens. Because protein A has such properties, it is used in immunochemistry for quantitative and qualitative purposes, and is It is a useful protein that can also be used for purification (J, J. Langone in “Ad
ances in Isnunology”,
Vol, 32. p, 157°Acadesic
Press, (1982)) *Fox1iL strain Protein A is a membrane-bound protein, so to obtain it as a soluble protein, heat-extract the cells of S. aureus (T., L6fkvist and J, 5j6q)
uist, ActaPath, Microbiol, 5
cand, B, 56.295 (1962)), extracted using cell membrane-digesting enzymes such as lysostaphin (J, Sj'oquist+B, Meloun and
H, Hjels.

Eurj, Biochem, 29.572 (19
72)) method has been adopted. A method for producing soluble protein using a mutant strain of S. aureus lacking the membrane-binding portion of protein is also known (R. Lindm
ark, J., Moritz, &J., 5joquist
, Eur, J., Biochem,, 7C623 (
1977)).

The protein A gene has been cloned using various microorganisms as hosts, and a method for producing protein A using genetic engineering techniques has been reported (C, J, Duggleby
and S, A, Jones+ Nucl, Ac1
dsRes, 11.3065 (1983);
M, Uhl'en, B, Guss.

B, Nilsson+F, Gi;tz and M, L
interberg.

J, Bacteriol, 159.713 (1
984) ; D, Colbert.

A., Anilionis, P., Ge1ep, J., Far.
ley and R, Breyer+J, Biol.
, Re5ponse Modifiers+ 3 , 2
55 (1984)).

S. aureus is a pathogenic bacterium and cannot be said to be a suitable bacterium for protein production.
Its yield is low, it is difficult to purify and it is therefore expensive.

Protein A produced by the above recombinant DNA method is 4
Alternatively, it is a large protein with a molecular weight of approximately 40,000 to 50,000, containing five IgG binding sites and a portion not involved in IgG binding, which is disadvantageous for industrial production.

Therefore, it is desired to supply low-molecular-weight proteins capable of binding to IgG in large quantities at low cost using a safe method that does not use pathogenic bacteria.

One of the IgG-binding sites of protein A that is used to bind to protein A is made up of 58 amino acids and has a molecular weight of approximately 7,000. have the ability. Each IgG binding site
Because it has the ability to bind to the Fc of G (J.

5j2idahl, Ff! BS Lett, 67,
62 (1976); M, Uhl'en.

M, Lindberg and L, Ph11ip
son+I+ssunology Today+5
, 244 (1984)), it is recommended to use a single IgG binding site when attempting to use the binding ability of Protein 8 for immunochemical measurements such as purification of IgG using an affinity column or complex with a labeled enzyme. is preferable to using large molecular weight proteins. In addition, the same binding site was
A new protein with a molecular weight of about 27,000 and a two-linked structure has the ability to cause an aggregation-sedimentation reaction in IgG, which is equivalent to Protein A, and has a lower molecular weight per unit weight than Protein A, which has a molecular weight of 40,000 to 50,000. It has the advantage of having a large IgG binding ability. As mentioned above, protein 8 Ig
Since proteins with 1 to 4 G-binding sites are useful, the researchers designed genes encoding these proteins, chemically synthesized them, and inserted these genes into Escherichia coli to mass-produce the proteins.

The present invention will be explained in detail below.

The present invention provides a protein having an amino acid sequence represented by the following formula 1, formula 13, or formula 15 and having IgG aggregation activity.

Ala (Formula 1) (Formula 13
) MetThrAlaAspAsnLysPheAsnL
ysGluG1nGlnAsnAlaPheTyrG
Iu I 1eLeuH isLeuProAs nL
euAs nG luG luG 1nA rgAsn
G1yPheIleGlnserLeuLysAspA
spProSerGInSerA1aAsnLeuLe
uA1aG1uAlaLysLysLeuAsnAsp
AlaGlnAlaProLysA1aAspAsnL
ysPheAsnLysG1uG1nGlnAsnA1
aPheTyrGlu1 1eLeuH isLeuP
roAsnLeuAsnG 1 uG 1uG l n
ArgAsnG 1yPhe11eG1nSerLeu
LysAspAspProSerG1nSerA1aA
snLeuLeuAlaG1uAlaLysLysLe
uAsnAspAlaG1nAlaProLysAla
AspAsnLysPheAsnLysG 1 uG
lnG 1 nAsnA 1aPheTyrG lu
I leLeuOldS LeuProAsnLeuAsn
G 1uG luG 1nArgAsnG lyPhe
I l eG l nSerLeuLysAspAs
pl'roserG1nSerA1aAsnLeuLe
uAlaGlu^1 aLys Lys LeuAs
nAs pA 1 aG 1nA 1 aProLys
A 1 aAs pAs nLysPheAsn Ly
sG 1 uG lnG lnAsnA 1 aPh
eTyrG 1 u 1 1 eLeuH isLeu
ProAsnLeuAsnG1uGluGlnArgA
snGlyPheI1eG1nSerLeuLysAs
pAspProSerGlnSerA1aAsnLeu
LeuA1aGIuA1aLys (Formula 15) (In the formula, the symbols represent the following amino acids. Met: methionine, Thr: threonine, Ala: alanine, As
p: aspartic acid, ^3n: asparagine, Ly
s: lysine, Phe: phenylalanine, G
lu: glutamic acid, Gln: glutamine, Tyr
:Tyrosine, ■le: Isoleucine, Leu: Leucine, Old S: Histidine, Pro: Proline, A
rg: Arginine, Guy: Glycine, Ser
The novel protein of the present invention can be obtained by introducing into a microorganism a recombinant DNA in which a DNA encoding the protein, for example, a DNA represented by the following formula 2, formula 14 or formula 16 is inserted into a vector. It can be produced by culturing using converted microorganisms. Accordingly, the present invention provides that the formula l. A DNA encoding a protein having an amino acid sequence represented by formula 13 or formula 15. ．．
Recombinant DNA incorporating said DNA, said recombinant DNA
A microorganism containing A and a method for producing the protein using the microorganism are provided.

(Formula 2) GTCGTCTTGC GTAAGATGCT CTA
GAACGTA GACGGCTTAATTCTTTG
ACT TGCTGCGAGT CCGCGGTT
ACTA11'Li1 (Formula 16) (A, G, C, and T in the formula each represent alanine, guanine, cytosine, and thymine. The same applies hereinafter) DNA represented by the nucleotide sequence of Formula 22 Formula 14 or Formula 16 is added to TCC. Hereinafter, it is also referred to as gene 2, gene 14, or gene 16. The method for producing the protein, DNA, recombinant DNA, and microorganism of the present invention is shown below. DNAs 3 to 12 in the following description are D having the nucleotide sequences of the following formulas 3 to 12, respectively.
Indicates NA.

5' CGATAAGCTT ATGACAGCTG
ATAACAAGTTCAACAAAGAG C3'
(Formula 3) % Formula % GTTGTTTCTCGTCGTCT 5' (Formula 4) 5' 8GCAGAACG CATTCTACG
A GATCTTGCATCTGCCGAATT T
3' C formula 5) % formula % GACGGCTTAA ATTTGCT 5' (
Formula 6) 5'^AACGAAG^^CAGCGTAACG
GTTTCATCCAGTCTCTGAA GG 3'
(Formula 7)%Formula% GGCTGAGGC3' (Formula 9)3'
GCTCGGTCTCGCGATTGGACGACC
GACTCCGA TTCTT 5' (21
0) 5' T AAGAAAACTGA ACGACG
CTCA GGCGCCAAAGGCCTAATAAC
3' (Formula 11)%Formula% CGGATTATTG GTAC5' (Formula 1
2) Kogoko (see Figure 2) The base sequence of chemical synthesis gene 2 is shown in Table 1.

Gene 2 encodes the B region, one of the IgG-binding regions of protein A, and its nucleotide sequence is determined by selecting the optimal codons for producing large amounts of protein in E. coli. In addition, in order to introduce gene 2 into the vector, cut ends with restriction enzymes Ban m and Nco 1 were provided at both ends.
Furthermore, cleavage sites for the restriction enzymes Pvun and 5tuT are introduced to link multiple genes encoding the BffI region.

Gene 2 is DNA 10 from 3 to 12 shown in Table 1.
The book's oligonucleotides are chemically synthesized and then enzymatically linked together.

DNAs 3 and 4 form a double strand with complementary base pairs, and the ends thereof form a double strand complementary to the ends of DNAs 5 and 6, which also formed a double strand. This DNA 3 and 4 and 5 and 6
The ends of the double strands can be joined using the enzyme DNA ligase. The oligonucleotides for DNAs 7 to 12 are the same as those for DNAs 3 to 6, and thus gene 2 is synthesized from the 10 oligonucleotides for DNAs 3 to 12.

Steps (See Figure 1) A plasmid vector incorporating gene 2 is created in order to express gene 2 encoding the Bg region of protein A and produce protein. As shown in Figure 1, plasmid pGll! contains the E. coli lipoprotein terminator! 1
(see Reference Example 1) using restriction enzymes Ban m and Neo I
, isolate the fragment containing the promoter (DNA1?), mix this fragment with previously synthesized DNAs 3 to 12 to form a double strand, and connect each fragment using DNA ligase. Plasmid pPr^1 is obtained.

The restriction enzyme map of pPrA I is shown in Figure 1.
The gene sequence and amino acid sequence encoding the protein AOB fiI region contained in the protein AOB fiI region are shown in Table 2 below.

eW L+1-ζvJ-N Engineering (see Figure 2.3) Next, pPrA1 is used to construct a plasmid containing a gene encoding a novel protein in which multiple Bil regions of protein A are linked. As shown in Figure 2, pPrA
1 was cut with restriction enzymes Pstl and Pvn II to obtain a fragment (DNA18) containing gene 2, and separately pPrA
Gene 2 was obtained by cutting 1 with restriction enzymes Pstl and Stu I.
A fragment containing (DNA19) is obtained. DNA obtained in this way
1B and DNA 19 are joined with DNA ligase to create gene 2.
Obtain a plasmid pPrA2 containing two linked genes Next, as shown in Figure 3, pPrA2 is cut with restriction enzymes Pstl and 5tuI to obtain a fragment containing gene 2 (
Take DNA20) and separately add pPrA 2 to restriction enzyme Ps.
Fragment containing gene 2 cut with tl and Pvu If (D
NA21) is obtained. Gene 2 shown in S/P in Figure 3
As shown in Figure 2, the connecting portion of
The cut ends of Pvu II and Pvu II were ligated together, but this part was no longer cleaved by either Stu 1 or Pvu If. DNA 20 and DNA 21 thus obtained were ligated with DNA ligase, and four genes of gene 2 were ligated together. A plasmid pPrA4 containing the gene is obtained. By using the method shown in FIGS. 2 and 3, it is possible to create genes in which any number of genes 2 are linked.

Engineered soil (see Figures 4 to 7) pPrA 1 and pPrA 4 created by the above method
A fragment containing gene 2 is cut out from the fragment and inserted into a plasmid containing a promoter to construct an expression vector.

As shown in Fig. 4, pPrA 1 was converted into
and old ndln to create a fragment containing gene 2 (DNA2
2) is obtained. Separately, plasmid pMTOI 4 (Reference Example 1
) is cut with the restriction enzymes Pstl and old ndl [[ to obtain a promoter-containing fragment (DNA23).

DNA22 is taken and NA23 is ligated with DNA ligase to obtain plasmid pPrAs1. Plasmid pMTOI
4 instead of pMTOII 4, pMTo4. p
MTOI [I 4 (Reference Example 1) was used to cut with the restriction enzymes Pstl and old ndln to obtain promoter-containing fragments (named DNA24.25゜26, respectively) (No. 5).
figure). DNA22 and DNA24 are ligated with DNA ligase to obtain plasmid pPrAT1. Similarly, DNA2
2 and DNA25 to pPrAW 1, DNA22 and DN
pPrALl is obtained from A26 (Figure 6). Plasmid pPrAl is cut with restriction enzymes Pstl and Ban III to obtain a fragment containing gene 2 (DNA27). Plasmid pupal (Reference Example 2) was digested with restriction enzyme Pstl.
and cut with Ban III to obtain a promoter-containing fragment (
DNA28) is obtained. DNAs 27 and 28 are ligated with DNA ligase to obtain plasmid pPrAP1 (Figure 7)
.

Engineering 1- (Refer to Figures 8 to IO) Plasmid pPrA 4 and restriction enzymes Pstl and old ndl
A fragment containing gene 2 (DNA29) is obtained by cutting with l.

Plasmid pPrAS 4 is obtained by ligating this DNA 29 and DNA 23 with DNA ligase (Fig. 8). Similarly, pPrAT 4 and DNA 2 are ligated from DNA 29 and DNA 24.
9 and DNA25 to pPrAW 4, DNA29 and DN
pPrAL 4 is obtained from A26 (Figure 9).

pPrA 4 was cut with restriction enzymes Pstl and BanI [I to obtain a fragment (DNA30) containing gene 2, and this DNA
A30 and DNA2B are ligated with DNA ligase to obtain plasmid pPrAP 4 (Figure 10).

After cutting the plasmid psGtl1M1 (Reference Example 4) with restriction enzyme 5all (see Figure 11), the DNA polymerase Kle
Using the now fragment, a complementary strand of the DNA end is synthesized to make a blunt end, and then cut with the restriction enzyme PstI to obtain a promoter-containing fragment (DNA31). Plasmid pP
rA 1 was similarly cut with the restriction enzyme Ban m, and DN
Complementary strand synthesis by A polymerase Klenov fragment, P
A fragment containing gene 2 (DNA32
). DNA31 and DNA32 are ligated with DNA ligase to obtain plasmid pPrAFW 1 (Figure 11).

Step 1: After introducing the plasmid created by the above method into Escherichia coli and culturing it, the Escherichia coli cells were collected, the cells were destroyed, and the produced protein was analyzed. As a result, the protein in the Bml region of protein A encoded by gene 2 was detected. was being produced in large quantities. Plasmid pPrAs1. pPrATl.

From pPrAWl, pPrALl, and pPrAPl, protein 1 in the BjI region of protein A, which consists of 61 amino acids and has a molecular weight of approximately 7.000, was transferred to plasmid pPrAS4.
．． pPrAT4. pPrAW4゜pPrAL4.
From pPrAP4, protein 15 consisting of 4 linked protein A B regions with a molecular weight of approximately 27,000 consisting of 235 amino acids was obtained, and from plasmid pPrAFWl, a portion of salmon growth hormone and the protein A B region (protein 1) were obtained.
A protein with a molecular weight of about 15,000 is obtained by fusion of the plasmids pPrAS 2 and pPrAW 2 , and a protein 13 consisting of 119 amino acids and a molecular weight of about 14,000 linked with two protein A Bwi regions is obtained.

Step 8 Add 60% ammonium sulfate (ammonium sulfate) to a solution containing protein obtained from E. coli into which plasmid pPrAS4 has been introduced.
and remove the precipitate that has formed. When ammonium sulfate is further added to the supernatant to give a concentration of 80%, protein 15, in which four BeJl regions of protein A are linked, is obtained as a precipitate. This precipitate is collected and subjected to gel column chromatography on Sephadex G-75 to obtain pure protein 15. Protein AOB SI thus obtained
Protein 15, which has four linked regions, aggregates IgG.

Using engineering plasmid pPrA112, Sephadex G-
Protein 13 is obtained in the same manner as in step 8 except that 50 is used. In this way, protein 13, in which two B regions of protein A are linked, aggregates IgG.

Using the chemically synthesized gene using the method described above, we succeeded in producing a new protein that can aggregate IgG.
This chemically synthesized gene also has the advantage of easy mutation introduction. Protein A specifically binds to IgG, but if its specificity can be changed so that it binds to other immunoglobulins, it can be expected to have a wide range of applications such as qualitative, quantitative, and purification of immunoglobulins. The specificity of protein A binding is determined by the structure of the binding site, which in turn is determined by the amino acid sequence of that site.

Therefore, by changing the amino acid sequence of the binding site, the structure changes and specificity can be changed. Recent advances in genetic engineering technology have made it possible to arbitrarily convert amino acids in specific regions of proteins (D, Bostein et al.
d D, 5hortle, 5science+ 229
1193 (1985)). This method is a mutagenesis method in which amino acids at specific sites in a protein are changed by introducing mutations into the base sequence of a gene that encodes the amino acid sequence of the protein. There are 4 natural protein A genes.
Or, since we have a gene encoding five binding sites, if we want to change the binding specificity of protein A, we need to introduce the same mutation into equivalent sites among these four or five binding sites. be. However, it is impossible to simultaneously introduce the same mutation into such multiple sites.

As a result of the introduction of different mutations into different binding sites, protein A ends up having several binding sites with different specificities and loses its usefulness altogether.

It is possible to introduce mutations into gene 2, which encodes one binding site of protein A synthesized in the present invention, to convert amino acids, and to obtain proteins with altered binding specificity for immunoglobulins from among the resulting proteins. can. By linking four genes encoding binding sites with altered specificity using the method of the present invention, a protein A-like protein with altered specificity for immunoglobulins can be obtained.

Several restriction enzyme cleavage sites were introduced into gene 2 to facilitate mutation introduction. Among these, the main restriction enzyme sites are shown below.

Hind m Pvu IIBgl II
Dral/Aha3Aval Ha
eU (Formula 1 %Formula %) The reaction conditions in the above recombinant technique are generally as follows.

The digestion reaction of DNA with restriction enzymes is usually 0.1 to 20
ul of DNA at 2-200 sM (preferably 10-200 sM)
40 sM) Tris-HCl, (pH 6.0-9.5
Preferably pH 7.0-8.0), 0-200+M NaCjl! or Kl, 2-30aM (preferably 5
~10mM) of MgCj! z, restriction enzyme 0.1-20+mM in a reaction solution containing mercaptoethanol.
100 units (preferably 1 to 1 for 11!g of DNA)
3 units) at 20 to 70°C (the optimum temperature varies depending on the restriction enzyme used) for 15 minutes to 24 hours.

Purification of DNA fragments generated by restriction enzyme digestion was performed using LG
This is performed using the T method, polyacrylamide gel electrophoresis, or the like.

The binding reaction of DNA fragments is carried out over a period of 2 to 200 d (preferably 1
0-40mM) Tris-HCl2 (pH 6,1-9
．． 5, preferably p117.0-8.0), 2-20m
M (preferably 5 to 10a+M) MgCj! z,
0.1-10-M (preferably 0.5-2.0+++M
) (7) ATP, 1-50mM (preferably 5-50mM)
In a reaction solution containing dithiothreitol (10mM), T
1-37° using 0.3-10 units of 4 DNA ligase
C (preferably 3 to 20'C) for 15 minutes to 72 hours (preferably 2 to 20 hours).

The recombinant plasmid DNA produced by the ligation reaction is
If necessary, the transformation method of Cohen et al.
Proc, Natl, Acad, Sci,), U
SA, Deaf, 2110 (1972)) into E. coli.

Isolation of recombinant plasmid DNA from Escherichia coli containing recombinant plasmid DNA is carried out using cesium chloride-ethidium bromide density gradient ultracentrifugation [D.B.
, C1e, et al.: Proceedings of
The National Academy of Science (Pr.
oc, Natl, Acad, Sci,), USA.

62, 1159 (1969)) or the method of Birnboim et al.
Birnboim, H.C. et al.: Nucleic Acid Research
s Res, ) 1.1513 (1979)).

After digesting the plasmid DNA with a restriction enzyme, the cleavage site is examined by agarose gel electrophoresis or polyacrylamide gel electrophoresis. Furthermore, when it is necessary to determine the base sequence of DNA, the Maxam-Gilbert method [Proceedings of the National Academy of Sciences (Proc.

Natl, Acad, Sci, ), 74.560
(1977)) or the Sanger method using M13 phage [Sanger et al.
Proceedings of the National Academy of Sciences (Proc. Natl. Aca.
d, Sci,), USA, ? C5463 (19
77) A+++eraham M13 Cloning and Sea Fencing Handbook
nghandbook)).

The protein of the present invention can be produced as follows.

That is, using a plasmid to infect E. coli with 12c600
E. coli containing the plasmid is selected from ampicillin-resistant colonies. By culturing E. coli containing a plasmid in a medium, proteins can be produced in the culture.

As the medium used here, either a synthetic medium or a natural medium can be used as long as it is suitable for the growth of E. coli and protein production.

Carbon sources include glucose, fructose, lactose, glycerol, mannitol, sorbitol, etc. Nitrogen sources include Nl(, Cf, (NHn)gso4
, casamino acids, yeast extract, polypeptone, meat extract,
Batato tryptone, corn staple liquor, etc. are other nutritional sources such as K! HPO,, uzpo
a, NaCj! 5Mg5Oa, vitamin B3, Mg
CIl, etc. can be used.

The culture is carried out at a pH of 5.5 to 8.5 and a temperature of 18 to 40''C with aeration and stirring.

Protein accumulates in the culture after 5 to 90 hours of culture, so the cells are collected from the culture, treated with lysozyme, frozen and thawed repeatedly to crush the cells, and then centrifuged. Protein is collected from the supernatant according to a conventional protein extraction method.

In addition, the protein can be detected directly from cultured bacterial cells using Laemmli (L
aessli) sample buffer [Laemmli
mmli), Nature 227.
680 (1970)), and then subjected to 5DS-polyacrylamide gel electrophoresis (Lae@s+Ii) method: Ibid.
Coos+assieBrilliant Blue)
Staining was performed using a dye (manufactured by Bio-Rad).

Examples of the present invention are shown below.

Example 1 Production of DNAs 3 to 12: DNAs 3 to 12 were synthesized by solid-phase synthesis using the phosphoramidide method (S, L. Beaucage et al., Tetra et al.
Tetrahedron Lett
)22. '1859 (1981), L, JlMcB
rie et al., 24.245 (1983)), an automated DNA synthesizer 38 from Applied Biosystems was used.
The following procedure was performed using 0A.

Using silica gel as a solid phase support, (1) nucleotides are condensed with the 5' hydroxyl group of the nucleotide bound via the 3' hydroxyl group by the phosphate amidite method, and (2) the phosphorous acid bond of the condensed nucleotide is removed with iodine. (3) The protecting group on the 5' hydroxyl group of the condensed nucleotide was removed with trifluoroacetic acid. Next, returning to step (1), the next nucleotide was similarly condensed. thus(
Steps 1) to (3) were repeated to synthesize DNA on the carrier. After completion of the synthesis, the DNA-bound carrier was left in a thiophenol solution for 1 hour at room temperature to remove the phosphoric acid protecting group, and then left in concentrated ammonia water at room temperature for 1 hour to release the DNA from the carrier. . Concentrated ammonia water containing DNA was heated in a sealed container at 60° C. for 12 hours to remove the protecting groups on the base.

Taking DNAAl0 as an example, synthesis was performed using a carrier bound with 1μ5ole of nucleotides, and the condensation reaction was completed with a total yield of 76%.Then, the protecting group was removed and the release reaction from the solid phase was performed to obtain a crude DNAAlO. Product 2750.0. Units (measured at 260 na+) were obtained.

140.0 of this crude product. The unit was purified by electrophoresis on a 10% polyacrylamide gel (2 layers thick, 13CIIX 13C11) using Tris-borate buffer (pH 18) containing 7M urea, the area of the gel containing DNA10 was taken, and 0.2M Triethylamine carbonate buffer (pH 8) (
(hereinafter referred to as TEAB) 1a+4! and DNA10 is 18
The DNA was extracted for a long time, and then the extract was passed through a Sephadex DE52 column (diameter 6 mm, length 5 mm).
10 was adsorbed. 2MTEA82ca1. Elute at 2.00. D. A pure product of DNA10 was obtained.

DNAs other than DNA10 were also synthesized with similar yields.

The 5′ hydroxyl group of these DNAs was removed using a conventional method using phage T4 polynucleotide kinase and [γ-”P)ATP [^,
M. Maxa et al., “Methods in Enzymology”
) Vol.

65, Part L p, 499. Academy
c Press (1980)) to give a radioactive label. The labeled DNA was subjected to 20% polyacrylamide gel electrophoresis using a Tris-borate buffer containing 7M urea to confirm the purity and chain length of the DNA. Furthermore, the base sequence of the labeled DNA was determined using the Maxas-Gilbert method (see the above-mentioned document).
It was confirmed that each DNA had the desired base sequence.

Example 2 Preparation of plasmid pPrA 1: Plasmid pGll produced in Reference Example 3! 12μg of restriction enzyme Ba
A solution (101IM Tris-H) containing 10 units of nnI (manufactured by Toyobo Co., Ltd.) and 15 units of Neo I (manufactured by Neo I)
Cl (pH 7,5), 7mM Mg (/!g,
6mM 2-mercaptoethanol, 100mM NaC
12) Dissolved in 30 μl and subjected to a digestion reaction at 37° C. for 2 hours. This reaction solution was subjected to agarose gel electrophoresis containing ethidium bromide, detected with ultraviolet light (wavelength: 302 ns), and a gel piece containing approximately 2.5 kb DNA 17 was excised. The gel pieces were frozen and dissolved by adding 0.5 mf of phenol, the aqueous layer was washed with chloroform, and DNA17 was recovered by ethanol precipitation.

DNA 3-12 each IQ pmole each in T44 polynucleotide kinase application buffer (50sM) Lis HC
j! (pH 7,5), 10sM MgC28,5
sM dithiothreitol (hereinafter abbreviated as DTT), l
IIMATP, 0.1mM spermidine, 0.1 sM
Dissolve 30 μ2 each of EDTA), add 3 units of T4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.), and incubate at 37°C.
After carrying out the phosphorylation reaction for 0 minutes, the enzyme was inactivated by heating at 65° C. for 15 minutes. Mix 4 μl of this reaction solution and add 0.08 pmole of the DNA 17 obtained above.
and 2811M Tris-HCl (pH 7,5)
, 9a+MMgCfz, 10s+MD
TT, 0.03mM EDTA.

The composition was adjusted to 0.7mM ATP, 0.03M H spermidine, and 2 units of T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.) was added to make 50ttl. The binding reaction was carried out at 4°C for 16 hours.

Using this reaction solution, E. coli strain H8101 [Polyvar (B
Gene, 2.75 (
1977)) using the method of Cohen et al.
, 115A 69.2110 (1972)) to obtain ampicillin-resistant (Ap') colonies. From this colony, an alkali treatment method [edited by Maniatis et al., Molecular Cloning, P. 3]
6B, Cold Spring Harbor
ing Harbor)
A was collected to obtain pPrA1. The structure of pPrA 1 is Bgl If, Pstl, old ndI[I, Stu
I, Bam1l I and confirmed by agarose gel electrophoresis. Restriction enzyme cleavage reaction is 10sM
) Squirrel-11cj! (pH 7, 5), 7mM MgC
j! g, 6mM 2-mercaptoethanol solution to the optimum concentration of each enzyme (0 to 200a
+M) Add NaCl2 or KCf to the reaction solution (
Only the concentration of the restriction enzyme reaction solution, NaCl or KCf, is shown below. ).

Furthermore, the literature [^, J, H, Sm1th, “Methods in Enzymology”
Enzys+ology”), ed, by L,
Grosssen and K, Mo1d.
ave Vol, 65+ p.

560 (1980), Academic Press
) The sequence of 402 bases including portions derived from DNAs 3 to 12 was determined by the method described in ), and gene 2 of interest was confirmed.

The Escherichia coli strain containing pPrAl is Escherichia
coliEPrAI FERM BP-1282 was transferred to the Institute of Microbial Technology (Feikoken), Agency of Industrial Science and Technology in 1932.
It was deposited on February 10, 2013.

Example 3 Preparation of plasmid pPr^2: 0.2 μg of plasmid pPrA1 obtained in Example 2 was dissolved in the restriction enzyme reaction solution shown in Example 2 (15 μn of 100 mM NaCj, 6 units of psi (manufactured by Takara Shuzo Co., Ltd.), 6 units of 5tul (manufactured by Takara Shuzo Co., Ltd.) were added to carry out a digestion reaction at 37°C for 2 hours.This was fractionated by agarose gel electrophoresis in the same manner as in Example 2, and a DNA fragment of approximately 1 kb (DNA19) was obtained. .

Separately, add 0.2 μg of pPrAl to restriction enzyme reaction solution (5
Dissolved in 0mM NaCj and 15tlN, Pstl
6 units, add 6 units of PvuII (manufactured by Takara Shuzo Co., Ltd.) and 3
Digestion reaction was carried out at 7°C for 2 hours. This was fractionated by agarose gel electrophoresis, and a DNA fragment of approximately 1.9 kb (
DNA18) was obtained.

50 ng of each of DNA18 and DNA19 thus obtained were mixed, and a reaction solution for T4 DNA ligase [20■H Tris-HCj! (pH 7,5), 10d MgCj!
□, 10mM DTT, 0.3mM ATP) 30
The mixture was dissolved in μl, 1 unit of T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.) was added, and a binding reaction was performed at 4° C. for 18 hours. E. coli strain 88101 was transformed using this reaction solution to obtain Apr colonies. Plasmid DNA was recovered from this colony to obtain pPrA2. The structure of this plasmid is
Pst I, Bawl I, Ban III, 5
It was cut with tul and confirmed by agarose gel electrophoresis.

Example 4 Preparation of plasmid pPr^4: 0.2 μg of plasmid pPrA2 obtained in Example 3 was added to the restriction enzyme reaction solution (100 sM) shown in Example 2 in the same manner as in Example 3.
Dissolve in 5 ul of NaCj, Pst [6 units, 5 tu
The mixture was digested with 6 units of 1 at 37°C for 2 hours, and fractionated by agarose gel electrophoresis to obtain DNA 20 of approximately 1.2 kb. In addition, 0.2 μg of pPrA 2 was dissolved in a restriction enzyme reaction solution (50 mM Na (15 μj/ml),
Add 6 units of Pstl and 6 units of Pvull to make 37
The DNA was digested at ℃ for 2 hours and fractionated by agarose gel electrophoresis to obtain DNA 21 of about 2.1 kb.

Q and 2 psole of each of these DNAs were mixed, dissolved in 30 μl of a reaction solution for T4 DNA ligase, 1 unit of T4 DNA ligase was added, and a binding reaction was performed at 4° C. for 18 hours. E. coli strain 88101 was transformed with this reaction solution to obtain 74 pF colonies, and plasmid D
NA was collected to obtain pPr^4. The structure of this plasmid is former ndll1%5tul, PvuII, Bawl
1 and confirmed by agarose gel electrophoresis.

Example 5 Preparation of plasmid pPrAS 1 1 μg of niblasmid pMTOHI 4 was added to a restriction enzyme reaction solution (10
Dissolved in 30 tl of 0mM NaCj, Ps
Add t16 units and old ndI [[6 units, perform a digestion reaction at 37°C for 2 hours, and obtain approximately 1.3 kb by agarose gel electrophoresis.
23 DNAs were fractionated and recovered.

Separately, 111g of plasmid pPrA1 was added to pMTOIl.
Digested with PstI, old ndl[[ in the same manner as 4, and digested with approx.
．． 22 pieces of 9kb DNA were recovered.

22.23 of these DNAs were mixed in 0.03 psole and dissolved in 0μ2 of T4DNA4DNA ligase.
Add 1 unit of T4 DNA ligase and perform a ligation reaction at 4°C for 18 hours. E. coli strain 88101 was transformed with this reaction solution to obtain Apr colonies and collect plasmid DNA.
PrAS I was obtained.

Example 6 Preparation of plasmid pPrAT 1 111 μg of niblasmid pMTO was digested with restriction enzymes PstI and 1ndlll under the same conditions as in Example 5, and then fractionated by agarose gel electrophoresis to obtain DNA 24 of about 1 kb.

DNA22 and DNA24 obtained by the method of Example 5 were 0.0
3 paroles were mixed, dissolved in 30μ2 of T4 DNA ligase reaction solution, added 1 unit of T4 DNA ligase and allowed to bind at 4°C for 18 hours.
8101 strain was transformed, Apr colonies were obtained, and plasmid DNA was collected to obtain pPrATl.

Example 7 Preparation of plasmid pPrAWl 41 μg of niblasmid pMTO was digested with restriction enzymes pstI and old ndIII under the same conditions as in Example 5, and fractionated by agarose gel electrophoresis to obtain DNA 25 of approximately 1 kb.

DNA22 and DNA25 obtained by the method of Example 5 were 0.0
Mix 3p+mole and make 30al of T4 DNA ligase reaction solution! 1 unit of T4 DNA ligase was added and a ligation reaction was carried out for 1 hour at 4°C.Escherichia coli H8101 strain was transformed with this reaction solution, colonies A and r were obtained, and plasmid DNA was recovered to obtain pPrAWl.

Example 8 Preparation of plasmid pPrAL 1 1 μg of niblasmid pMTo I [I4 was digested with restriction enzymes Pstl and 1lindnl under the same conditions as in Example 5, and then fractionated by agarose gel electrophoresis to obtain approximately 1 kb DNA26.
I got it.

DNA22 and DNA26 obtained by the method of Example 5 were 0.0
3 pn + ole were mixed, dissolved in 30 μl of T4 DNA ligase reaction solution, added 1 unit of T4 DNA ligase, and allowed to react for 1 hour at 4°C. E. coli H8101 strain was transformed with this reaction solution, and colonies of /i pr The resulting plasmid DNA was recovered to obtain pPrALl.

Example 9 Preparation of plasmid ρPrAP 1 1 μg of niblasmid pPrA1 was dissolved in the restriction enzyme reaction solution shown in Example 2 (100 aiM NaC, 130 μl, Ps
Add t16 units and 6 Banm units, digest at 37°C for 2 hours, and fractionate by agarose gel electrophoresis to approx.
27 pieces of 9kb DNA were obtained. Plasmid pUPA11
ug was digested with PstI and BanI under the same conditions.
Approximately 2.2 kb of DNA was fractionated by agarose gel electrophoresis.
A28 was obtained.

Mix 0.03 psole of DNA27.28,
Dissolve in 30 μl of T4 DNA ligase reaction solution and add T4D
One unit of NA ligase was added and the binding reaction was carried out for 18 hours at 4°C.Escherichia coli strain H8101 was transformed with this reaction solution, and Ap
IF colonies were obtained, plasmid DNA was collected, and pP
rAP 1 was obtained.

Example 10 Preparation of plasmid pPrAS 4 41 μg of niblasmid pPrA was digested with restriction enzymes pstl and 1lindlll under the same conditions as in Example 5, and fractionated by agarose gel electrophoresis to obtain DNA 29 of approximately 2.4 kb.

DNA23 and DNA29 obtained by the method of Example 5 were
Mix 3 pmole each, dissolve in 30 µi of T4 DNA ligase reaction solution, add 1 unit of T4 DNA ligase, perform binding reaction at 4°C for 1 hour, and use this reaction solution to incubate E. coli H.
The 8101 strain was transformed, Apr colonies were obtained, and plasmid DNA was collected to obtain pPrAS4.

Escherichia coli containing pPrAS4 is Hscherichia
coli F! It was deposited as PrAS4FERM BP-1283 with the Microtech Institute on February 10, 1986.

Example 11 Preparation of plasmid pPrAT 4: DNA24 obtained by the method of Example 6 and D obtained by the method of Example 1O
Mix 0.03 pmole of NA29 and add T4DN
Dissolve in 30μ2 of reaction solution for A ligase, add 1 unit of T4 DNA ligase, perform a binding reaction for 1 hour at 4°C, transform E. coli strain H8101 with this reaction solution, obtain colonies of QpP, collect plasmid DNA, and collect pPrAT4.
I got it.

Example 12 Preparation of plasmid pPrAG14: DNA25 obtained by the method of Example 7 and D obtained by the method of Example 1.
Mix 0.03 pmole of NA29 and add T4DN
Reaction solution for A ligase 30ttl! Dissolve T4 DNA in
One unit of ligase was added and the binding reaction was carried out for 18 hours at 4°C.Escherichia coli HB101 strain was transformed with this reaction solution, and Ap'
Colonies were obtained, plasmid DNA was collected, and pPr4
I got it.

Example 13 Preparation of plasmid pPrAL 4: DNA26 obtained by the method of Example 8 and D obtained by the method of Example 1.
Mix 0.03 pmole of NA29 and add T4DN
Dissolved in A ligase reaction solution 3091, add 1 unit of T4 DNA ligase, perform a binding reaction for 1 hour at 4°C, transform E. coli strain H8101 with this reaction solution, obtain Apr colonies, recover plasmid DNA, and transform pPrAL.
I got 4.

Example 14 Preparation of plasmid pPrAP 4 1 μg of niblasmid pPrA4 was incubated with Ps under the same conditions as in Example 9.
tI, Ban1 [digested and fractionated by agarose gel electrophoresis to yield approximately 2.4 kb DNA30. 0.03 pmol of DNA2B and DNA30 obtained in Example 9
E. coli strain H8101 was transformed with this reaction solution to obtain Apr colonies. plasmid DNA
was collected to obtain pPrAP4.

Example 15 Preparation of plasmid pPrAFW 1 12 μg of niblasmid pPrA was dissolved in the restriction enzyme reaction solution shown in Example 2 (100 mM NaCj 30I11, 8 units of Ban11 was added, and the digestion reaction was carried out at 37°C for 2 hours. Add DNA polymerase-Kl to the reaction solution.
2 units of eno-fragment (manufactured by Takara Shuzo) and dATP.

dGTP, dCTP, and dTTP were each added to a concentration of 0.4 sM, and the reaction was carried out at 16°C for 2 hours.Next, 30μ2 of phenol was added to the reaction solution to extract the enzyme, and 75μ2 of ethanol was added to the aqueous layer, and the mixture was incubated at -70"C. After cooling, the plasmid was recovered by centrifugation.This plasmid was mixed with the restriction enzyme reaction solution shown in Example 2 (100sM Na
Dissolve in 30ul of C/li, add Pstl B unit and add 3
The digestion reaction was carried out at 7°C for 2 hours. This was fractionated by agarose gel electrophoresis in the same manner as in Example 2, and approximately 1.1 kb DNA 32 was obtained.

12 μg of plasmid psGHM was mixed with the restriction enzyme reaction solution (175 mM NaCf) shown in Example 2.
/I was dissolved, 8 units of 5ail (manufactured by Takara Shuzo Co., Ltd.) were added, and a digestion reaction was carried out at 37°C for 2 hours. Add DN to this reaction solution.
2 units of A polymerase Klenow fragment and dATP.

dGTP, dCTP, and dTTP were each added at a concentration of 0.4 mM, and the reaction was carried out at 16°C for 2 hours. This was subjected to phenol extraction and plasmid recovery in the same manner as above, and digested with the restriction enzyme Pstl to obtain DNA31.

0.03 pmole of the DNA 31.32 thus obtained
The mixture was mixed, dissolved in 30 μl of T4 DNA ligase reaction solution, 1 unit of T4 DNA ligase was added, and a 4"C binding reaction was carried out for 18 hours. This reaction solution was used to transform Escherichia coli strain 18101 to obtain colonies of Ap' and transform the plasmid. DNA
was collected to obtain pPrAFW 1.

Escherichia coli containing pPrAFW1 is Escherichia
a coltEPrAFWI Ft! RM BP-12
No. 84, it was deposited with the Institute of Fine Technology on February 10, 1986.

Example 16 Production of protein 1 by E. coli using plasmid pPrAS 1: Production of protein 1 by E. coli using pPrAS 1 obtained in Example 5
1105trA strain (FERM BP-732) was transformed. The obtained Apr colony was added to 8 ml of LG medium (1% Batatotributone, 0.5% yeast extract, 0.5
% NaCl, 0, 1% glucose, 50 μg/ml tryptophan, 50 μg/-2 ampicillin pH 7,5)
and cultured at 30°C for 16 hours. This culture solution 400
μf is 10mj! of MCG medium (0.6%, NatHP
Oa, 0.3% Kll! PO, 0.5% NaCl1,
0.1% NH, Cf.

0.5% glucose, 0.5% casamino acids, 1mM Mg
SO4,4μg/-j! Vitamin B+5 pH 7,2) with 50μg/all tryptophan and 50μg/l
An was inoculated into a medium supplemented with ampicillin and cultured at 30°C. The turbidity (ODsso) of the culture solution was measured and was 0.9.
(about 4 hours later), indole acetic acid 200
μg was added, and the culture was continued for an additional 4 hours. 7 of the culture solution
．． The cells were collected by centrifugation at 00 rpm for 5 minutes. The bacterial cells were collected using the method of Laemwli et al. [Laemwli et al.; Nature, 227.680 (1
After dissolving in the sample buffer of 970) ) and heating, 5DS-polyacrylamide gel electrophoresis was performed according to the same method, and as a result of staining with Coomassie brilliant blue, a polypeptide band was detected at a site with a molecular weight of about 7.000. E. coli-3110 without pPrAS 1
There was no corresponding band in the strain, so pPrAs
It was revealed that E. coli W31105trA, which carries protein 1, produces protein 1.

Example 17 Production of protein 1 by E. coli using plasmid pPrAT 1: Production of protein 1 by E. coli using plasmid pPrAT 1: E. coli W3 using pPrAT 1 obtained in Example 6
1105trA strain was transformed. The obtained A and P colonies were cultured in the same culture method as in Example 16, and the culture solution was centrifuged at 7.00 rpm for 5 minutes to collect bacterial cells.

The cells were dissolved in the sample buffer of Laemmle et al.'s method, heated, and then subjected to 5DS-polyacrylamide gel electrophoresis according to the same method, and stained with Coomassie Priliant Blue. detected. Since there was no corresponding band in E. coli-3110■0 strain, which does not contain pPrATl, E. coli-31105tr, which contains pPrAT1,
It became clear that A produced protein 1.

Example 18 Production of protein 1 by E. coli using plasmid pPrAW 1: Production of protein 1 by E. coli using pPrAI41 obtained in Example 7
110 str^ strain was transformed. The obtained Apr colony was cultured using the same culture method as in Example 16, and the culture solution was centrifuged at 7.00 rpm for 5 minutes to collect bacterial cells.

After dissolving this bacterial cell in the sample buffer of Laemmle et al.'s method and heating, 5DS-polyacrylamide gel electrophoresis was performed according to the same method, and as a result of staining with Coomassie brilliant blue, a polypeptide band was found at a site with a molecular weight of approximately 7.000. was detected.

Since there was no corresponding band in E. coliformes 1105trA strain that does not contain pPrlV1, ρPr1W
It was revealed that E. coli-31105trA, which carries protein 1, produces protein 1.

Example 19 Production of protein 1 by E. coli using plasmid pPrAL 1: Production of protein 1 by E. coli using plasmid pPrAL 1: E. coli W3 using pPrAL 1 obtained in Example 8
1105trA strain was transformed. The obtained Apr colony was cultured using the same culture method as in Example 16, and the culture solution was centrifuged at 7.00 rpm for 5 minutes to collect bacterial cells.

After dissolving this bacterial cell in the sample buffer of Laemmle et al.'s method and heating, 5DS-polyacrylamide gel electrophoresis was performed according to the same method, and as a result of staining with Coomassie brilliant blue, a polypeptide band was found at a site with a molecular weight of approximately 7,000. was detected. Since there was no corresponding band in E. coli W31jO5trA strain, which does not contain pPrAL1, E. coli W3110, which carries pPrAL1,
It was revealed that the 5trA strain produces protein 1.

Example 20 Production of protein 1 by E. coli using plasmid pPrAP 1: Production of protein 1 by E. coli using pPrAP 1 obtained in Example 9
1105trA strain was transformed. Culture was started under the same conditions as in Example 16, and the turbidity (ODss.) of the culture solution was 0.
．． 5, the culture temperature was raised to 42°C and cultured for an additional 2 hours.

The culture solution was centrifuged at 7,000 rp+s for 5 minutes to collect bacterial cells. After dissolving this bacterial cell in the sample buffer of Laemmle et al.'s method and heating, 5DS-polyacrylamide gel electrophoresis was performed according to the same method, and as a result of staining with Coomassie brilliant blue, a polypeptide band was found at a site with a molecular weight of approximately 7,000. was detected. Since no corresponding band was present in the E. coli 1113110 concavity strain that does not contain pPrAP 1, it was revealed that the E. coli -31105trA strain harboring pPrAP 1 was producing protein 1.

Example 21 Production of protein 15 by E. coli using plasmid pPrAT 4: Production of protein 15 by E. coli using pPrAT 4 obtained in Example 11
3110 str^ strain was transformed. The obtained Ap
The r colony was cultured using the same culture method as in Example 16, and the culture solution was centrifuged at 7,000 rpta for 5 minutes to collect bacterial cells. After dissolving this bacterial cell in the sample buffer of Laemmle et al.'s method and heating, 5DS-polyacrylamide gel electrophoresis was performed according to the same method, and as a result of staining with Coomassie brilliant blue, a polypeptide band was found at a site with a molecular weight of approximately 27,000. was detected.

Since there was no corresponding band in the E. coli-31105trA strain that does not contain pPrAT4, pPrAT
Escherichia coli W31105trA strain harboring protein 4 has protein 1.
It has been revealed that they are producing 5.

Example 22 Production of protein 15 by E. coli using plasmid pPrAW 4: Production of protein 15 by E. coli using pPrAW 4 obtained in Example 12
31105trA strain was transformed. The obtained Ap′
Colonies were cultured using the same culture method as in Example 16, and the culture solution was centrifuged at 7.00 rpm for 5 minutes to collect bacterial cells. After dissolving this bacterial cell in the sample buffer of Laemmle et al.'s method and heating it, SO3-polyacrylamide gel electrophoresis was performed according to the same method, and as a result of staining with Coomassie brilliant blue, a polypeptide band was found at a site with a molecular weight of approximately 27,000. was detected.

Since there was no corresponding band in the E. coli-31105trA strain that does not contain pPrA114, pPrA@
Escherichia coli W31105trA strain that maintains protein 15
It was revealed that they were producing.

Example 23 Production of protein 15 by E. coli using plasmid ρPrAL 4: Production of protein 15 by E. coli using pPrAL 4 obtained in Example 13
31105trA strain was transformed. Obtained ＾p′
Colonies were cultured using the same culture method as in Example 16, and the culture solution was centrifuged at 7.00 rpm for 5 minutes to collect bacterial cells. After dissolving this bacterial cell in the sample buffer of Laemmle et al.'s method and heating, 5DS-polyacrylamide gel electrophoresis was performed according to the same method, and as a result of staining with Coomassie brilliant blue, a polypeptide band was found at a site with a molecular weight of approximately 27,000. was detected.

Since there was no corresponding band in the E. coli-31105trA strain that does not contain pPrAL4, pPrAL
E. coli-31105trA strain carrying protein 4 has protein 1.
It has been revealed that they are producing 5.

Example 24 Production of protein 15 by E. coli using plasmid pPrAP 4: Production of protein 15 by E. coli using pPrAP 4 obtained in Example 14
3110 str^ strain was transformed. Obtained 4p
The r colony was cultured using the same culture method as in Example 20, and the culture solution was centrifuged at 7.000 rρ for 5 minutes to collect the bacterial cells. After dissolving this bacterial cell in the sample buffer of Laemmle et al.'s method and heating, 5DS-polyacrylamide gel electrophoresis was performed according to the same method, and as a result of staining with Coomassie brilliant blue, a polypeptide band was found at a site with a molecular weight of approximately 27,000. was detected.

There was no corresponding band in the E. coli-31105trA strain that does not contain pPrAP4, so pPrAP
E. coli-31105trA strain carrying protein 4 has protein 1.
It has been revealed that they are producing 5.

Example 25 Protein production by E. coli using plasmid pPrAFW 1: Using pAFW 1 obtained in Example 15, E. coli W3
1105trA strain was transformed. The obtained Apr colony was cultured using the same culture method as in Example 16, and the culture solution was centrifuged at 7.00 rpm for 5 minutes to collect bacterial cells.

After dissolving this bacterial cell in the sample buffer of Laemmle et al.'s method and heating, 5DS-polyacrylamide gel electrophoresis was performed according to the same method, and as a result of staining with Coomassie brilliant blue, a polypeptide band was found at a site with a molecular weight of approximately 15,000. was detected.

There was no corresponding band in the E. coli-3110str^ strain that does not contain pPrAF, so pPrAF
It has been revealed that the E. coli-31105trA strain harboring W1 produces a fusion protein of a portion of chum salmon growth hormone and protein 1.

Example 26 Production and purification of protein 15 by E. coli using plasmid pPrAS 4: pPrAS 4 obtained in Example 10 was used to transform E. coli W31105trA strain. Obtained A
Colonies of pr were inoculated into 8-2 LG medium and incubated at 30°
The cells were cultured for 16 hours at C. This culture solution 2tan is 100
The cells were inoculated into a tan MCG medium supplemented with 50 μg/lll1 tryptophan and 50 μg/ml ampicillin, and cultured at 30°C. Turbidity (ODs) of culture solution
So) was measured, and when it reached 0.9 (about 4 hours later), 2 ml of indole acetic acid was added, and the culture was continued for an additional 4 hours. The culture solution was centrifuged at 700 rpm for 5 minutes to collect bacterial cells. After washing the bacterial cells with PBS, they were suspended in 25 mA PBS, and the bacterial cells were disrupted by ultrasonication (10
Centrifuge at 7,000 rpm for 1 hour, collect the supernatant, add ammonium sulfate to the supernatant to a saturation concentration of 60%, let stand at 0°C for 1 hour, and centrifuge at 7,000 rpm again.
The mixture was centrifuged for 1 hour at m. Add ammonium sulfate to this supernatant.
The mixture was further added to achieve a 0% saturation concentration, left to stand at 0°C for 1 hour, and then centrifuged at 7000 rpm for 1 hour to obtain protein 1r15 as a precipitate. This protein 15 was dissolved in 2 ml of distilled water, dialyzed in distilled water for 16 hours, dissolved in sample buffer according to the method of Laemmle et al., heated, and subjected to SO3-polyacrylamide gel electrophoresis according to the same method. As a result of staining and quantifying using blue, the purity of protein 15 obtained was 38%.
Met. Sephadex G-
75 column chromatograph 4 (2,4X84c
m) and 501IIM Trisu HC/! (pH7
, 5), the eluate was fractionated into approximately 4rals, and fractions 39-50 were concentrated to obtain a pure protein 15 of 4μ.

Example 27 Ig of protein 15 produced by E. coli
G aggregation activity: Ig of purified protein 15 obtained in Example 26
G aggregation activity was measured as follows.

Activity measurement reaction solution (20mM HEPPSO(N-(
2-hydroxyethyl)piperazine-N'-2-hydroxypropane-3-sulfonic acid) (pH 8),
1.00mM KCj! , 6% Pluronic F
1a (manufactured by Asahi Denka)] Protein A in 2.5 mft
(manufactured by Lebrisien) aqueous solution (concentration 0.0.05.0.
1.0.2.0.5.0.8゜1.0 mg/tan
) was added and heated at 37°C for 3 minutes. After measuring the absorbance at a wavelength of 340 nm (A, 4°), 20 all of protein calibrator [containing 18 μl/l IgG] (manufactured by Midori Juji Co., Ltd.) was further added.
Warm at 7°C for 15 minutes. A 34° was measured, and the previously measured value of A34° was subtracted to obtain the true A 34° to prepare a calibration curve of protein A concentration-A, 4°.

A 0.5 μ/cal aqueous solution of protein 15 purified in Example 26 was prepared, and the reaction was carried out in exactly the same manner except that this was added instead of the protein A aqueous solution, A 34° was measured, and its value was determined from the calibration curve. When the protein A concentration corresponding to the aggregation activity was determined, it was found to be 0.58 .mu./lag, which confirmed that the IgG aggregation activity was almost equivalent to that of protein A.

Example 28 Preparation of plasmid pPrAS 2 177 g of niblasmid pPr^2 was digested with restriction enzymes pstl and old ndI[I under the same conditions as in Example 5, and then fractionated by agarose gel electrophoresis to obtain approximately 2.1 kb DNA32.
I got it. DNAs 23 and 32 obtained by the method of Example 5 were diluted with 0.
03 pwole were mixed, dissolved in 30 μl of T4 DNA ligase reaction solution, added 1 unit of T4 DNA ligase, and allowed to undergo a binding reaction at 4°C for 18 hours. With this reaction solution, E. coli 8
8101 strain was transformed, Ap' colonies were obtained, and plasmid DNA was recovered to obtain pPrAS2.

Example 29 Preparation of plasmid pPrAW 2: DNA32 obtained by the method of Example 28 and D obtained by the method of Example 7
Mix 0.03 plole of NA25 and add T4DN
Dissolve in 30 μl of reaction solution for A ligase, add 1 unit of T4 DNA ligase, perform a binding reaction at 4°C for 18 hours, transform E. coli strain 88101 with this reaction solution, obtain Apr colonies, collect plasmid DNA, and transform pPrAW. 2
I got it.

Example 30 Production of protein 13 by E. coli using plasmid pPrAS 2: Production of protein 13 by E. coli using pPrAS 2 obtained in Example 28
31105trA strain was transformed. obtained/l
The p' colony was cultured using the same culture method as in Example 16, and the culture solution was centrifuged at 7.00 rpm for 5 minutes to collect bacterial cells. After dissolving this bacterial cell in the sample buffer of Laemmle et al.'s method and heating, 5DS-polyacrylamide gel electrophoresis was performed according to the same method, and as a result of staining with Coomassie brilliant blue, a polypeptide band was found at a site with a molecular weight of approximately 14,000. was detected.

E. coli W3110 5trA without pPrAS 2
Since no corresponding band was present in the strain, pPrA
E. coli-31105trA strain carrying S2 has protein 1
It has been revealed that they are producing 3.

Example 31 Production of protein 13 by E. coli using plasmid pPrAW 2: Production of protein 13 by E. coli using pPr/V 2 obtained in Example 29
311O5trA strain was transformed. The obtained Apr colony was cultured using the same culture method as in Example 16, and the culture solution was centrifuged at 7.00 rpm for 5 minutes to collect bacterial cells.

After dissolving this bacterial cell in the sample buffer of Laemmle et al.'s method and heating, 5DS-polyacrylamide gel electrophoresis was performed according to the same method, and as a result of staining with Coomassie brilliant blue, a polypeptide band was found at a site with a molecular weight of approximately 14,000. was detected.

There was no corresponding band for E. coli-3110str eight strains that do not contain pPrlV2, so pPrAW
E. coli-31105trA strain harboring protein 13
It was revealed that they were producing.

Example 32 Production and purification of protein 13 by E. coli using plasmid pPrlV 2: pPrlV 2 obtained in Example 29 was used to transform E. coli W3110 str^ strain. 8 ml of the obtained 4pr colony! inoculated into LG medium of 3
Cultured at 0°C for 16 hours. 2 lll of this culture solution to 1
The cells were inoculated into a medium containing 001Il MCG medium supplemented with 50°C g/111 tryptophan and 50°C g/lag ampicillin, and cultured at 30°C. Turbidity of culture solution (OD
ss*) and when it reached 0.9 (about 4 hours later)
Then, 2 μm of indole acetic acid was added, and the culture was continued for an additional 4 hours. The culture solution was centrifuged at 7000 rpm for 5 minutes to collect bacterial cells. After washing this bacterial body with PBS, 25 mj
l! The cells were suspended in PBS and disrupted by ultrasonication (10 minutes), centrifuged at 7000 rpm for 1 hour to collect the supernatant, ammonium sulfate was added to the supernatant to a saturation concentration of 60%, and the cells were incubated at O''C for 1 hour. 700 again after leaving it for an hour.
Centrifugation was performed at 0 rpm for 1 hour. Ammonium sulfate was further added to this supernatant to reach a saturation concentration of 80%, and after standing at 0° C. for 1 hour, centrifugation was performed at 7000 rpm for 1 hour to obtain protein 13 as a precipitate. This protein 13 was dissolved in distilled water 2-2 and subjected to Sephadex G-50 column chromatography (2.4 x 84 cm).
0a+M) Squirrel-HCj! (pH 7,5),
The eluate was fractionated into approximately 4 tan fractions.
Protein 46 was concentrated to obtain a purified product of protein 13 weighing about 3 ml.

Example 33 Ig of protein 13 produced by E. coli
G aggregation activity Ig of purified protein 13 obtained in Example 32
G aggregation activity was measured as follows.

Activity measurement reaction solution (20+M H1l! PP5O (pH
8), 100mW KCl1.6% Pluroni
c P6B (manufactured by Asahi Denka)] 2.5mj! Protein A (manufactured by Replisien) aqueous solution (concentration 0.0.05
．． 0.1.0.2.0.5.0.8゜1,0■/-2)
20a1. and warmed at 37°C for 3 minutes. After measuring the absorbance (A, 4°) at a wavelength of 340 n-, 20 μl of protein calibrator (containing 1B/wail IgG) (manufactured by Midori Juji Co., Ltd.) was further added, and 37
Warm at °C for 15 minutes. A34° was measured, and the previously measured A3. . Subtract the value of true A 24°,
A calibration curve of protein A concentration-A at 4° was prepared. A 0.1 μ/1all aqueous solution of protein 13 purified in Example 30 was prepared, and the reaction was carried out in exactly the same manner except that this was added instead of the protein A aqueous solution, A, 4° was measured, and from the calibration curve When the protein A concentration corresponding to the aggregation activity was determined, it was found to be 0.21/-2, confirming that the IgG aggregation activity was almost equivalent to that of protein A.

Reference Example 1゜1) Construction of ATG vector pTrs20 (Figure 13): According to the procedure shown in Figure 13, the distance between the SD sequence and the ATG start codon is 14 bases, and the 5acl site is placed immediately after the ATG codon. ATG vector p T r with
Created S20.

First, pKYPlo 3, which was prepared by the method described in JP-A-58-110600, was dissolved in 30 μg of Y-100 buffer, and 6 units each of restriction enzyme Ban■ and restriction enzyme Nrul (manufactured by New England Biolabs) were added. In addition, a cleavage reaction was performed at 37°C for 3 hours. Approximately 3.8 K containing Ptrp was extracted from this reaction solution by the LGT method.
DNA fragment b (Bann[-Nru! fragment) approximately 0.5
I got a moth.

On the other hand, in order to provide an ATG initiation codon downstream of Ptrp, the following DNA linker was synthesized by the phosphotriester method.

Synthetic NA of 19-+++er and 17-aver (10 pmole each) was added to 50 mM) Lis-HC1.
(pH 7,5), 10mM MgC1z, 5mM
Dithiothreitol, 0.1mM EDTA and 1
Dissolve in a total volume of 20 liters of solution containing mM ATP and add T4
Three units of polynucleotide kinase (Takara Shuzo Co., Ltd. 1) were added, and a phosphorylation reaction was carried out at 37°C for 60 minutes.

Next, BannI-Nr derived from pKYP10 obtained above
ul fragment (approximately 3.8 Kb) and approximately 5 pmole of the above DNA linker in T4 ligase buffer for 20 min.
2 units of 7'4 DNA ligase were added, and the binding reaction was carried out at 4°C for 18 hours.

Using the obtained mixture of recombinant plasmids, E. coli H.
Strain 8101 [Bolivar et al.; Gene (
Gene) 2, 75 (1977)) was transformed to obtain Ap' colonies. Plasmid DNA was recovered from the culture medium II cells of this colony. Obtained -
The structure of the plasmid was prepared using restriction enzymes EcoRI, Band,
After cleavage with HindIII, 5acI, and Nrul, it was confirmed by agarose gel electrophoresis.

This plasmid was named pTr820.

The nucleotide sequence near the Banm and Hindm sites of pTrS20 was confirmed as shown below using the dideoxy-seefence method using M13 phage.

2) Production of DNA3-6.13 and 14: DNA3-6.
6.Synthesis of 13 and 14 was carried out using a solid-phase synthesis method using the phosphoamidide method (S, L. Beaucage et al., Tetrahedron Letters).
) 22.1859 (1981), L, J.
McBrie et al., 24.245 (1983)), using an automated DNA synthesizer 380A manufactured by Applied Biosystems, as described below.

Using silica gel as a solid support, 1) nucleotide is condensed with the 5' hydroxyl group of the nucleotide bound via the 3' hydroxyl group by the phosphate amidite method, and (2) the phosphorous bond of the condensed nucleotide is oxidized with iodine. (3) The protecting group on the 5' hydroxyl group of the condensed nucleotide was removed with trifluoroacetic acid. Next, returning to step (1), the next nucleotide was similarly condensed. thus(
Steps 1) to (3) are repeated and DNA is synthesized on the carrier. After the synthesis is complete, the carrier with the DNA bound is left in a thiophenol solution at room temperature for 1 hour to remove the phosphoric acid protecting group. After that, the DNA was released from the carrier by standing in concentrated ammonia water at room temperature for 1 hour. Concentrated ammonia water containing DNA was heated in a sealed container at 60° C. for 12 hours to remove the protecting groups on the base.

Taking DNA3 as an example, synthesis was performed using a carrier bound with 1 μH of nucleotides, and the condensation reaction was completed with a total yield of 81%. Next, the protecting group was removed and the release reaction from the solid phase was performed to obtain the crude DNA3. Product 2420.0. Unit (260n
s) was obtained. 220.0 of this crude product. 10% polyacrylamide gel (2+u+thickness, 13 cm) using Tris-borate buffer (pH 8) containing 7M urea
The area of the gel containing DNA3 was taken, and the DNA3 was extracted with 0.2M triethylamine carbonate buffer (pH 8) (hereinafter referred to as TEAB) 1-1 for 18 hours, and then the extraction Transfer the solution to a Sephadex DE52 column (diameter 6111% length 5s+a+
) to adsorb DNA 3. 2M TEA8
Elution with 2 ml was 3.90. D. A pure product of 3 units of DNA was obtained.

DNAs other than DNA3 were also synthesized with similar yields.

The 5′ hydroxyl group of these DNAs was isolated using a conventional method using phage T4 polynucleotide kinase and (γ-”P)ATP (A,
M. Maxam et al., “Methods in Enzymology”
) Vol, 65. Part I, p, 499.
Academic Press (1980)) and phosphorylated it with a radioactive label. labeled DNA
The DNA was subjected to 20% polyacrylamide gel electrophoresis in a Tris-borate buffer containing 7M urea to confirm the purity and chain length of the DNA. Furthermore, the base sequence of the labeled DNA was determined by Maxaae-Gilbert.
) method (mentioned above), and it was confirmed that each DNA had the desired base sequence.

3) Preparation of plasmid pMTA1 (Figure 14) Niblasmid pTr320 2n was mixed with a solution containing 10 units of restriction enzyme C1al (manufactured by Boehringer Mannheim) and 15 units of 5acI (manufactured by Takara Shuzo Co., Ltd.) [10 h Tris-HCl]
(pH 7,5), 7mM MgC1z, 6+M2
-mercaptoethanol] and a digestion reaction was performed at 37°C for 2 hours. This reaction solution was subjected to agarose gel electrophoresis containing ethidium bromide, and ultraviolet rays (wavelength 3
02rv+) and cut out a gel piece containing approximately 3.8 Kb DNA7. Phenol 0.5m on gel piece
After freezing and thawing the aqueous layer and washing the aqueous layer with chloroform,
DNA7 was recovered by ethanol precipitation.

Add 10 pmole each of DNA 3 to 6 to T44 polynucleotide kinase application buffer (50+mM)
(pH 7,5), 10mM MgClz 1
5mM dithiothreitol (hereinafter abbreviated as DTT)
, 1mM ATP, 0.1+M spermidine, 0,1
nM EDTA), dissolved in 30 fractions, added 3 units of T4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.), and incubated at 37°C.
After carrying out the phosphorylation reaction for 0 minutes, the enzyme was inactivated by heating at 65° C. for 15 minutes.

This reaction solution was mixed with 4 IttI each, and the DNA obtained above was
Add 70,08 pmole, 28mM) Lis-H
Cl (pH 7,5), 9mM Mg Clz
, 10d DTT, 0.03n+M EDTA,
The composition was adjusted to 0.7a+MATP and 0.03a+M spermidine, and 2 units of T 4 DNA IJ gauze (manufactured by Takara Shuzo Co., Ltd.) was added to make 50 IItl. The binding reaction was carried out at 4°C for 16 hours.

Using this reaction solution, E. coli) (8101 strain [Polyvar (
Bolivar et al; Gene 2.
75 (1977)) to the method of Cohen et al.
Sci, ), LISA 69+2110 (197
2) Apr colonies were obtained by transformation. From this colony, the alkali treatment method [Mania Nice (
Maniatis et al., eds.; Molecular Cloning, P, 36
8゜Cold Spring Harbor
Plasmid DNA
was collected to obtain pMTAl. The structure of pMTAl is B
It was cleaved with glII, Pstl, 5acl, and BamHI and confirmed by agarose gel electrophoresis. Restriction enzyme cleavage reaction was carried out using 10 wM ToIJ-HCj (pH 7.5), 7
A solution with the composition of mM MgC1z, 6n+M 2-mercaptoethanol was added to 0 to give the optimal concentration of each enzyme.
~200mM NaCl or KCI was added to prepare a reaction solution (reaction solution for restriction enzymes, hereinafter only the concentration of NaCl or KCI is shown).

Furthermore, the literature (A, J, H, Sm1th, “Methods in Enzymology”)
zyIlology”) ed, by L, Gro
sswen and, Mo1dave Vol,
65+p, 560 (1980), Academy
A sequence of 115 bases including portions derived from DNAs 3 to 6 was determined by the method described in J.C.Press), and the desired "Leu-motilin gene" was confirmed.

4) Preparation of plasmid pMTA2 (Fig. 15): Dissolve 0.2 mm of plasmid pMTA1 obtained in the previous section in the restriction enzyme reaction solution (100 mM NaCZ) shown in Example 2, and add Pstl (manufactured by Takara Shuzo Co., Ltd.) 6 6 units of BglI [(Toyo Jozo Co., Ltd. 11) were added, and the digestion reaction was carried out at 37°C for 2 hours. This was fractionated by agarose gel electrophoresis similar to Example 2, and a DNA fragment of approximately 2.8 Kb (DNA8
) was obtained.

Separately, pMTAl 0.2n restriction enzyme reaction solution (100
aeM KCZ) 15 units, Pst 16 units,
Six units of BamHI (manufactured by Takara Shuzo Co., Ltd.) were added and a digestion reaction was carried out at 37°C for 2 hours. This was fractionated by agarose gel electrophoresis and a DNA fragment of approximately 1.2 Kb (DNA9) was obtained.
I got it.

50 ng each of DNA8 and DNA9 thus obtained were mixed, and a reaction solution for T4 DNA ligase [20 IIM Tris-HCl (pH 7,5), 10 N HMgC1z
, 10mM DTT-0, 3mM ATP) 30M
1 unit of T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.) was added, and a binding reaction was performed at 4°C for 18 hours. E. coli HB I O1 strain was transformed using this reaction solution to obtain A pr colonies. Plasmid DNA was recovered from this colony to obtain pMTA2. The structure of this plasmid is
It was cleaved with Ps t I, BamHI, and Bgln, and confirmed by agarose gel electrophoresis.

5) Preparation of plasmid pMTA4 (Figure 16): Plasmid pMTA2 0.2x obtained in the previous section was mixed with the restriction enzyme reaction solution (IQOmM NaC) shown in section 3 in the same manner as in the previous section.
1) Dissolved in 15I11, Pst16 units, BgllI
6 units were added, the mixture was digested at 37°C for 2 hours, and fractionated by agarose gel electrophoresis to obtain a DNA fragment (approximately 2.8 Kbc). In addition, pMTA20.2n was added to the restriction enzyme reaction solution (100I
IIM KCZ) was dissolved in 15 ml of water, added with 16 units of Pst and 6 units of BamHI, digested at 37°C for 2 hours, and fractionated by agarose gel electrophoresis to yield approximately 1.3 Kb.
A DNA fragment was obtained. Each of these DNA fragments is 0.2
Mix pmole by pmole, dissolve in 30 ml of T4 DNA ligase reaction solution, add 1 unit of T4 DNA ligase, and add 4 pmole of T4 DNA ligase.
The binding reaction was carried out for 18 hours at °C. With this reaction solution, E. coli H81
01 strain was transformed, Apr colonies were obtained, and plasmid DNA was collected to obtain pMTA4. The structure of this plasmid was confirmed by cutting with Pstl and BamHISBglII and agarose gel electrophoresis.

6) Preparation of plasmid pMTL4 (Fig. 17) Niblasmid psGHIM11n was dissolved in 30% of restriction enzyme reaction solution (100+mM KCZ), and Ps
Add 6 units of tI and 16 units of BamH, perform a digestion reaction at 37°C for 2 hours, and analyze with agarose gel electrophoresis. IK
The DNAlr fragment of b was fractionated and collected.

Plasmid pGHD7 1x was digested with psGF (Pstl as well as IMl, and BamHI), and a DNA fragment of approximately 1.7 Kb was recovered.

These DNA fragments were mixed in an amount of 0.03 pmole and dissolved in 30 ml of T4 DNA ligase reaction solution.
Add 1 unit of NA ligase and heat at 4°C.

The binding reaction was carried out for 18 hours, and this reaction solution was used to bind Escherichia coli) IB 1.
01 strain was transformed to obtain A pr colonies, and plasmid DNA was collected to obtain psGHIMEI.

The thus obtained plasmid psGHIME10.5N was dissolved in 30% of restriction enzyme reaction solution (NaCj, KCl not added), 50 units of 5acl (manufactured by Takara Shuzo Co., Ltd.) was added,
Digestion reaction was carried out at 7°C for 2 hours. Next, 2 rinses of 2M NaCl were added, 15 units of Bgln were added, and the digestion reaction was continued at 37°C for 2 hours. This was fractionated by agarose gel electrophoresis, and a DNA fragment of approximately 3.2 Kb was recovered.

pMTA4 0.54 obtained in Section 5 was added to psGH [MEI and total (same as Bgl
Approximately 0.3 Kbf7) after digestion and agarose gel electrophoresis
Collect DNA fragments.

These DNA fragments were mixed in an amount of 0.03 pmole and dissolved in 30It1 of reaction solution for T4 DNA ligase.
One unit of 4 DNA ligase was added and incubated at 4°C for 15 hours. E. coli strain H8101 was transformed with this reaction mixture to obtain colonies of A11) P, and plasmid DNA was recovered to obtain pMTL4.

7) Creation of plasmid pMTN4 1 (Figure 18): 1.5 liters of plasmid pMTL4 obtained in Section 6 was added to restriction enzyme reaction solution (100sM NaCZ) 20/4! dissolved in
Pstl B units, BglI [7 units added, 37°C
The mixture was digested for 2 hours and fractionated by agarose gel electrophoresis to recover a DNA fragment of about 2.2 Kb (DNA15). In addition, pMTL4 was added to the restriction enzyme reaction solution (100
mMNaCj)20. Dissolve in ccj2, add 8 units of PstI and 6 units of HindI [[(manufactured by Takara Shuzo) to make 37
Digestion reaction was carried out at ℃ for 2 hours, fractionated by agarose gel electrophoresis, and approximately 1. A DNA fragment (DNA16) of OKb was recovered.

21 parole each of DNA15 and DNA16 as the third
It was dissolved in the T4 polynucleotide kinase reaction buffer shown in section 50, added with 4 units of T4 polynucleotide kinase, and carried out a phosphorylation reaction at 37°C for 40 minutes.
The enzyme was inactivated by heating at ℃ for 15 minutes. This reaction solution 2
Mix each part, add DNA 15 and 16, and add 28-
M)'JX-HCI (pH 7,5), 9mM
MgCl, 10mM DTT, 0.03mM E
The composition was adjusted to include DTA, 0.7mM ATP, and 0.03mM spermidine, and 2 units of T4 DNA ligase was added to make 40 precepts. The binding reaction was carried out at 4°C for 16 hours. E. coli strain 88101 was transformed using this reaction solution to obtain Apr colonies, plasmid DNA was collected, and p
Obtained MTN4.

8) 7' Rasumi F p M T O4 (by D!!
(Figure 19) Niblasmid pMTN4 2.5n was added to Tr
Reaction solution for restriction enzyme CNa containing i tonXO001%
C1゜KCI not added) dissolved in 40, 5ac11
Two units were added and the digestion reaction was carried out at 37°C for 2 hours. After extracting this reaction solution with phenol-chloroform, pMTN4, which had been cleaved by 5 acl, was recovered by ethanol precipitation, and 20 mM
) Lis-HCI (pH 7,8), 7mM M
g C7z, 6IIM 2-mercaptoethanol, d
NTP (dATP.

dTTP, dCTP, dGTP) were dissolved in solution 40 with a composition of 0.25 mM each, and E. coli DNA polymerase I
Four units of Klenow fragment (manufactured by Takara Shuzo Co., Ltd.) were added and the reaction was carried out at 20°C for 1 hour.

This reaction solution was subjected to phenol-chloroform extraction f& and ethanol precipitation to recover the pMTN 4 fragment treated with polymerase. Add this to the restriction enzyme reaction solution (100a+M
Dissolve 30 units of NaCj), add 15 units of Pst, and add 2
A time digestion reaction was performed and fractionation was performed by agarose gel electrophoresis to obtain a DNA fragment of about 1.3 Kb (DNA20).

2 g of plasmid pArgE1 was added to restriction enzyme reaction solution (1
50d Dissolved in NaCl>20I11, Pstll
O units and 10 units of EcoRV (manufactured by Takara Shuzo Co., Ltd.) are added to make 3
Digestion reaction was carried out for 2 hours at 7°C, and a DNA fragment of approximately 1.7 Kb (DNA19) was fractionated by agarose gel electrophoresis.
I got it.

0.06 pmole each of this DNA19 and DNA20
Mix and dissolve in T4 DNA ligase reaction solution 25,
Three units of T4 DNA ligase were added and a binding reaction was carried out at 4°C for 20 hours. E. coli strain 88101 was transformed using this reaction solution to obtain Apr colonies. Plasmid DNA was recovered from this colony to obtain pMTO4.

The structure of pFITO4 is Ps t I, ECoRI%Hi
This was confirmed by cutting with nd11[, 5all, Bgl■, and Mlul.

9) Preparation of plasmid pMTOI4 (Figure 20) Niblasmid pMT04 2n was added to restriction enzyme reaction solution (100mM
Dissolved in NaCj) for 30 hours, added Bindl [I66 units, Pst16 units, and allowed to undergo a digestion reaction at 37°C for 2 hours.
Approximately 3.0% was fractionated by agarose gel electrophoresis and contained the '3L eu-motilin polymer gene. A DNA fragment (DNA21) of OK b was recovered. In addition, plasmid pKY
P10 (Japanese Unexamined Patent Publication No. 58-110600) was dissolved in restriction enzyme reaction solution (100sM NaCj) 30P1, 9 units of Hind and 19 units of Pst were added, and 37
Digestion reaction was carried out for 2 hours at ℃, fractionated by agarose gel electrophoresis, and approximately 1. IK b DNA
A fragment (DNA22) was recovered. Approximately 0.1 n of these DNA fragments were dissolved in a reaction solution for T4 DNA ligase at 30°C, and 1 unit of T4 DNA ligase was added thereto, followed by a ligation reaction at 4°C for 18 hours.

This reaction solution was used to transform E. coli strain 88101, and plasmid DNA was recovered from the resulting Apr colonies to obtain the "Leu-motilin polymer expression plasmid pMTOI4, which has a tryptophan promoter. pM
The structure of TOI4 is PstI, BanI[[, Hin
It was confirmed by cutting with dIII and MluI.

10) Creation of plasmid pMTOII4! l (21st
Figure) Niblasmid pMT04 2n was added to the restriction enzyme reaction solution (
100mM NaCf)30. Dissolved in ccl, Hi
Add 6 units of ndnI and 16 units of Pst at 37°C.
Digestion reaction was allowed for 2 hours. It was fractionated by agarose gel electrophoresis, and approximately 3 cells containing the '3L eu-motilin polymer gene were
．． A DNA fragment (DNA21) of OK b was recovered.

In addition, plasmid pGEL1 (JP-A-6L-93197
)3n into restriction enzyme reaction solution (l OO+M NaC1)
Dissolved in the 30 precepts, H4ndll [9 units and Pst
19 units were added and the digestion reaction was carried out at 37°C for 2 hours. Fractionated by agarose gel electrophoresis, approximately 1 containing the promoter
．． A DNA fragment (DNA23) of IKb was recovered.

Approximately 0.1 n of these DNA fragments were dissolved in T4 DNA ligase reaction solution 30, 1 unit of T4 DNA ligase was added, and a ligation reaction was carried out at 4°C for 18 hours. E. coli strain H8101 was transformed with this reaction solution, and the resulting A p
Plasmid DNA was collected from the 1' colony to obtain a ``Leu-motilin polymer expression plasmid pMTOII4 having a 2-linked tryptophan promoter and a distance of 14 bases between the SD sequence and the ATG start codon.

The structure of pMTOII4 is PstI, BanI[, Hi
It was confirmed by cutting with ndI[I and Mlul.

11) Construction of plasmid pMTOIII4 (Figure 22)
2g of Niblasmid pMT04 was added to restriction enzyme reaction solution (lQ
Dissolved in OmM NaC7)30I11 and diluted with HindI
6 units of II and 16 units of Pst were added, and a digestion reaction was carried out at 37°C for 2 hours. Fractionated by agarose gel electrophoresis,
About 3. containing the Leu-motilin polymer gene. OK b
DNA fragments were recovered. In addition, plasmid pGHA2
(JP-A-660-221091) 3 was dissolved in restriction enzyme reaction solution (100aM NaCl) 30/4I, and Hi
Add 19 units of ndI and 19 units of Pst at 37°C.
Digestion reaction was carried out for 2 hours. The DNA fragment was fractionated by agarose gel electrophoresis and a DNA fragment of approximately 0.9 Kb containing the promoter was recovered. Approximately 0.1~ each of these DNA fragments are
Dissolve T4DNA in 30 liquids of reaction solution for 4DNA ligase.
One unit of ligase was added and the binding reaction was carried out at 4°C for 18 hours.

E. coli strain H8101 was transformed with this reaction solution, and plasmid DNA was collected from the resulting Apr colonies.
A "Leu-motilin polymer expression plasmid pMTOm4 having the let promoter was obtained.

The structure of pM1'om4 is Ps t l5BanI[[
, HindI[1, Mlul, and BglI]. (See Figure 11) Reference Example 2゜1) Preparation of poly (A) RNA from eel pituitary gland: Poly (A) RNA was prepared from eel pituitary gland using the guanidine thiocyanate-lithium chloride method [Cathala et al., DNA DNA), 2.329 (1983))
RNA having poly(A) was prepared as follows.

0.5 g of frozen eel pituitary gland (approximately 1,000 individuals) was mixed with 5M guanidine thiocyanate, 10mM EDTA, and 50%
mM Tris-HCf (pH 7) and 8% (V/V
) A solution consisting of β-mercaptoethanol
The mixture was crushed and solubilized using a Teflon homogenizer (5 rpm). Transfer this lysate to a centrifuge tube and
After adding 70ml+1 of the solution and stirring, the mixture was allowed to stand at 4°C for 20 hours.

9.00 with Hitachi RP R10 rotor
Orpm.

After centrifugation for 90 minutes, RNA was collected as a precipitate.

The RNA precipitate was suspended in 100 ml of a solution consisting of 4 M urea and 2 M lithium chloride, and
After centrifugation for 60 minutes at 9.00 Orpm in a C)-tar, RNA was recovered as a precipitate.

Precipitate the RNA with 0.1% sodium lauryl sulfate, 1 m
M EDTA, 10sM) Lis-HCj (pH 7
, 5), extracted with phenol-chloroform, and recovered by ethanol precipitation.

Approximately 1 μm of the obtained RNA was added to 10 mM Tris-HCl (
Solution 1 consisting of pH 8,0) and 1mM EDTA
Dissolved in a+1. Incubate at 65°C for 5 minutes,
0.11 of 5M N'aCl was added. The mixture was subjected to oligo(dT) cellulose column chromatography (manufactured by PL Biochemical) (column volume 0.5 ml).

mRNA with adsorbed poly(A) was treated with lQmM Tris-HCI (pH 7,5) and lnIME.
Elution was performed with a solution consisting of DTA to obtain about 7n of mRNA containing poly(A).

2) cDNA synthesis and insertion of the DNA into a vector: Okayams-Berg method [Molecular and Cellular Biology (Mo.
1. Ce11. Biol, ), 2.161 (19
82)), cDNA was synthesized and a recombinant plasmid incorporating it was constructed. The outline of the process is shown in FIG.

pcDVl (Okaya?-and Barb)
o+a & Berg): Molecular and Cellular Biology (Mo1. Ce11. Biol
), 3, 280 (1983) ) 400
n 10mM) Lis-HCff1 (pH 7,5),
6 mM MgCl2 and lOmMNac
In addition to 300 μf of a solution consisting of 1, 500 units of Kpnl (manufactured by Takara Shuzo Co., Ltd.; unless otherwise specified, all restriction enzymes are manufactured by Takara Shuzo Co., Ltd.) were added, and the mixture was reacted at 37°C for 6 hours to cleave at the KpnI site in the plasmid. . After phenol-chloroform extraction, DNA was recovered by ethanol precipitation. Approximately 200 μg of the Kpnl-cleaved DNA was
0mM sodium cacodylate, 30mM) Lis-HCl
(pH 6,8), 1mM CaC1z and 0.1m
Add to a solution 200P1 in which dTTP was added to a buffer solution (hereinafter abbreviated as TdT buffer) consisting of M dithiothreitol (hereinafter abbreviated as DTT) to a concentration of 0.25 mM,
In addition, 81 units of terminal deoxynucleotidyl transferase (hereinafter abbreviated as TdT) (P
-L Biochemicals), 37
℃ for 11 minutes. Here, Kp of p CDVl
Approximately 67 poly(dT) chains were added to the 3' end of the nl cleavage site. Phenol-chloroform extraction from the solution,
By ethanol precipitation, p with added po17(dT) chain was
Approximately 100n of CDVI DNA was recovered. 1 of the DNA
0mM) Lis-HCl (pH 7,5), 6mM
MgCj,.

In addition to 150/J of a buffer consisting of 100 mM NaCl, 360 units of EcoRI were added, and the mixture was reacted at 37°C for 2 hours. After treating the reactant with the LGT method, about 3.
The I K b DNA fragment was recovered and a poly(d
T) Chain-added pCDV1 was obtained. The DNA was diluted with 10mM) Lis-MCI (pH 8,0) and 1mM EDTA.
After incubation at 65°C for 5 minutes, it was cooled with water and dissolved in 50ItII of 5M NaC.
Added f. The mixture was subjected to oligo(dA) cellulose column chromatography (manufactured by Collaborative Research). Poly (dT) with a sufficient length of 1 m is adsorbed onto the column, and this is added to 10 mM Tris-HCl (pH 8.0).
and eluted with a solution consisting of 1mM EDTA to obtain 27#g of pcDVl (hereinafter abbreviated as vector primer) to which a poly(dT) chain was added.

Next, linker DNA was prepared.

pLl (Okayama and Barb (Okayan+
a & Berg): Molecular and Cellular Biology (Mo1. Ce11. Biol,)
, 3, 280 (1983)) approximately 14IlfI to 1
0mM) Lis-HC1 (pH 7,5), 6 m
Buffer 200II consisting of M
p L I DNA and reacted for 4 hours at 37°C.
It was cut at the Pst1 site inside. After the reaction product was extracted with phenol-chloroform, ethanol precipitation was performed, and Pst
Approximately 13 g of pLI DNA cut with l was recovered.

Approximately 13x of the DNA was added to TdT buffer at a final concentration of 0.25mM.
In addition to 50It1 of a solution containing dGTP of T d
T (manufactured by P-L Biochea+1cals) 5
Add 4 units and incubate at 37°C for 13 minutes, pL
Approximately 14 oligo(dG) chains G were added to the 3' end of the PstI cleavage site of 1. After phenol-chloroform extraction, DNA was recovered by ethanol precipitation.

The DNA was mixed with 10mM 1-Lis-HC1 (pH 7.5),
6 m M M g Cj! , and 60mM
In addition to 100 fi of a buffer consisting of NaCl, an additional 80
One unit of BindnI was added, incubated at 37°C for 3 hours, and the pLI DNA was cleaved at the Hindm site. The reaction product was fractionated by agarose gel electrophoresis, and DNA fragments of approximately 0.5 Kb were separated using the DEAE paper method [Oretzen et al., Analytical Biochemistry (Anal, Biochem,
2.295 (1981)), linker DNA with oligo (dG) chains (hereinafter simply referred to as linker DNA)
(abbreviated as DNA) was obtained.

Approximately 2IN of poly(A) RNA prepared above and approximately 1.4N of vector primer were added to 50mM) Lis-HCII (p
H8,3), 8mM MgCfz, 30mM
KCI! , 0.3 m M D T T ,
2 m MdNTP (dATP, dTTP, dG
TP and dCTP) and 10 units of glue bonuclease inhibitor (P-L Biochea+1ca
ls company! 1), dissolved in 22.37J of a solution consisting of 10
Add unit of reverse transcriptase (Seikagaku Kogyo Co., Ltd.) and heat at 37°C.
Incubate for 0 min, and incubate complementary to mRNA A.
A was synthesized. The reaction product was subjected to phenol-chloroform extraction and ethanol precipitation to recover vector primer DNA to which RNA-DNA duplex material had been added. The DN
A was dissolved in 20 μl of TdT buffer containing 66 μM dCTP and 0.2 poly(A), and 14 units of TdTr
(Manufactured by P-L Bioche+5icals)
and incubate for 8 minutes at 37°C.
Twelve (d C) chains were added to the end. The reaction product was extracted with phenol-chloroform, and the cDNA-vector primer DNA with the (dC) strand added was recovered by ethanol precipitation. 1tDNA (10mM) Lis-HC
1 (pH 7,5), 6mM Mg (1, and 60mM
It was dissolved in a solution consisting of NaCj2 400ml, added with 20 units of Hindllr, incubated at 37°C for 2 hours, and cleaved at the Hind■ site. The reaction product was extracted with phenol-chloroform and precipitated with ethanol to give 0.5 p.
A mole (dC)-adducted cDNA-vector primer DNA was obtained. t3DNAO, o 8 pmol
e and 0.16 pmole of the above linker DNA at 10 mM. J Su-HC1 (pH 7,5), 0.
Dissolved in 40d of solution consisting of I M Na C1 and 1mM EDTA, + at 65°C, 42°C, and 0°C.
Incubate for 10 minutes, 25 minutes, and 30 minutes, respectively.

20mM) Lis-HCl (pH 7,5), 4mM
g C1t+ l OmM (NH4)zS 0
A reaction solution was prepared with a composition of 4,0.1M KCl and 0.1rnMβ-NAD to a total volume of 400. IO units of E. coli DNA ligase (NesvE) were added to the reaction solution.
Add ngland 8io1abs M) and heat to 11℃-
I inked it at night. The reaction solution was added with 40 μM of dNT.
P, 0.15mM β-NAD, 5 units of E. coli DNA ligase, 7 units of E. coli DNA polymerase I (P-L Biochemical
als) and 2 units of E. coli ribonuclease H.
(manufactured by P-L Biochemicals) and 1
The cells were incubated sequentially at 2°C and 25°C for 1 hour each. In the above reaction, circularization of recombinant DNA including cDNA and R
NA-DNA double double material NA part is replaced with DNA,
A complete double-stranded DNA recombinant plasmid was generated.

3) Selection of recombinant DNA containing eel growth hormone cDNA: Using the recombinant plasmid obtained in the previous section, Escherichia coli c600s
F8 strain [Cataeron: Proceedings of the National Academy of Sciences (Proc, Natl, Acad.

Sci.) USA, 72.3416 (1975)
) 5cott et al.'s method C. Katsuya Shigesada: II Cell Engineering, 2
．． 616 (1983)). Approximately 2,400 colonies obtained were immobilized on nitrocellulose. 27th position from the N-terminus of eel growth hormone
The synthetic NA corresponding to the 32nd azino acid sequence is 3'5' (T) (G) (C) (C) (G) (G
) (3rd base is A, T, G1 6th base is A or G1 9th base is T or C1 12th base is T or C5 15th base is A or G, We selected three strains that strongly associated with the 0P-labeled probe at 38°C [Grunstein Hognes
Proceedings of the National Academy of Sciences (Proc, Natl., Acad, Sci.)
USA, 72.3961 (1975)), and Southern's method [Journal of Molecular Biology (J, Mol.

Biol,) Nira, 503. (1975)), the above probe and a synthetic NA probe corresponding to the amino acid sequence near the C-terminus, namely 5'3' (C) (G) (T) (G) (G) (C) (3rd The base is T or C1, the 6th base is A or G1, the 9th base is any of A, T, G, or C, and the 12th base is A or G, making a mixture of 32 synthetic NAs in combination. ) A meeting was confirmed with both parties. These plasmids are pEGH
They were named 8, 9, and 15, and because they all had DNA sequences predicted from the amino acid sequence of eel growth hormone, they were considered to contain eel growth hormone cDNA.

4) Base sequence of plasmid pEGH15 The three plasmids obtained above were digested with various restriction enzymes, c
A cleavage map of the DNA portion was determined. Plasmid pEGH
The cDNA 11 restriction enzyme map in No. 15 is shown in FIG.

Next, pE that shows strong association with the synthetic NA probe performed in Section 3 and is thought to contain almost full-length cDNA.
The entire nucleotide sequence of the translated region of GH15 was determined using the Sanger method using M13 phage [
Sanger et al.: Proceedings of the National Academy of Sciences (P.
roc, Natl.

Acad, Sci,), USA, 74.5463
(1977) Asersham
) M13 Cloning and Sea Fencing
handbook (cloning and sequence)
ing handbook)).

The nucleotide sequence is shown in formula 18. In formula 18, the number of bases is 1
-57 encodes the signal peptide, and 58-627 encodes the mature peptide of eel growth hormone. pEGH15
The amino acid sequence predicted from the cDNA sequence contained in It was confirmed that there is.

Colonic seedlings containing formula 18 pEGH15 are Escherichia
colt EEG) 115 (FERM BP
-824) and was deposited with the Institute of Fine Technology on July 2, 1985.

5) Construction of recombinant plasmid pUPA1 encoding eel growth hormone (GH-1): Plasmid pEGH153n containing DNA encoding eel growth hormone
Dissolved in 30 liters of Y-100 buffer, restriction enzyme B, an
I [(manufactured by Toyobo Co., Ltd.) 8 units and restriction enzyme BamHI
(manufactured by Toyobo Co., Ltd.) 8 units were added, and a digestion reaction was performed at 37°C for 2 hours. From the reaction solution, pEGH1 was obtained by LGT method.
The mature eel growth hormone translation region encoded by 5, 3
'--approximately 5 sobp including untranslated region and part of the vector
A DNA fragment of about 0.1~ was obtained.

Separately, pGLM12 was dissolved in 30% Y-100&1 buffer, and the restriction enzyme BanI[I (Toyobo Co., Ltd. 1) 8
unit and restriction enzyme Bam1-11 8 units, and
Digestion reaction was carried out at 7°C for 2 hours.

The PL promoter and c
Approximately 4. containing 1857 genes. Approximately 1n DNA fragment of OK b was obtained.

On the other hand, the following DNA linker was synthesized for the purpose of adding a translation initiation codon ATG necessary for the expression of the DNA encoding mature eel growth hormone, and further connecting the vector DNA and the above DNA.

First, the heavyweight DNA 21s+er and 15mer were separated using the usual triester method [R, Crea et al.
Proceedings of the National Academy of Sciences (Proc, Natl, Aca)
d.

Sci, ), USA, 75.5765 (197B
)). 21mar and 15a+er
50mM) of each 3Qpmole of DNA
HCl (pH 7,5), 10mM MgClt,
It was dissolved in solution 39p1 containing 10mM DTT and 1mM ATP, 3 units of T4 polynucleotide kinase (Takara Shuzo Co., Ltd.) was added, and a phosphorylation reaction was performed at 37°C for 40 minutes.

BanII-BamH fragment of pEGH15 obtained above (approximately 850 bp) 0.03 pmole, pGL
BamHI-Banl [I fragment of Ml (approximately 4. OK
b) 0,006pmole 50mM) Lis-HC
j (pH 7,5), 10mM MgCl, 10mM
A solution containing DTT and 1mM ATP was dissolved at 30°C, and the above-mentioned synthetic NA phosphorylation reaction solution IPI was added thereto. Add 74 DNA ligase (manufactured by Takara Shuzo Co., Ltd.) to this mixture.
Add 3 units and heat to 4°C.

The binding reaction was carried out for 18 hours. E. coli strain 294 was transformed using the reaction mixture to obtain A pr colonies, and plasmid DNA was recovered from these colonies to obtain pUPAl shown in FIG. 25.

The structure of pUPAl is EcoRI, BanI [[.

Hindm, Banff, Hpa I, Bam HI.

It was cleaved with Bgln, Pstl, and Xhol, and confirmed by agarose gel electrophoresis.

Reference example 3゜Preparation of plasmid pGE1 Plasmid pGBL1 (Japanese Patent Application Laid-Open No. 61-93197) 2
Dilute the eyebrows with restriction enzyme reaction solution (100mM NaCj 30
8 units of Pstl and 8 units of Hindl were added, and the digestion reaction was carried out at 37°C for 2 hours.

This was fractionated by agarose gel electrophoresis and approximately 2.4Kb
DNA fragment 31 was obtained. Separately pBR322CBoli
var et al., Gene, 2.95-113 (1
977) ) 21rIl was added to the restriction enzyme reaction solution (1
00mM NaCl) dissolved in 30% Pstl
8 units of Hindl[I] were added, and the digestion reaction was carried out at 37t for 2 hours. This was fractionated by agarose gel electrophoresis to obtain DNA fragment 32 of approximately 0.75 Kb. The DNA fragments 31 and 32 thus obtained were mixed in an amount of 1100n each, dissolved in T4 DNA ligase reaction solution 30P1, and
One unit of 4DNA ligase was added and the ligation reaction was carried out at 4°C for 18 hours. E. coli HB 101 strain was transformed using this reaction solution to obtain Apr colonies. Plasmid DNA was recovered from this colony to obtain pGEl. The structure of this plasmid was confirmed by cutting with Hindnr and Pstl and agarose gel electrophoresis.

Reference Example 4゜1) Construction of recombinant plasmid pscH1 encoding mature salmon growth hormone; HIB2: 5 g of plasmid pscH1 (manufactured by the method described in JP-A-61-15699) containing DNA encoding salmon growth hormone at 20 mM ) Lis-HC1 (pH 7,5),
A solution containing 10mM MgCl1z and 10mM NaCl (hereinafter abbreviated as "Y-10 buffer") was dissolved in 40 order, and restriction enzyme M bo II (New En
37 by adding 10 units (manufactured by grand Biolabs)
The digestion reaction was carried out at ℃ for 3 hours. Next, NaC of the solution
1 concentration to 175mM, 5aI1110
Units were added and a digestion reaction was carried out at 37°C for 3 hours. From this reaction solution, 163, which corresponds to the vicinity of the N-terminus, was determined by the LOT method.
A bp DNA fragment of approximately 0.2 trg was obtained.

Next, 5n of psGHl was dissolved in Y-100@buffer 40%, 10 units of BamHI was added, and a digestion reaction was performed at 37°C for 3 hours. Next, Na Cj! of the reaction solution was added. Adjust the concentration to 175mM and add 10 units of 5afI.
Digestion reaction was carried out at 7°C for 3 hours. Approximately 90Qb containing the C-terminal side and 3'-untranslated region was extracted from the reaction solution by the LGT method.
Approximately 0.5 n of DNA fragments of p were obtained.

Separately, pGEL15 was dissolved in 40% Y-100 buffer, 10 units each of BamHI and HindII were added, and a digestion reaction was performed at 30°C for 3 hours. Approximately 2.7 K containing the tryptophan promoter was extracted from this reaction solution.
Approximately 1n DNA fragment of b was obtained.

On the other hand, the following DNA linker was synthesized for the purpose of adding a translation initiation codon ATC necessary for the expression of the DNA encoding mature salmon growth hormone, and further connecting the vector DNA and the above DNA.

First, the heavy chain DNA % 17mer and 12mer were separated using the usual triester method [R, Crea
) et al.: Proceedings of the National Academy of Sciences (Proc. Natl.

Acad, Sci,) USA,...? 5.5765
(197B) ) One of *17s+er and 12mer 1! DNA each l'l pso
le 50mM) Lis-HCl (pH 7,5), 10
Dissolved in 20 g of a solution containing 42 g of mM MgC, 10 mM dithiothreitol and 1 mM ATP, T4
Six units of polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.) were added, and a phosphorylation reaction was carried out at 37°C for 60 minutes.

0.1 pmole of the psGH1-derived MboII-3all fragment (163 bp) obtained above, Sal I-Ba
mHI fragment (approximately 900 bp) 0.06 paiol
e.

HindI[-BamHI fragment of pGELl (approximately 2,
7 K b) 0.02 pmole in 50mM) Lis-HC1 (pH 7,5), 10mM MgC1z
.

It was dissolved in a solution 30PR containing 10mM dithiothreitol and 1mM ATP, and the above synthetic NA zolination reaction solution 5 was added thereto. Add T4 ligase (
6 units (manufactured by Takara Shuzo Co., Ltd.) were added, and a binding reaction was carried out at 4°C for 18 hours.

E. coli H8101 strain was transformed using the reaction solution and A
I) A colony of P was obtained, and plasmid DNA was recovered from this colony to obtain psGHIB2 as shown in FIG. The structure of psGHIB2 is EcoRI, HindDl,
C1al, BglII.

3311, was cut with BamHI and confirmed by agarose gel electrophoresis. Using M13 phage, it was determined that the sequence near the N-terminus of the DNA encoding salmon growth hormone in psGHIB2 was
ger) method. As a result psGHIB2
D encodes the mature salmon growth hormone polypeptide
It was found to contain NA. Plasmid psGHIB2
Escherichia coli containing Bscherichia coli E
It has been deposited as SGHI B 2 (FERMBP-612) with the Institute of Fine Technology on September 20, 1981.

2) pGEL where the distance between 5O-ATG is tobp
Construction of IO: pGELl (approximately 3.4 Kb):lsr was dissolved in 40 volumes of Y-100 buffer, 5 units of Bann [(Toyobo Co., Ltd. W) was added, and a digestion reaction was performed at 37°C for 3 hours.

The reaction solution was extracted with phenol, and Ba was precipitated with ethanol.
pGEL1 cleaved at the n111 site - approximately 2.44
I got it. Approximately 2.4 kg of this DNA fragment was added to 50 mM)
A solution containing HCl (pH 7,8), 7mM MgCl, and 6mM mercaptoethanol (hereinafter referred to as “DNA
Polymerase buffer (abbreviated as “Polymerase Buffer”))
ATP.

Add dTTP to 1mM each, add 5 units of DNA polymerase (manufactured by A. England Biolabs), and react at 37°C for 30 minutes.
The protruding end was trimmed.

Approximately 2.0 DNA fragments were recovered by phenol extraction and ethanol precipitation. The DNA fragment 1n was dissolved in 20mM) Lis-HCl (pH 7,6), 10mM MgCft.
, 10mM dithiothreitol and 1mM ATP
(hereinafter abbreviated as "T4 ligase buffer") containing 2 units of T4 DNA ligase (Takara Shuzo i!) was added and incubated at 4°C for 18 hours.

Using the reaction solution, E. coli strain 88101 (Bolivar
et al.: Gene, 2.75 (1977)
) to the method of Cohen et al.
,al,: Proc, Natl, Acad.

Sci, USA, 69.2110 (1972)
) to obtain AI) W colonies.

Plasmid DNA was extracted from this transformed strain using a known method ().
1. C. Birn-boim et al.: Nu
cleic Ac1ds Res, L 1513 (
(1979)) and pGELlo (ca.
4Kb) was obtained. The structure of pGELlo is EcoR■,P
It was cut with stl, Hindul, and BamHI and confirmed by agarose gel electrophoresis. The base sequence from the SD sequence downstream of the trp promoter to the translation start codon ATG of the interferon-T gene was determined using the Maxam-Gilbert method (Proc, Natl, Acad, Sci,
USA, +7C5603) Construction of salmon growth hormone expression plasmid psGHI: pGELlo 5x obtained in the previous section was mixed with Y-100 buffer 4
10 units each of HindlI and BamHI were added to carry out a cleavage reaction at 37°C for 3 hours. Approximately 2 DNA fragments of approximately 2.7 Kb containing a portion of the trp promoter, a replication origin, and a lipoprotein terminator were recovered from the reaction solution by a freeze-thaw method.

Separately the psGHIB2 (about 3.8Kb) about 5 eyebrows 4
10 units of Ba dissolved in 0fi11 Y-100 buffer
Add mHI and react at 37°C for 3 hours to completely cleave.
Further, 11 units of HindI were added and the reaction was carried out at 37° C. for 30 minutes to perform partial cleavage with HindI. Approximately 1.0% of the reaction solution encoding mature salmon growth hormone was obtained by freezing and thawing the reaction solution. Approximately 0.7 n of IKb DNA fragment was recovered.

The DNA fragment of pGELlo recovered as described above was about 0.
1 Dissolve approximately 0.2 parts of the tttr and psGHIB2 DNA fragments in 30ml of T4 ligase atIK solution, add 2 units of T4 DNA ligase, and incubate at 4°C.

The binding reaction was carried out for 18 hours. Using the reaction solution, E. coli 8
8101 strain was transformed, and plasmid DNA was collected from the resulting colonies to obtain ps GHI M1. The structure of psGHIMl is EcoRI, Hindn[,
BamHI.

It was cleaved with Pstl and confirmed by agarose gel electrophoresis.

Effects of the Invention According to the present invention, a novel protein having protein human-like activity can be supplied at an industrial cost.

[Brief explanation of the drawing]

FIG. 1 shows the construction process of plasmid pPrA1. FIG. 2 shows the construction process of plasmid pPrA2. FIG. 3 shows the construction process of plasmid pPrA4. FIG. 4 shows the construction process of plasmid pPrAs1. FIG. 5 shows the steps for creating DNAs 24-26. Figure 6 shows plasmid pPrAT1. The construction steps of pPrAW1 and pPrAL1 are shown. FIG. 7 shows the construction process of plasmid pPrAP1. FIG. 8 shows the construction process of plasmid pPrAS4. Figure 9 shows plasmid pPrAT4. The construction steps of pPrAW4 and pPrAL,4 are shown. FIG. 10 shows the steps for constructing plasmid pPrAP4. FIG. 11 shows the construction process of plasmid pPrAFW1. Figure 12 shows plasmids pPrAS2 and pPrAW2.
The construction process is shown below. FIG. 13 shows the construction process of ATG vector pTrS20. Figure 14 shows the steps for constructing plasmid pMTA1. Figure 15 shows the steps for constructing plasmid pMTA2. Figure 16 shows the steps for constructing plasmid pMTA4. Figure 17 shows the steps for constructing plasmid pMTL4. Figure 18 shows the steps for constructing plasmid pMTN4. Figure 19 shows the steps for constructing plasmid pMTO4. Figure 20 shows the steps for constructing plasmid pMTOI4. FIG. 21 shows the construction process of plasmid pMTOn4. FIG. 22 shows the construction process of plasmid pMTOI[[4. Figures 23 (1) and (2) outline the process of cDNA synthesis by the Okayama-Barb method and the construction of a recombinant plasmid containing the DNA. FIG. 24 shows a restriction enzyme map of the cDNA encoding eel growth hormone contained in pEGH15. Figure 25 shows the steps for constructing plasmid pUPA1. FIG. 26 shows the construction process of plasmid psGHIM1. Figure 27 shows the steps for constructing plasmid pGEL10. Patent applicant (102) Kyowa Hakko Kogyo Co., Ltd. Figure 16 Psfl 8gm Bom Figure 18 Hlnd III
H1nduFigure 23 (1) IHlndlI[;*IkoFigure 26πρp Figure 27

Claims (1)

Claims: (1) A protein having an amino acid sequence represented by the following formula 1, formula 13, or formula 15 and having IgG aggregation activity. [There is an amino acid sequence] (Formula 1) [There is an amino acid sequence] (Formula 13) [There is an amino acid sequence] (Formula 15) (In the formula, the symbols indicate the following amino acids, Met: methionine, Thr: threonine, Ala :Alanine, Asp:
Aspartic acid, Asn: asparagine, Lys: lysine, Phe: phenylalanine, Glu: glutamic acid, Gln: glutamine, Tyr: tyrosine, Ile:
(2) Encodes a protein having the amino acid sequence represented by Formula 1, Formula 13 or Formula 15. DNA having a nucleotide sequence. (3) The nucleotide sequence is the following formula 2, formula 14 or formula 1
6. The DNA according to claim 2, which is represented by [There is an amino acid sequence] (Formula 2) [There is an amino acid sequence] (Formula 14) [There is an amino acid sequence] [There is an amino acid sequence] (Formula 16) (A, G, C, and T in the formula are each alanine , represents guanine, cytosine, thymine, the same applies hereinafter) (4) Formula 1, Formula 13
Or a recombinant DNA incorporating DNA encoding a protein having the amino acid sequence of Formula 15. (5) A microorganism containing a recombinant DNA incorporating DNA encoding a protein having the amino acid sequence of Formula 1, Formula 13, or Formula 15. (6) DNA having the nucleotide sequence of formula 3 below. [There is an amino acid sequence] (Formula 3) (7) DNA having the nucleotide sequence of Formula 4 below. [There is an amino acid sequence] (Formula 4) (8) DNA having the nucleotide sequence of Formula 5 below. [There is an amino acid sequence] (Formula 5) (9) DNA having the nucleotide sequence of Formula 6 below. [There is an amino acid sequence] (Formula 6) (10) DNA having the nucleotide sequence of Formula 7 below. [There is an amino acid sequence] (Formula 7) (11) DNA having the nucleotide sequence of Formula 8 below. [There is an amino acid sequence] (Formula 8) (12) DNA having the nucleotide sequence of Formula 9 below. [There is an amino acid sequence] (Formula 9) (13) DNA having the nucleotide sequence of the following formula 10
. [There is an amino acid sequence] (Formula 10) (14) DNA having the nucleotide sequence of the following formula 11
. [There is an amino acid sequence] (Formula 11) (15) DNA having the nucleotide sequence of the following formula 12
. [There is an amino acid sequence] (Formula 12)