JPH0355111B2 - - Google Patents

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to bovine growth hormone releasing factors (hereinafter abbreviated as GRF) known to have growth hormone secretion regulating activity. A new group that enables the production of dihydrofolate reductase (hereinafter abbreviated as DHFR), which has a mutant in which the 27th methionine (Met) of the peptide fragments up to 29th position is converted to isolosine (Ile) at the carboxy terminal side. This relates to replacement plasmids. pSG1
-12 has the DNA sequence shown in FIG.
pSG1-12 and E. coli containing pSG1-12 are
Suitable for fields such as fermentation industry and pharmaceutical industry.

[Prior Art and Problems] The technical background of the present invention includes so-called genetic manipulation technology. Recently, with advances in genetic engineering technology, it has become possible to produce interesting polypeptides using microorganisms. For example, DNA, which is a gene corresponding to a polypeptide, is isolated from a living organism by a method called cloning, and then it is inserted into a suitable plasmid called an expression vector. The target polypeptide is introduced into microorganism cells, expressed in the microorganism, and the target polypeptide is isolated and purified from the microorganism. Under these circumstances, the most important issue is to construct a recombinant plasmid that contains the target polypeptide gene and allows for efficient expression. Furthermore, different target polypeptides naturally require different methodologies, and this is a technical problem that must be solved.

CRF is a peptide that stimulates the secretion of growth hormone. Bovine GRF consists of 44 amino acids, and its sequence is tyrosine - alanine - from the amino terminus.
Aspartic acid - Alanine - Isoleucine - Phenylalanine - Threonine - Asparagine - Serine - Tyrosine - Arginine - Lysine - Valine - Leucine - Glycine - Glutamine - Leucine - Serine - Alanine - Arginine - Lysine - Leucine -
Leucine - Glutamine - Aspartic acid - Isoleucine - Methionine - Aspartic acid - Arginine - Glutamine - Glutamine - Glycine - Glutamic acid - Arginine - Asparagine - Glutamine - Glutamic acid - Glutamine - Glycine - Alanine - Lysine - Valine - Arginine - Leucine, and carboxy The terminal is amidated.

GRF exists in the hypothalamus, but its content is small, and it is only necessary to isolate and purify a few milligrams from 500 cows, and the development of efficient production methods is expected. Bovine GRF consists of 44 amino acids, of which the peptide portion from the first tyrosine to the 29th arginine (hereinafter referred to as GRF1-29) alone has approximately one-fourth the activity of GRF. (N.Ling et al., Biochem Biophys.Res.
Commun., vol.123, pp.854 (1984)). Also,
Methionine, the 27th amino acid of GRF,
It is not important for the expression of GRF activity, and it is known that mutating to isoleucine or leucine, which are structural analogs of methionine, has GRF activity (GM Clore, et al. J. Mol. Biol.,
vol.191, 553 (1986)). If the 27th methionine of GRF is replaced with another amino acid, since GRF is a methionine-free peptide,
It is also unaffected by cleavage specific to the amino acid sequence at the methionine position by bromcyane treatment.

Regarding recombinant plasmids incorporating DNA encoding GRF1-29, pGRF28-29 (Japanese Patent Application Laid-open No. 115287-1987) constructed by the present inventors and
pGRF2-15 (Japanese Unexamined Patent Publication No. 1-144978) is known. However, in E. coli cells into which pGRF28-29 has been incorporated, although the target polypeptide is expressed, the amount accumulated within the cells is extremely small. Also
pGRF2-15 is a plasmid that was successfully expressed as a fusion protein with DHFR in order to stably express GRF1-29.
From fusion protein of DHFR and GRF1-29
A problem has arisen in the efficiency of specifically excising GRF1-19. That is, pGRF2−15 is DHFR and
It encodes a fusion protein that binds GRF1-29 through an amino acid sequence that can be recognized and cleaved by an enzyme called Factor Xa, but Factor Xa is expensive and the efficiency of cleavage by the enzyme reaction is not very high. There were some difficulties such as:

[Object of the invention] In order to solve the above-mentioned problems, the object of the present invention is to develop a recombinant plasmid that enables mass production of a derivative of GRF1-29, which is said to have GRF activity. . Furthermore, the present invention was carried out as part of the development of a method for mass-producing GRF1-29 derivatives using genetic engineering techniques.

As a result of intensive research, the present inventors discovered that methionine, the 27th amino acid of GRF, can be converted to isoleucine, a structural analogue, and that DHFR and
We devised a method of binding a GRF1-29 derivative via methionine, producing it as a fusion protein, and cutting it out from the fusion protein by bromcyan treatment, and developed a recombinant gene and its incorporation that would make this possible. A recombinant plasmid was constructed and the present invention was completed.

[Configuration of the Invention] Figure 1 shows the amino acid sequence of the fusion protein of DHFR and GRF1-29 derivative (hereinafter abbreviated as DHFR-GRFM) incorporated into pSG1-12 of the present invention and the sequence encoding it. Showing the DNA sequence. DHFR-GRFM is a protein consisting of 191 amino acids, of which the sequence from 1 to 159 counting from the amino terminal side has a single amino acid substitution substitution in the wild-type DHFR of E. coli (Cys-152 ( wildtype)→Clu-152) sequence, with methionine at position 162, and by final treatment with bromcyan, it is possible to excise a derivative of GRF1-29 (hereinafter abbreviated as GRFM). The 162nd to 191st array is
This is the amino acid sequence of GRFM.

FIG. 2 shows the entire base sequence of pSG1-12 of the present invention. The figure shows one side of the double-stranded DNA.
Only the DNA strand sequence is described from the 5' end to the 3' end. Furthermore, pSG1-12 is a circular DNA, but for convenience of description, it is shown as a linear DNA. Therefore, the first and last exist next to each other. pSG1−12 is 4715 base pairs in size;
Trimethoprim and ampicillin resistance can be imparted to host E. coli. pSG1−12
is introduced into E. coli and kept in a stable state,
Escherichia coli containing pSG1-12 was sent to the FIKEN using FERM.
Deposited as BP-2149.

pSG1-12 contains sequences encoding DHFR-GRFM. In FIG. 2, the sequence from 57th to 629th is the sequence encoding DHFR-GRFM. Upstream of the sequence encoding DHFR-GRFM, there is a sequence that allows efficient expression of this gene (Japanese Unexamined Patent Publication No. 46193/1983). i.e. 43
The sequence from position 4673 to position 50 is called the SD sequence, and is useful for efficient translation.
The sequence up to position 4701 is a consensus transcription promoter and contributes to efficient transcription. From this, pSG1-12 produces a large amount of DHFR-GRFM when introduced into E. coli. DHFR− made
GRFM accumulates in large amounts within the bacterial body, accounting for 15-20% of the bacterial body protein. Also, accumulated
DHFR-GRFM exhibits dihydrofolate reductase activity, which makes E. coli harboring pSG1-12 resistant to trimethoprim.

As described in the Examples, pSG1-12 is a recombinant plasmid that has already been created by the present inventors.
It can be created by replacing the sequence between the restriction enzyme BamHI and XhoI sites of pMEK2 (described in JP-A-1-252289) with a sequence encoding GRFM. pMEK2 contains a gene that confers resistance to the antibacterial agent ampicillin, and the expression of this gene is unaffected by the sequence replacement between the BamHI and XhoI sites. That is, E. coli harboring pSG1-12 exhibits resistance to ampicillin.

pSG1-12 having such features can be constructed according to the Examples, but the present invention is not limited by the method of constructing the recombinant plasmid.

Next, examples of the present invention will be shown.

Example Creation of pSG1-12 The DNA to encode GRFM is 1.5′-
GATCCTGATGTACGCTGATGCTACTTC−
3′ 2.5′−
ACCAACTCGTACCGTAAAGTTCTGGGTC
AGCTGTCGGCTCG-3'3.5'-
TAAACTGCTGCAGGATATCATCAACCGT
TAAC-3'4.5'-
TCGAGTTAACGGTTGATGATATCCTGCA
GC-3'5.5'-
AGTTTACGAGCCGACAGCTGACCCAGAA
CTTTACGG-3'6.5'-
TACGAGTTGGTGAAGATAGCATCAGCGT
Six DNAs of ACATCAG-3' were chemically synthesized according to the phosphoramidite method.

As a vector for incorporating chemically synthesized DNA, pMEK2 (described in JP-A-1-252289) is used.
was used. After phosphorylating the 5' ends of six chemically synthesized DNAs, annealing was performed, and this
After cutting with BamHI and XhoI, it was mixed with pMEK2 treated with alkaline phosphatase, and T4-
Chemically synthesized DNA and vector were ligated using DNA ligase. All such reactions are referred to as “Molecular Cloning ALoboratory
Manual” (T. Maniatis, EFFritsch, J.
Sambrook, eds.Cold Spring Harbor Labora.
-tory (1982), hereinafter referred to as Reference 1. ). The obtained reaction product is
It was introduced into E. coli according to the transformation method (described in Document 1 above). The treated bacterial cells were transferred to a nutrient agar medium containing 50 mg/ampicillin sodium and 2 mg/trimethoprim (in medium 11, 2 g glucose,
Contains 1g dipotassium phosphate, 5g yeast extract, 5g polypeptone, and 15g agar. )
By coating on the top and culturing at 37℃ for 24 hours,
Approximately 50 colonies were obtained. Select 10 of these colonies and incubate them overnight at 37°C in 1.5 ml of YT+Ap medium (medium 11 contains 5 g of NaCl, 5 g of yeast extract, 8 g of tryptone, and 50 mg of ampicillin sodium). The body was cultured. Each culture solution was placed in an Etzpendorf centrifuge tube, centrifuged at 12,000 rpm for 10 minutes, and the bacterial cells were collected as a precipitate. Add 0.1 ml of electrophoresis sample preparation solution (0.0625M Tris-HCl, PH6.8,
2% sodium lauryl sulfate (SDS), 10% glycerin, 5% 2-mercaptoethanol,
Contains 0.001% Bromophenol Blue. ) was added to suspend the bacterial cells, and this was kept in boiling water for 5 minutes to dissolve the bacterial cells. A sample that has undergone this treatment
SDS-polyacrylamide gel electrophoresis (U.
K. Lammli; Nature, vol.227, p.680−685
(1970)). As molecular weight markers, lactalbumin (molecular weight 14200), trypsin inhibitor (molecular weight 20100), trypsinogen (molecular weight 20400), carbonic anhydrase (molecular weight 29000), glyceraldehyde 3-phosphate dehydrogenase (molecular weight 36000), egg albumin (molecular weight 45000) ), and bovine serum albumin (molecular weight 66,000) were run on a 10 to 20% polyacrylamide gradient gel. As a result, it was revealed that 5 of the 10 colonies were newly producing large quantities of proteins with increased molecular weight (estimated to have a molecular weight of about 25,000). By analyzing with a densitometer, it was estimated that the amount of newly appeared protein was about 20% of the bacterial body protein.

Select one of the colonies that produce a new protein with a large molecular weight and use it as YT+
Cultured in Ap medium, using the method of Tanaka and Weisblum (T. Tanaka, B. Weisblum; J. Bacteriology,
vol. 121, p. 354 (1975)). The obtained plasmid was named pSG1-12. When the obtained plasmid was reintroduced into E. coli, a transformed strain with exactly the same properties as pSG1-12 was found to have a concentration of 10 ⁵ to 10 ⁶ /μg plasmid.
Obtained with DNA frequency. The thing is pSG1−12
This shows that the properties of are stable. Also,
This shows that pSG1-12 is stably replicated within E. coli cells.

In pSG1-12, the sequence between the BamHI and XhoI sites of pMEK2 should be replaced with a sequence derived from synthetic DNA. About 400 nucleotides long, obtained by cutting pSG1-12 with EcoRI and SalI.
For DNA, the dideoxy method using M13 phage (J. Messing; Mehtodsin Enzymology,
vol. 101, p. 20 (1983)). As a result, the 471st to 937th sequences of the entire base sequence of pGRFM1-29 shown in FIG. 2 were revealed. The base sequence of pMEK2 has been revealed by the present inventors (described in JP-A-1-252289). The sequence of pSG1−12 is that of pMEK2.
The sequence between the BamHI and XhoI sites located between the EcoRI-SalI sequence was a sequence in which 101 nucleotides of DNA (a sequence encoding GRFM) derived from chemically synthesized DNA was bound.

In addition, the approximately 4.2 kilobase pair DNA obtained by EcoRI-SalI digestion of pSG1-12 is Pst,
The results of restriction enzyme cleavage experiments using Hind, Hpa, Aat, Pvu, Bgl, and Cla showed that the DNA was completely identical to the approximately 4.2 kilobase pair DNA obtained by EcoR-Sal cleavage of pMEK2. Ta.

From the above results, it is clear that the entire base sequence of pSG1-12 is the sequence shown in FIG.

[Effect of the invention] As mentioned above, the novel recombinant plasmid pSG1-
12 encrypts DHFR-GRFM and
E. coli harboring pSG1-12 accumulates and produces large amounts of DHFR-GRFM. Furthermore, the generated DHFR
-GRFM has a structure in which DHFR and GRFM are bonded via methionine, and the two can be easily separated by bromcyane treatment. Because of these properties, the novel recombinant plasmid pSG1-12 of the present invention and E. coli containing it are useful for the production of DHFR-GRFM and the production of GRFM using it.

[Brief explanation of drawings]

Figure 1 shows DHFR- present in pSG1-12.
FIG. 2 is a diagram showing the base sequence of the portion encoding GRFM and the amino acid sequence of the protein. The symbols in the figure represent nucleobases and amino acids, A for adenine, C for cytosine, G for guanine, T for thymine, Ala for alanine, Arg for arginine,
Asn represents asparagine, Asp represents aspartic acid, Cys represents cysteine, Gln represents glutamine,
Clu is glutamic acid, Gly is glycine, His is histidine, Ile is isoleucine, Leu is leucine, Lys is lysine, Met is methionine,
Phe stands for phenylalanine, Pro stands for proline,
Ser represents serine, Thr represents threonine, Trp represents tryptophan, Tyr represents tyrosine, and Val represents valine. The numbers in the figure indicate the numbers counted starting from "A" of the ATG codon that encodes the first amino acid, methionine. FIG. 2 is a diagram showing the entire base sequence of pSG1-12, in which only one DNA strand sequence of the double-stranded DNA is written in the direction from the 5' end to the 3' end. The symbols in the figure represent nucleic acid bases, A is adenine, C
indicates cytosine, G indicates guanine, and T indicates thymine. The numbers in the figure indicate restriction enzymes among the two restriction enzyme Cla cleavage recognition sites that exist in pSG1-12.
The numbers are shown starting from the first "A" of 5'-ATCGAT-3', which is the Cla cleavage recognition site closer to the Hind cleavage site.

Claims (1)

[Scope of Claims] 1. A peptide fragment of bovine growth hormone-releasing factor that is stably replicated in Escherichia coli and can confer trimethoprim resistance and ampicillin resistance to the host Escherichia coli, and that has the amino acid sequence shown in 1 below and divitro The fusion protein with folate reductase is encoded and shown in note 2 below.
Novel recombinant plasmid pSG1- with DNA sequence
12. [Table] [Table] [Table] [Table] [Table] [Table] 2 Stably replicates in E. coli, can confer trimethoprim resistance and ampicillin resistance to the host E. coli, and has the amino acid sequence shown in 1 below. A fusion protein of a peptide fragment of bovine growth hormone-releasing factor and divitrofolate reductase, which has
Novel recombinant plasmid pSG1- with DNA sequence
E. coli containing 12. [Table] [Table] [Table] [Table] [Table] [Table]