JPH01273591A - Human growth hormonal secretory plasmid, transformant using the same plasmid and method for secreting protein - Google Patents

Abstract

PURPOSE:To enable mass production of natural type human growth hormones by using a plasmid consisting of a conjugate of a DNA fragment in which mature human growth hormonal gene is linked to a part just after special regions with a vector. CONSTITUTION:A DNA fragment in which a mature human growth hormonal gene is bound to a part just after a promoter and a ribosome-binding part participating in expression of a gene and a region (preferably derived from extracellular protease gene of bacteria belonging to the genus Bacillus) capable of coding a secretion signal is bound to a vector DNA to afford a human growth hormonal secretory plasmid, which is used to transform a host microorganism.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a DNA DNA in which a mature human growth hormone gene is bound immediately after a promoter involved in gene expression, a ribosome binding site, and a region encoding a secretion signal.
The present invention relates to a human growth hormone secretion plasmid in which the A fragment is linked to the vector DIJA, a transformant using the same, and a protein secretion production method. In particular, DNA in which the human growth hormone (mature hG++) gene, excluding its own secretion signal, is linked immediately after the promoter, ribosome binding site, and secretion signal region of the neutral protease gene of Bacillus amyloliquefaciens.
This invention relates to an hGI+ secretion plasmid containing the fragment, a transformant using the same, and a protein secretion production method.

Here, the adult hGll gene is an hGll gene consisting of 191 amino acid residues starting from naturally occurring phenylalanine.
Refers to the DNA base sequence encoding Gll.

In addition, the present invention particularly provides a natural hGH characterized in that a Bacillus bacterium is transformed using this hG11 secretion plasmid, the obtained transformed strain is cultured, and natural hGH is recovered from the culture supernatant. The present invention relates to a method for producing type hG)I.

Human growth hormone (hGll) is a type of peptide hormone secreted from human pituitary eosinophil cells.
The molecular weight is approximately 22,000 amino acid residues.
It is. The physiological activities of hGll are known to include, in addition to growth promoting effects, protein anabolism, lipid metabolism, and sugar metabolism. Prior to the advent of Mi recombinant DNA technology, hGH was produced by extracting it from the pituitary gland of cadavers.

hG)l was known to be effective in the treatment of burns, wound healing, dystrophies, bone adhesions, disseminated gastric hemorrhages and pseudoarthrosis. Nevertheless, due to the production method described above, only a small amount of hGll can be obtained, and it has not been possible to sufficiently supply hGll to patients with pituitary dwarfism.

Furthermore, it has been pointed out that a major problem associated with conventional hG11 production is death of administered patients due to virus contamination from cadavers (Reference ■).
Establishment of a production method for ll has been eagerly awaited.

(Prior art and problems to be solved by the invention) In recent years, recombinant DNA technology has made it possible to express the hGH gene and produce hGll using microorganisms as hosts.

hGll produced by microorganisms can be broadly classified into intracellular production and exocrine production. In the former case, it is possible to efficiently produce hGH within the bacterial body, but the produced hGll is usually 1nclusi with no biological activity.
It has been pointed out that hGH often forms an insoluble mass called on body, and that it is difficult to return hGH to its natural higher-order structure after recovery from the bacterial body (Reference ■
).

Furthermore, methionine (Met) derived from a translation initiation signal is usually not removed from recombinant DNA-derived heterologous proteins produced within bacterial cells. Therefore, it is widely known that the produced protein is of the methionyl type with Met added to the N-terminus. Even in the case of hGIf, methionyl-type hGH, which has Met added to the N-terminus of natural hGll, is produced, and this is said to increase the level of anti-hGH antibody production in patients (References ).

Therefore, the establishment of an efficient production method for natural hGl+ without Met added to the N-terminus has great medical significance. Based on this, research has been conducted on the process of producing natural hGH, including a method of enzymatically cleaving the additional amino acid of hGI+ that has an additional amino acid (Reference ■), and a method of enzymatically cleaving the additional amino acid of hGI+ that has an additional amino acid, and a method of enzymatically cleaving the additional amino acid of hGI+ that has an additional amino acid. A method for obtaining this has been reported (Reference ■). The former method uses aminopebutidase, which is expensive and difficult to obtain as a commercial product, after hG)l purification (Reference ■), which makes the process complicated and reduces economic efficiency. It is necessary to establish a method for obtaining Tase. On the other hand, regarding the latter method, a method using E. coli, pseudomonas, @mammalian cells as a host has been reported (Reference ■, Reference ■, Reference ■). This method is characterized by the following principle (
This is based on Reference ■). That is, the secreted protein is
It is synthesized intracellularly as a precursor protein with a secretion signal added to the N-terminal upstream of the mature protein, but the secretion signal is removed during the secretion process. Mature h from this point
If G11 is expressed in cells as a precursor protein linked to the C-terminal downstream of the secretion signal, natural hG
) Secretory production of I can be expected.

From the above viewpoint, as mentioned above, natural hGl+
There have been reports of attempts to secrete

When Escherichia coli is used as a host, the precursor hG)I passes through the inner membrane and becomes Gl+ in its natural form, but it accumulates in the periplasm that exists between the inner and outer membranes. Therefore, natural hGH
In order to recover E. coli, it is necessary to disrupt the outer membrane of the E. coli used as a host (Reference ■).

In this case, there is a high possibility of contamination with bacterial outer membrane components, which poses a problem in terms of contamination with pyrogens originating from the outer membrane components. Therefore, in addition to the normal purification process, it becomes necessary to remove the contaminated outer membrane components using a complicated process.
Accordingly, productivity and economic efficiency are reduced. On the other hand, Pseu
When domonas aerginosa is used as a host, there is a big problem in terms of safety because the bacterium itself is a pathogenic bacterium known as Pseudomonas aeruginosa. Furthermore, when mammalian cells are used as a host, there are problems in terms of productivity and economy because it is difficult to culture mammalian cells in large quantities and the culture medium used for culture is expensive.

In order to solve these problems, the present inventors investigated a method using Bacillus bacteria as a host. Among Bacillus bacteria, Bacillus subtilis has attracted attention because of its genetic, biochemical, molecular biological, and applied microbiological findings, as well as its high safety profile.
Efforts have been made to create a host DNA system for extracellular secretion of Gl+. However, even when Bacillus subtilis is used as a host, it is difficult to secrete large amounts of animal-derived proteins (References ), there have been no reports of secretion of naturally occurring animal-derived proteins.As an example of human-derived protein secretion using Bacillus subtilis as a host,
in C, H, etc. are - of the extracellular enzyme gene α
- They have reported that they attempted to secrete natural IFN by linking a mature interferon (IFN) gene immediately after the secretion signal region of the amylase gene. However, in this case, it has been reported that a large amount of precursor IFN remained within the cells, and only a small amount was secreted into the medium (Reference ■).

As this example shows, it is not possible to secrete a naturally occurring human-derived protein simply by attaching a gene encoding a mature human-derived protein immediately after the region encoding the secretion signal of the extracellular enzyme gene. .

(Means for Solving the Problems) In view of the above points, the present inventors searched for a gene having a promoter, a ribosome binding site, and a secretion signal capable of secreting natural hGH, and finally achieved the present objective. They discovered a DNA fragment and completed the present invention.

That is, the present invention provides a method in which the mature human growth hormone gene is linked immediately after the promoter involved in gene expression, the ribosome binding site, and the region encoding the secretion signal. This is a protein secretion production method in which the human growth hormone secretion plasmid is used to transform a transformed strain, and the transformed strain is cultured, and human growth hormone is recovered from the culture supernatant.

The present invention provides a gene promoter, a ribosome binding site, and a region encoding a secretion signal derived from an extracellular protease gene of a bacterium of the genus Bacillus, and the bacterium of the genus Bacillus is Bacillus amyloliquefaciens; A human growth hormone-secreting plasmid whose extracellular protease is a neutral protease, in particular, the region encoding the promoter, ribosome binding site, and secretion signal involved in gene expression is derived from the neutral protease gene of Bacillus amyloliquefaciens A human growth hormone-secreting plasmid, a transformed strain using the plasmid, and a method for secreting and producing human growth hormone protein using the transformed strain.

The present invention will be explained in detail below.

The promoter referred to in the present invention is I? The term "ribosome binding site" refers to the region that NA polymerase recognizes and binds to, and the term "ribosome binding site" refers to the region where mRNA synthesized by RNA polymerase binds to ribosomes.

These regions play important roles in gene expression.

Furthermore, it is widely known today that the gene sequences of these regions are related to the amount of gene expression (References [
phase]).

When expressing a gene encoding a desired protein using a bacterium of the genus Bacillus as a host, the RNA of the bacterium of the genus Bacillus
Because polymerases and ribosomes have strict specificity for promoters and ribosome binding sites (Reference ■), these regions are preferably derived from Bacillus bacteria (Reference @).

Furthermore, the secretion signal refers to a polypeptide present at the N-terminal upstream portion of the extracellular protein precursor.

Since this polypeptide is removed during the secretion process, it is thought to play an important role in the passage of extracellular protein precursors through the cell membrane (Reference ■).

From a practical point of view, it is possible to insert and link a gene encoding a heterologous protein downstream of the gene's promoter, ribosome binding site, and region encoding the secretion signal, and moreover, it is possible to insert and link a gene encoding a heterologous protein that can be replicated in Bacillus bacteria. When creating a protein-secreting plasmid, it is important to use an extracellular enzyme gene that can be secreted and produced outside the bacterial cell within a short period of time.

Such enzymes include alkaline protease, alpha
-Amylase, levansucrase, etc. are known.

However, the characteristics of a secretion plasmid are not satisfied by using the promoter, ribosome binding site, and secretion signal of these genes, as previously reported by Manen et al.
(Reference ■) It should be noted that the report by et al.

In the present invention, the neutral protease gene of Bacillus amyloliquefaciens shows excellent results as a gene encoding an extracellular enzyme capable of secreting a large amount of natural hG11 to the outside of the cell. It is used as a. That is, a plasmid is constructed in which the successful human growth hormone gene is ligated immediately after the neutral protease gene promoter, ribosome binding site, and secretion signal.

Further, as the vector DNA constituting the natural hGH secretion plasmid of the present invention, any DNA can be used as long as it can be replicated in bacteria of the genus Bacillus. The commonly used ones are ptl [1110, prps, pcI94, pDB9] derived from Staphylococcus bacteria.
, pBD64, pBc16, pE194, and derivatives thereof. All of the above plasmids were purchased from the Ohio University Bacillus Stock Center (address:
484 West 12th Avenue Colu
mbus, 0hio 43210 USA) and will be distributed to everyone.

The human growth hormone secretion plasmid thus constructed is used to generate transformants. For example, C
A transformed strain can be obtained by the method of Hang et al., that is, by treating Bacillus subtilis treated with Lysotium and a plasmid with a 40x polyethylene solution.

In order to obtain natural hGH using the thus obtained transformed strain, the strain may be cultured in liquid by a conventional method. Natural hGH is secreted into the culture supernatant with high efficiency. Natural hGH can be prepared from a culture solution by collecting and purifying the culture supernatant using a conventional method.

By culturing this transformed strain, for example, as shown in the Examples, a DNA fragment in which the adult PhGH gene is linked immediately after the promoter, ribosome binding site, and secretion signal of the neutral protease gene is present. After constructing the natural hG11 secretion plasmid, it was introduced into Bacillus subtilis, and the resulting transformed strain was used to produce natural hG11.
The natural hGI+ secreted and accumulated in the Bacillus subtilis culture supernatant is I mg/E ・A66
A hGll of 0 was reached. This unit Ctng/l・A66
0) is the hGll expression (mg/
1) divided by the absorbance (A660) of the culture solution. Furthermore, hGH in this bacterial cell supernatant accounted for about 20% of the total bacterial protein.

(Function) As shown as one aspect of the present invention, a natural hGH secretion plasmid is constructed using the promoter, ribosome binding site, and secretion signal of the neutral protease gene of Bacillus amyloliquefaciens, and this is used. By obtaining a transformant and culturing it, the natural hG
It became possible to secrete If into the culture supernatant. That is, according to the present invention, hGll is extracted from the culture supernatant by nW.
A method has been established that allows for recovery and purification using 'II' methods.

The present invention also provides that if a gene encoding another mature heterologous protein is selected as the gene to be linked immediately after the promoter, ribosome binding site, and secretion signal of the neutral protease gene, the gene encoding the mature protein will correspond to the gene encoding the mature protein. Indicates that a natural protein is obtained. Therefore, by using the method presented in the present invention,
That is, if it is possible to secrete and produce a desired natural heterologous protein by constructing a natural heterologous protein secretion plasmid in which a gene encoding a mature heterologous protein is ligated immediately after the region encoding the secretion signal. This shows that the desired natural heterologous protein can be recovered and purified more easily.

(Example) Hereinafter, the present invention will be explained using specific examples, but the present invention is not limited to these examples in any way.

Example 1 Natural bGH secretion plasmid phGI! Construction of 324 First, a heterologous protein expression secretion hector pNPA225 capable of inserting and ligating a gene encoding a heterologous protein immediately after the promoter, ribosome binding site and secretion signal region of the neutral protease gene of Bacillus amyloliquefaciens was constructed according to the method shown in Figure 1. Plasmid pNPA84 is an amylase secretion plasmid having a region in which the mature α-amylase gene is linked downstream of the region encoding the promoter of the neutral protease gene, the ribosome binding site and secretion signal, and the upstream region of propeptide. Transformed strain MT-8400 strain containing 84 (
Tab pNPA84 from FRPM-BP-923).
Prepared using the method of ak et al. (Reference @). This pNPA84ON8 was digested with restriction enzyme tlpalT (manufactured by Takara Shuzo) and restriction enzyme Bam1l (manufactured by Takara Shuzo), resulting in an approximately 7.8 Kb DNA fragment (hereinafter referred to as DNA fragment A).
) was purified by gel electrophoresis using agarose gel.

This DN^ fragment A contains the promoter, ribosome binding site and secretion signal lacking the C-terminal region.

On the other hand, in order to create a restriction enzyme Stu■ cleavage site immediately after the secretion signal region, two types of synthetic oligonucleotides, 16mer and 18mar, were synthesized by the modified triester method (Reference ■).

1 μg of each of the two synthetic oligonucleotides was phosphorylated using T4 vorinucledokinase (manufactured by Takara Shuzo) and curp (manufactured by Almasia) (References @). next,
These reaction products were mixed, heated in boiling water for 3 minutes, and then slowly cooled to anneal the two synthetic oligonucleotides. After that, DNA fragment A (0.5 μg
) and the annealed synthetic oligonucleotide (1 μg) were ligated using T4 ligase (manufactured by Takara Shuzo) to obtain plasmid pNPA225. Next, the natural hG11 secretion plasmid phGI+324 was constructed according to the method shown in FIG. First, five types of oligonucleotides were synthesized in order to construct a DNA fragment (Fig. 3) encoding the amino acids from the N-terminus to the 22nd acid group of mature hGH.

Next, 1 μg of each of the obtained synthetic oligonucleotides was phosphorylated and then annealed. pNPA225 [INA] was cut with restriction enzyme 5tul and BamHI.
using T4 ligase (manufactured by Takara Shuzo), and immediately after the region encoding the secretion signal of the neutral protease gene, h
2 amino acid residues) is combined with hybrid plasmid ρh
GH317 was obtained. Furthermore, by digesting the hGl+ expression plasmid phGH20 DNA with restriction enzyme Pvu II and restriction enzyme EcoRV, DNA containing the region encoding hGll from position 23 to the C-terminus was obtained.
The fragment (DNA fragment B) was purified by preparative agarose gel electrophoresis. This DNA fragment and phGH317 were digested with the restriction enzyme Pvu II, resulting in approximately 5.8K
The DNA fragment of b (DNA fragment C) was ligated using T4 ligase to create a natural hGH secretion plasmid phG+132.
4 was constructed. This phGI+324 is a DNA in which the mature hG11 gene is bound immediately after the promoter, ribosome binding site, and region encoding the secretion signal of the neutral protease gene of Bacillus amyloliquefaciens.
This is a hybrid plasmid containing an NA fragment. In addition, h
The entire base sequence of the Gll gene has already been reported. Therefore, the DNA fragment containing the hGH gene used in this example was prepared from the hGH expression plasmid phGI+20 containing the chemically synthesized hG)I gene.

Example 2 Secretory production of hGll using natural hGI + secretion plasmid Using plasmid phcH324 constructed in Example 1, high production strain MT-430 (FERM BP-1079)
was transformed using the method of Chang et al. (Reference [Phase]). The obtained transformed strain MT-324 (FERM
BP-1399) in a micro jar (actual volume 1400
The secreted and accumulated amount of hGll was examined by enzyme immunoassay using an antibody against hGH (Reference @). hGH in the medium was determined by enzyme immunoassay using the culture supernatant after culturing at 30°C for 10 hours (stirring rate: 400 rpm, aeration rate: 11/min) using double concentration LB medium. As a result of measuring the concentration, 10 mg of hGll per 1 liter of medium.
Accumulation of 12 was recorded. In addition, the results of SO3-PAGE analysis of the precipitate obtained by adding trichloroacetic acid to the culture supernatant revealed that the molecule LE22, which has the same size as natural hGI+,
,000 Dalton protein was found to accumulate in large amounts in the culture supernatant. Moreover, since it reacted with an antibody against human growth hormone, it was confirmed that this molecule was i22,000 protein; hGll. Also,
As a result of analysis using a gel scanner densitometer, MT
The amount of hG11 present in the culture supernatant of the -324 strain was as high as 20%. Surprisingly, hG
The abundance ratio of hGl increased to 50%, and there were very few contaminant proteins. Next, in order to investigate the molecular size of hGH secreted into the supernatant, we purified hGl secreted into the medium by the MT-324 strain. Culture fi (542) was concentrated using a hollow fiber (molecular weight fraction 10,000, manufactured by Brace Japan), acetone precipitation (40% V/V), and isoelectric focusing (buffer pl+ was 5.5%). It was adjusted to 0.

), hGll was purified by ion exchange chromatography, and 5O
A specimen with one band was obtained by 5-PAGE (Fig. 4).

From this result, hGll secreted by the MT-324 strain is
It was found to have the same electrophoretic mobility as natural hGI+. Therefore, the N-terminal amino acid sequence was determined using purified hGIl (In 5ole) (Reference ■)
However, it was Phe-Pro-The-11e-Pro. From this, the MT-324 strain has natural hGI
It was found to have an N-terminal amino acid that exactly matches the +.

From the above results, it was shown that secretory production of natural hG11 is possible in the Orchid subtilis system.

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[Brief explanation of the drawing]

Figure 1 shows the natural heterologous protein expression secretion vector pNPA.
225 is a diagram showing a construction method of 225. Figure 2 shows the natural hGH secretion plasmid phGI+32.
4 is a diagram showing a construction method of No. 4. FIG. 3 shows a synthetic oligonucleotide encoding the 22nd amino acid from the N-terminus of mature hGll. Figure 4 shows the 5DS of hGll secreted by the elbow-324 strain.
- It is a figure showing a PAGE pattern. In addition, in FIG. 1, FIG. 2, and FIG. 3, A represents adenine, C represents cytosine, G represents thymine, and G represents guanine. In addition, in FIGS. 1 and 2, the promo ter is
The promoter region necessary for the expression of the neutral protease gene is expressed as l? 11 is the ribosome binding site of the neutral protease gene, pre is the region encoding the secretion signal of the neutral protease gene, Δpro is the upstream region of the propeptide of the neutral protease gene, α-amyl
Ase indicates the alpha amylase gene, hG11 indicates the mature human growth hormone gene, and Δprotease indicates the latter half of the neutral protease. Furthermore, in FIG. 4, lane 1.4 is the molecular weight size marker, lane 2 is the natural hG11 preparation,
Lane 3 is l'1T-324 strain Yamaki hGl-1. Patent applicant: Director of the Agency of Industrial Science and Technology, Soko Iizuka (No change in the contents of the 2FC drawings) No. 1 Zuka 4 Commentary 2 (Ho Seikichi (spontaneous) Date of June 3, 1988 Commissioner of the Japan Patent Office Yoshi 1) Moon Tsuyoshi 1 , Indication of the case 1988 Patent Application No. 204104 2 Title of the invention Human growth hormone secretion plasmid, transformant using the same and protein secretion production method 3 Amendment person 1 Kita 2fb>>-1 5. Contents of correction Replace all drawings with drawings clearly drawn in sufficiently deep black using appropriate paper (no changes in content). that's all

Claims (1)

[Scope of Claims] 1) A DNA fragment in which a mature human growth hormone gene is bound immediately after a promoter involved in gene expression, a ribosome binding site, and a region encoding a secretion signal is bound to vector DNA. A human growth hormone secretion plasmid characterized by: 2) The human growth hormone secretion plasmid according to claim 1, wherein the gene promoter, ribosome binding site, and region encoding a secretion signal are derived from an extracellular protease gene of a bacterium belonging to the genus Bacillus. 3) The human growth hormone secretion plasmid according to claim 2, wherein the Bacillus bacterium is Bacillus amyloliquefaciens. 4) The human growth hormone secretion plasmid according to claim 2, wherein the extracellular protease is a neutral protease. 5) Human growth according to claim 1, wherein the region encoding the promoter, ribosome binding site, and secretion signal involved in gene expression is derived from the neutral protease gene of Bacillus amyloliquefaciens. Hormone secretion plasmid. 6) A DNA fragment in which the mature human growth hormone gene is linked immediately after the promoter involved in gene expression, the ribosome binding site, and the region encoding the secretion signal is a human growth hormone secretion plasmid linked to the vector DNA. A transformed strain characterized by being transformed. 7) The transformed strain according to claim 6, wherein the gene promoter, ribosome binding site, and region encoding a secretion signal are derived from an extracellular protease gene of a bacterium belonging to the genus Bacillus. 8) The transformed strain according to claim 6, wherein the Bacillus bacterium is Bacillus amyloliquefaciens. 9) The transformed strain according to claim 6, wherein the extracellular protease is a neutral protease. 10) The transformed strain according to claim 6, wherein the microorganism to be transformed is a bacterium belonging to the genus Bacillus. 11) The transformation according to claim 6, wherein the region encoding the promoter, ribosome binding site, and secretion signal involved in gene expression is derived from the neutral protease gene of Bacillus amyloliquefaciens. KK. 12) The transformed strain according to claim 11, wherein the microorganism to be transformed is a bacterium belonging to the genus Bacillus. 13) A DNA fragment in which the mature human growth hormone gene is linked immediately after the promoter involved in gene expression, the ribosome binding site, and the region encoding the secretion signal is a human growth hormone secretion plasmid linked to the vector DNA. A method for secretory production of human growth hormone, which comprises culturing a transformed strain and recovering human growth hormone from the culture supernatant. 14) Secretory production according to claim 13, wherein the promoter involved in gene expression, the ribosome binding site, and the region encoding the secretion signal are derived from the neutral protease gene of Bacillus amyloliquefaciens. Law.