JP2000078989A - Production by secretion of human growth hormone - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing 20 K hGH by secretion using microorganisms more effectively than conventional one, and to provide a method for producing 20 K hGH by secretion using microorganisms, including a less quantity of protein impurities in periplasm extract solution than conventional one. SOLUTION: This 20 KhGH is produced in a state secreted in the periplasm of E. coli by constructing a transformed plasmid having a DNA sequence made by ligating DNA encoding 20 K hGH immediately behind a gene encoding the secretion signal of E. coli OppA protein or its variants and DNA encoding signal peptidase 1, by transforming E. coli with the plasmid and by incubating the E. coli transformant. The 20 K hGH is secreted in the periplasm of E. coli more efficiently than in any conventional method, and is easy to be purified, because the periplasm extract solution includes only a less quantity of protein impurities than in any conventional method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for secretory production of human growth hormone having a molecular weight of about 20,000 using Escherichia coli. Specifically, the present invention relates to the secretion signal of E. coli or Salmonella OppA or E. coli Opp.
A DNA fragment encoding a human growth hormone having a molecular weight of about 20,000 is linked in series to the 3 'end of a DNA fragment encoding an amino acid variant of the A secretion signal.
The present invention relates to a method for producing human growth hormone having a molecular weight of about 20,000 using a culture solution, cells, or a processed product thereof obtained by culturing an Escherichia coli transformant transformed with a recombinant plasmid having an NA fragment.

The present invention also relates to a culture obtained by culturing a transformant of Escherichia coli transformed with a recombinant plasmid having a DNA fragment encoding human growth hormone having a molecular weight of about 20,000 and a DNA encoding Escherichia coli signal peptidase 1. Using liquids, cells, or their processed products,
A method for producing human growth hormone having a molecular weight of about 20,000. Further, the present invention provides a method for culturing a transformant transformed with the recombinant plasmid having the above-described structure and culturing the transformant with a molecular weight of about 200.
The present invention also relates to a method of increasing the production amount of human growth hormone by changing the medium composition when producing the same.

[0003]

BACKGROUND OF THE INVENTION Pituitary-derived human growth hormone (hG
H) include those having a molecular weight of about 22,000 (hereinafter, 22 Kh)
GH) and those having a molecular weight of about 20,000 (hereinafter, 20KhG)
H) is known.

[0004] 22KhGH is a drug produced by a genetic recombination technique, and is used for treatment of pituitary insufficiency short stature, childhood chronic renal failure, and the like. In recent years, it has been revealed that hGH has excellent effects such as a growth stimulating activity, an immunostimulatory effect and a body lipolysis promoting effect, and it is greatly expected that hGH will be applied in the future.

[0005] On the other hand, in recent years, the risk of the occurrence of side effects in the clinical use of 22KhGH has been frequently reported, and this has become a major problem in assuring the safety of GH in the clinic and expanding the indications in the future. 22KhGH so far
Reports on the risk of
Diabetic pathogenicity and the like. Under the expectation of expanding the indications of GH, GH having a low risk of side effects is required in clinical practice.

[0006] 20KhGH has a sequence in which, out of 191 amino acids constituting 22KhGH, 15 amino acids at positions 32 to 46 from the N-terminal of the amino acid sequence are missing. This 20KhGH is 22KhGH
In comparison, it has been reported that the proliferation promoting activity of leukemia cells is low, and that glucose tolerance abnormal induction, which is an indicator of diabetes pathogenicity, is low, and that the risk of developing side effects pointed out by 22KhGH was found to be low. ing. Also, in vitro
In recent years, it has become clear from the experimental results of o that 20KhGH is different from 22KhGH in the binding mode to the GH receptor. That is, M. Wada et al., 22KhGH
Binds to a GH-binding protein (GH receptor extramembrane domain protein) present in serum in the physiological concentration range, thereby reducing the binding activity to the GH receptor on the cell surface.
Since 0KhGH hardly binds to a GH binding protein at physiological concentrations, there is no decrease in the binding ability of the GH binding protein to the GH receptor, and further, a 1: 2 ratio required when GH acts via the GH receptor. Regarding the formation of a coalescence (an aggregate of a GH1 molecule and two molecules of a GH receptor), 22KhGH forms a 1: 1 aggregate (inactive type) at a pharmacological concentration (high concentration range), whereas 20KhGH has a GH It has been clarified that the properties are different in forming a 1: 2 aggregate (active form) with the receptor (Mol. Endo. 12, 1, p146-156 (199).
7)). From these results, 20KhGH was converted to 22KhGH.
Expected to have a stronger activity than H. Also,
Regarding the lipolysis accelerating action that was unknown so far,
It has also been clarified that 20KhGH has the same as 2KhGH (Japanese Patent Application Laid-Open No. 9-216832 or European Patent Publication No. 0733307).

As described above, 20 KhGH is 22 KhG
It has been clarified that it is a growth hormone having a lower risk than H and having an activity equal to or higher than that of H, and thus its usefulness as a new growth hormone as a pharmaceutical is expected.

In recent years, advances in recombinant DNA technology have made it possible to produce heterologous proteins using microorganisms as hosts. Microbial protein production includes intracellular expression and secretory production. In the intracellular expression method, a protein having methionine added to the N-terminus accumulates in the cytoplasm. In order to obtain a protein having no N-terminal methionine residue, it is necessary to enzymatically cleave the amino acid sequence containing the N-terminal methionine residue using, for example, peptidase. Furthermore, the protein obtained by the intracellular expression method has a three-dimensional structure that is not active,
Refolding operation is required. In many cases, the intracellular expression method cannot be said to be an efficient protein production method.

On the other hand, in the secretory production method using Escherichia coli, the target protein is secreted and accumulated in the periplasm. Purification from this periplasmic extract is not only easy because the amount of contaminating proteins is small compared to the intracellular expression method, but the secreted protein does not contain methionine at the N-terminus and has a three-dimensional structure of natural type. This is an excellent method in that it is an active substance.

[0010] Nevertheless, the secretory production method of proteins using microorganisms requires a high level of technology, and there are few examples of practical use of such methods. The reason is that it is necessary that the precursor protein synthesized in the cytoplasm penetrates the cytoplasmic membrane and that the secretory signal is correctly cleaved and removed by processing.

Regarding the secretion of proteins in microorganisms,
The mechanism, factors involved and their functions have been elucidated, and various attempts have been made on efficient secretion methods of proteins based on those findings.

The first one is to examine secretory signals. The secretion signal plays a very important role in protein secretion, which leads the target protein to the cytoplasmic membrane. In order for a secretory signal to act, it has been clarified that a positive charge in the N-terminal positively charged region and hydrophobicity in the central hydrophobic region are essential (J. Bio).
l. Chem., 267, pp. 4882-4888, 1992). Udaka et al., Based on their knowledge of the structural characteristics of this secretory signal, found that in secretory production using Bacillus brevis as a host, MWP derived from Bacillus brevis was used.
(Middle Wall Protein) Secretion efficiency of flounder growth hormone was modified by adding a basic amino acid (Arg) to the N-terminal positive charge region of the secretion signal and adding a hydrophobic amino acid (Leu) to the central hydrophobic region. 60 mg / L
(Nippon Nogeikaga
ku kaishi, 67 (3), p.372, (1993)).

Nevertheless, there is no universal guiding principle for altering the amino acid sequence of the secretion signal, and at present it is necessary to find a preferable sequence by trial and error. Further, there is no clear guideline to date on which secretion signal from which secretory protein should be selected for a protein to be secreted and produced, and a suitable secretory signal must be found for each protein to be secreted and produced.

The above is described, for example, in JP-A-6-2964.
No. 91, which is understood. The publication states that 22KhGH
It has been described that a secretion signal derived from alkaline phosphatase or enterotoxin, which was effective for secretory production of E. coli, is not always effective for secretory production of some eukaryotic proteins. That is, there have been reported cases in which the protein was not synthesized depending on the combination of the secretory signal and the secretory protein, and cases in which the protein was expressed in the cytoplasm but not secreted, and the precursor protein remained in the cytoplasm. .

As described above, it is necessary to select an appropriate secretion signal for each secretory protein.
No secretion signal has been found that can be used to secrete any protein.

As a second attempt, there is a study of improving secretory productivity by co-expression of a protein. To date, regarding the secretory production of proteins other than GH, secretory production of interleukin-6 has been confirmed by SecE and prlA4 (Se
(BIO / cY mutant protein)
TECHNOLOGY, 12, pp.178-180 (1994)), Human Granulo
Improvement of secretory productivity of cyte-colony Stimulating factor (G-CSF) by co-expression of chaperone protein (Biochem. Biophys. Res. Commun., 210 (2), p.
p.524-529 (1995)).

Regarding the secretory production of 20KhGH, a method of co-expressing glutathione reductase with 20KhGH (Japanese Patent Laid-Open No. 8-322586, or European Patent Publication 07)
35140), the E. coli Sec protein was
Method for co-expression with hGH (JP-A-9-313191,
Or EP-A-0 798 380).

[0018]

The above-mentioned JP-A-8-32
20K by co-expression of glutathione reductase of 2586 (or EP 0735140)
According to the method for producing hGH, about 70 m / L culture solution is used.
Although g production has become possible, higher productivity is desired.

Although the secretory productivity was improved by co-expression of glutathione reductase as described above, according to the study of the present inventors, in this method, periplasm obtained from these cells by osmotic shock was used. It was found that the extraction solution contained a considerable amount of contaminating proteins other than the target protein, 20KhGH. Therefore, purification of 20 hGH from the periplasmic extraction solution is complicated, and enormous cost is required to purify 20 KhGH to a high purity of a pharmaceutical grade.

An object of the present invention is to provide a method using a microorganism for 20 Kh.
Another object of the present invention is to provide a more efficient method for secretory production of GH, and it is another object of the present invention to provide a 20K-based microorganism which contains less impurity protein in a periplasmic extraction solution.
An object of the present invention is to provide a method for secretory production of hGH.

An object of the present invention is to provide a plasmid comprising a DNA fragment in which the secretion signal of Escherichia coli OppA is modified and the 5 'end of the DNA encoding 20KhGH is linked to the 3' end of the DNA encoding the secretion signal. Another object of the present invention is to provide a transformant of Escherichia coli transformed with the plasmid, and a method for secretory production of 20KhGH using the transformant of Escherichia coli.

Another object of the present invention is to provide a recombinant plasmid having a DNA encoding 20KhGH and a DNA encoding Escherichia coli signal peptidase 1, an Escherichia coli transformant transformed with the plasmid, and an Escherichia coli transformant. An object of the present invention is to provide a method for secretory production of 20KhGH used.

[0023]

Means for Solving the Problems In order to solve the above problems, the present inventors have used a known strain described in Japanese Patent Application Laid-Open No. 8-322586 (or European Patent Publication No. 0735140). 20K using a secretory signal obtained by modifying a secretory signal derived from a neutral protease of Bacillus amyloliquefaciens, and using a glutathione reductase derived from Escherichia coli as a co-expressed protein.
Escherichia coli transformant MT-107 capable of secretory production of hGH
65 (FERM BP-5020).

First, a new secretion signal capable of more efficiently secreting and producing 20KhGH was examined.

In order to confirm the effectiveness of a new secretion signal, it is necessary to confirm the effect of each secretion signal on the secretory production of 20KhGH. The method is 20
DN in which the 3 'end of DNA encoding various secretion signals is linked to the 5' end of DNA encoding KhGH
It comprises a series of steps of constructing a plasmid containing the A fragment, isolating Escherichia coli transformed with the plasmid, culturing the resulting Escherichia coli transformant, and measuring the secretory production of 20KhGH.

The present inventors have developed a 20K hG G protein by utilizing the secretion signal of a protein secreted in large amounts by microorganisms.
An attempt was made to improve the productivity of H. However, this method requires 20K
It has not been possible to find a secretion signal that enhances the productivity of 20K hGH more than conventionally known methods for secretory production of hGH.

[0027] Protein secretion is a physicochemical phenomenon in which proteins pass through cell membranes in an energy-dependent manner.
Therefore, the present inventors have made a hypothesis that a secretion signal of a protein having a high structural similarity to 20KhGH is preferable, and verified this. As proteins having high structural similarity to 20KhGH, proteins having high amino acid sequence homology and proteins whose behavior in a purification column was similar to 20KhGH were selected. As a protein having high homology in amino acid sequence, TRBC (Meneewannakul, S. et al., J. Bacteri
173, 3872-3878 (1991)) and DPPA (Periplasm
ic Dipeptide Transport Protein Precursor (Dipeptid
e-Binding Protein)) (Olson, ER et al., J. Bacterio
l., 173, 234-244 (1991)). In addition, as a protein whose behavior in a purification column is similar to that of 20 KhGH, Escherichia coli OppA (Periplasmi) whose behavior in an ion-exchange column and affinity chromatography column has been found to be similar to that of 20 KhGH by examination by the present inventors has been found.
c Oligopeptide Binding Protein) (K. Kashiwagi et al.
J. Biol. Chem., 265, 8387-8391 (1990)).

The effect of the above three types of secretory signals on the secretory productivity of 20KhGH was examined.
When the secretory production of KhGH is 1 mg / L or less, 20 KhG
There was no effect of improving the secretory productivity of H. However, surprisingly, when using the E. coli OppA secretion signal,
JP-A-8-322586 (or European Patent Publication No. 0735)
No. 140) and MT-10765 (FERM).
BP-5020) was obtained.

Furthermore, the present inventors tried to further improve the secretory productivity of 20KhGH by modifying the secretory signal derived from OppA. As a result, the secretion signal obtained by inserting an appropriate number of amino acids, such as lysine, which is a basic amino acid, into the positively charged region of the N-terminal sequence and Leu, which is a hydrophobic amino acid, into the central hydrophobic region is a secretory signal derived from natural OppA The secretory productivity of 20KhGH was higher than that of the signal, and a secretory productivity of 110 mg or more per 1 L of the culture solution was achieved.

In secretory production in Escherichia coli, it is already known that the OppA secretion signal of Escherichia coli performs a function effective for protein secretion, which is outside the scope of the claims of the present application. Modification of Signals It is impossible to infer from the known information that secretion signals are extremely effective.

As a second attempt to solve the problem of the present invention, the present inventors examined an improvement in co-expression of a protein.

As a result, it has been found that Escherichia coli-derived signal peptidase 1 gives a secretory production of 20 KhGH which is almost equivalent to glutathione reductase described in JP-A-8-322586 (or European Patent Publication No. 0735140). Was.

Moreover, surprisingly, when co-expressing signal peptidase 1, the periplasmic solution extracted from the cultured cells by the osmotic shock method was compared to the case where glutathione reductase was co-expressed. It was found that the proportion of contaminating proteins other than 20KhGH present therein was very low. The cause of this is unknown, but signal peptidase 1 rather than glutathione reductase.
It is presumed that the effect of reducing the stress exerted on the cells by the expression of 20K hGH is stronger, and as a result, the tendency of the cultured cells to lyse is reduced, and the amount of the intracellular protein mixed into the periplasmic extract is reduced. Was done. This reduction in contaminating proteins in the periplasmic solution has the effect of facilitating the purification of 20KhGH from the periplasmic fraction extraction solution, and greatly contributes to the reduction of the production cost of 20KhGH such as improvement in purification efficiency.

Signal peptidase 1 is an enzyme that cleaves a secretory signal when a precursor protein produced in the cytoplasm permeates a membrane. Jan Maarten van Dijl et al. Have studied the effect of overexpressing signal peptidase 1 on secretory production of β-lactamase to which various secretion signals are connected (Jan Maarten van Dijl eta).
l, Mol. Gen. Genet, 227, p40-48 (1991)). Among them, it was reported that the effect of improving the processing (signal cleavage) efficiency by co-expression of signal peptidase 1 varies depending on the combination of the secretory signal sequence in the precursor and the secreted target protein. are doing.

However, it is completely unclear from the literature whether or not co-expression of signal peptidase 1 has an effect on improving the secretion efficiency of 20K hGH. It is completely unknown that the content of contaminating proteins in the extracted periplasmic solution is low.

Further, the present inventors changed the culture conditions,
An attempt was made to further improve the secretory production of 20KhGH. As a result, MT-10765 (FERM BP-) which is a known strain described in Japanese Patent Application Laid-Open No. 8-322586 (or European Patent Publication No. 0735140) is conventionally known.
5020) succeeded in improving the production of 20KhGH up to 82 mg / L by increasing the concentration of the carbon source in the culture solution. Therefore, this method was further applied to MT-10852 (FERM BP-6290) and MT-10853 (FERM BP-6291) of the present invention. Then, for example, when glycerol is 3.6% and succinic acid is 1.5%, MT-10852 (FERM BP-
6290), 360 mg / l, MT-10853 (F
ERMBP-6291) is 350 mg / l, which is 4
We succeeded in increasing the production up to 5 times. Further, at this time, 20K for the total protein in each periplasmic extract was used.
Japanese Patent Application Laid-Open No. 8-322586 (or,
MT-10765 (FERM BP-502) which is a known strain described in European Patent Publication No. 0735140).
The periplasmic extract extracted from 0) by the method described in the specification was compared with 1. Then, MT-10853 (FERM BP-) which co-expressed glutathione reductase was obtained.
6291) and MT-10852 (FERM BP-629) co-expressed with signal peptidase 1.
In 0), it was 8.3. Thus, the MT-10 of the present invention
852 (FERM BP-6290) and MT-10
20 extracted from 853 (FERMBP-6291)
The production amount of KhGH and the easiness of purification are greatly improved as compared with known methods, and the production cost of 20 KhGH can be reduced. Thus, the present inventors have accomplished the present invention.

That is, the present invention relates to (1) the E. coli Oppp
A secretion signal having a homology of 60% or more with A secretion signal (however, excluding Escherichia coli OpppA secretion signal and Salmonella OppA secretion signal), (2) DNA encoding the secretion signal of the above (1), (3) DNA encoding a secretion signal having a homology of 60% or more with E. coli OpppA secretion signal and a molecular weight of about 200
A recombinant plasmid containing a DNA encoding human growth hormone of No. 00, wherein the DNA encoding human growth hormone having a molecular weight of about 20,000 is bound immediately after the DNA encoding the secretion signal. Recombinant plasmid, (4) Escherichia coli transformant transformed with the recombinant plasmid of (3), (5) Escherichia coli transformant of (4) above was cultured, and a periplasm fraction was obtained from the resulting cells. A method for producing a human growth hormone having a molecular weight of about 20,000, which comprises purifying a periplasmic fraction solution obtained by extracting a human growth hormone having a molecular weight of about 20,000, (6) a DNA encoding a secretory signal, DNA encoding human growth hormone having a molecular weight of about 20,000, and Escherichia coli signal peptidase 1
A recombinant plasmid comprising DNA encoding
DNA encoding the secretion signal, wherein the 5 'end of the DNA encoding human growth hormone having a molecular weight of about 20,000
(7) a transformant of Escherichia coli transformed with the recombinant plasmid of the above (6), (8) a transformant of Escherichia coli of the above (7) The transformant is cultured, and a periplasmic fraction solution obtained by extracting a periplasmic fraction from the obtained cells is purified to obtain a human growth hormone having a molecular weight of about 20,000, which is a molecular weight of about 20,000. 20,000 human growth hormone production method. Further, the present invention provides
Changes in the medium composition when producing a human growth hormone having a molecular weight of about 20,000 by culturing the transformant transformed with the recombinant plasmid having the above constitution, that is, 0.01 to 10% of glycerol and 0.01 to succinic acid Included is a method for increasing the production by using a medium containing at least one of 5%.

[0038]

BEST MODE FOR CARRYING OUT THE INVENTION The secretion signals used in the present invention include Escherichia coli or Salumonella typhimurium OppA protein secretion signals (E. coli OppA secretion signals or Salmonella OppA secretion signals), and E. coli secretion OppA. A secretion signal (modified OppA secretion signal) obtained by modifying the amino acid sequence of a signal while maintaining the function as a secretion signal with homology of 60% or more.

The E. coli OppA secretion signal has the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing.
Examples of the ppA signal include the following modifications: (1) insertion of 1 to 3 basic amino acids into positions 2 to 7 of SEQ ID NO: 1, (2) positions 2 to 7 of SEQ ID NO: 1 (1) insertion of 1 to 9 hydrophobic amino acids into the 9th to 23rd portions of SEQ ID NO: 1 (4) Substitution of at least one of the non-hydrophobic basic amino acids in the portion from No. 9 to 23 of SEQ ID NO: 1 with hydrophobic amino acids.

Examples of the basic amino acids in the modifications (1) and (2) include Lys and Arg, and at least one of them can be used. Further, as the hydrophobic amino acid in the modification of (3) and (4), at least one amino acid selected from the group consisting of Leu, Ile, Val and Ala can be used.

Specific examples of the modified OppA signal include:
A modified OppA signal having the amino acid sequence of SEQ ID NOs: 2 to 27 can be mentioned. Preferred modified OppA described in SEQ ID NOs: 2 to 27 in these sequence listings
Among the secretion signals, the modified O described in SEQ ID NO: 23
The ppA secretion signal can be exemplified as a more preferable one showing the highest secretory productivity.

The secretion signal of Salmonella OppA has a very high homology with the OppA secretion signal of Escherichia coli. The Salmonella OpppA secretion signal is known and outside the scope of the present application, but it may be a Salmonella OppA modified secretion signal (eg, Salmonella OppA secretion signal and 60 amino acids in amino acid sequence).
% Of the modified secretion signal having homology of at least%) has the same effect as the E. coli OppA secretion signal or the modified secretion signal thereof, and the Salmonella OppA modified secretion signal is also within the scope of the present invention.

The recombinant DNA of the present invention has a structure in which the 5 'end of the DNA encoding 20KhGH is directly linked to the 3' end of the DNA encoding the Escherichia coli or Salmonella OppA secretion signal, or a variant thereof. ,
By expressing this in a host, a precursor in which an E. coli or Salmonella OppA secretion signal or a variant thereof is bound to the N-terminal side of 20KhGH can be obtained.

DNAs encoding 20KhGH include those constructed by chemical synthesis, and 20KhGH.
Any known cDNA such as a cDNA based on the mRNA sequence can be used. In addition, 20KhG
Regarding the 14th amino acid from the N-terminus of the amino acid sequence of H, two types of serine and methionine have been reported. That is, Masuda, N. et al, Biophyisica Acta, Vol.
In 949, p.125, (1988), the cDNA base sequence
(Codes serine), Martia1, J.A, eta1 Scie
nce, Vol, 205, p. 602, (1979), the RNA sequence encoding the 14th amino acid from the N-terminus of the mRNA is A
UG (encoding methionine). DNAs encoding any of them can be used in the present invention. Further, as the 20KhGH in the present invention, those in which 1 to 5 amino acids in the amino acid sequence have been substituted, deleted, inserted, or deleted within a range not impairing the characteristics and functions of 20KhGH are also included in the scope of the present invention. .

This recombinant DNA is combined with a promoter that enables its expression in the host, and used as a plasmid having a replication origin capable of replicating in the host, such as pBR322 and pUC19 for Escherichia coli. By integration, the recombinant plasmid for secretion of 20KhGH of the present invention can be obtained. As a promoter,
Any recombinant DNA can be used as long as it allows expression in a host, and it can be selected and used according to the type of host used and the like.

As a host, an Escherichia coli strain which has been used and is highly safe is preferable.
09 strains, HB101 strains, W3110 strains (ATCC 27
325). ATCC is the American Type Culture Collection number.

As an example of the recombinant plasmid, a DNA encoding a promoter, a ribosome binding site, a secretion signal and a DNA encoding 20KhGH are 5 '
To 3 ′ in this order.

On the other hand, signal peptidase 1 used for secretory production of 20KhGH by the co-expression system is derived from Escherichia coli. In addition, Escherichia coli-derived signal peptidase 1
The amino acid sequence of
Within a range where the effect of increasing the secretion amount of 0KhGH is obtained,
Two substitutions, insertions and deletions are also within the scope of the present invention.

The signal peptidase 1 gene is originally present in the chromosome of Escherichia coli, and a DNA encoding Escherichia coli-derived signal peptidase 1 can be used. Therefore, when Escherichia coli is used as a host in the present invention, an effect obtained by co-expression is obtained by expressing, in addition to the host's original one, those additionally introduced by artificial manipulation.

As a promoter for expressing the signal peptidase 1, any promoter can be used as long as it functions in a host. Since the co-expression level of signal peptidase 1 affects the secretion level of 20KhGH, it is preferable that the expression level is not excessively high. Therefore, for example, as described in the Examples, the expression level of signal peptidase 1 by the tac promoter is
It is desirable to use a method such as suppressing the ACI ^q. In addition, co-expression of signal peptidase 1
A method of incorporating a DNA encoding signal peptidase 1 into the same plasmid as a DNA encoding KhGH, a method of incorporating DNA encoding 20KhGH and DNA encoding signal peptidase 1 into separate plasmids, or Escherichia coli originally possessed Apart from the signal peptidase 1 gene, it can be carried out by a method of additionally incorporating DNA encoding signal peptidase 1 into the chromosome of Escherichia coli.

In the same plasmid, DNA encoding 20KhGH and DN encoding signal peptidase 1
Examples of a recombinant plasmid of a coexpression system incorporating A include, for example, a promoter, a ribosome binding site, a DNA encoding a secretion signal, a DNA encoding 20KhGH, a promoter and a signal peptidase 1
Has a configuration in which DNAs encoding are linked in this order in the 5 ′ to 3 ′ direction.

The secretion signal to be incorporated into the recombinant plasmid for the co-expression system may be any signal that enables secretion of 20 KhGH, and the E. coli or Salmonella OppA secretion signal described above or a modified version thereof is preferable. It can be mentioned as. In addition, 20K
As the DNA encoding hGH, the promoter for expressing it, the replication origin, and the like, those described above can be used.

The transformant of the present invention may be introduced into a host by transformation using the above-mentioned recombinant plasmid by a method known per se. Transformation of the host with the recombinant plasmid includes, for example, a method of preparing competent cells by the rubidium chloride method, adding the plasmid, and subjecting the host to heat shock at 42 ° C. for 1 minute to transduce the plasmid into the host cells. It can be easily implemented by an ordinary method.

In the obtained transformant, a precursor in which a secretion signal and 20KhGH are bound is expressed in the cytoplasm by a command from the promoter and the ribosome binding site sequence, and when the precursor passes through the inner membrane, The secretion signal is cleaved by signal peptidase 1 to secrete native 20K hGH. When Escherichia coli is used as a host, 20KhGH of a natural type is secreted and accumulated in the periplasm of Escherichia coli.

The transformant of Escherichia coli of the present invention which secretes and produces 20K hGH is MT-10853 (FERM B).
P-6291) or MT-10852 (FERM)
BP-6290). These strains were filed on March 11, 1998 with the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry, located at 1-3 1-3 Higashi, Tsukuba, Ibaraki Prefecture. Under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms,
Deposited with the above deposit number.

When Escherichia coli was used as a host, 2
As a method for culturing a transformant of Escherichia coli for secretory production of 0KhGH, a culture method using a flask, a culture method in a culture tank, and the like can be suitably selected. The culture temperature is preferably 25 to 37 ° C, and more preferably 28 to 30 ° C. As the medium composition, those usually used for culturing Escherichia coli can be used, but a double concentration LB medium (polypeptone 20 g / L, yeast extract 10 g / L), glycerol 0.01 to 10%, preferably 0 to 10% A medium containing 0.1 to 5%, 0.01 to 5% of succinic acid, preferably 0.1 to 4%, more preferably 0.1 to 3% is preferable. Such effects of glycerol and succinic acid are remarkable when glycerol and succinic acid are simultaneously added, but are also observed when glycerol or succinic acid is independently added to the medium.

Preparation of 20K hGH from a transformant of Escherichia coli can be easily performed by a conventional method for extracting and purifying a protein from a periplasm. For example, an osmotic shock method (Nossal G. N; J. Biol. Chem., 241 (13),
pp. 3055-3062, 1966). The extracted periplasmic fraction may be purified by a conventional protein purification method, for example, a column chromatography method. At this time, in the ion exchange column chromatography method, it is necessary to support the protein once on the carrier, and since a periplasmic fraction solution containing less impurity protein other than 20 KhGH is obtained as in the present invention, the capacity of the column can be reduced. There are advantages.

[0058]

The present invention will be described in more detail with reference to the following Examples, which do not limit the present invention in any way.

[Example 1] Examination of usefulness of OppA secretion signal

[1] 20K containing OppA secretion signal
Preparation of hGH secretion plasmid The oligonucleotides shown in SEQ ID NO: 28 and SEQ ID NO: 29 in the Sequence Listing were subjected to PCR (Polymerase Chain Reactor).
ion method) to form a double strand, and use the restriction enzymes SacI and Pst
And cut with Bacillus amyloliquefaciens ( Baci
llus amyloliquefacien s) DNA fragments encoding the front half portion of the promoter and OppA secretion signal from neutral protease (1) was obtained. Also, Japanese Patent Application Laid-Open
The MT-10765 strain (F
ERM BP-5020) has a secretory plasmid pG
Using HR10 as a template and the primers shown in SEQ ID NO: 30 and SEQ ID NO: 31, the latter half of the OppA secretion signal and a DNA fragment encoding 20 KhGH were amplified by PCR. Fragment (2) was obtained by digesting this PCR product with restriction enzymes PstI and XbaI. Furthermore, the secretory plasmid pGH
R10 was digested with restriction enzymes SacI and XbaI to obtain a vector fragment (3). By ligating the above DNA fragments (1), (2) and (3), a secretory plasmid pGHR52 was constructed. These steps are shown in FIG.

[2] Preparation of 20KhGH Secretory Plasmid Containing Modified OppA Secretion Signal Using pGHR52 prepared in [1] as a template,
The oligonucleotide shown in SEQ ID NO: 32 and SEQ ID NO:
PCR was carried out by selectively using various oligonucleotides shown in 33 to 39 to amplify a DNA fragment encoding a promoter derived from a neutral protease of Bacillus amyloliquefaciens and a modified portion of the OppA secretion signal. Each PCR product was treated with the restriction enzymes SacI,
Cleavage with PstI, fragments (4), (5),
(6), (7), (8), (9) and (10) were obtained. Thereafter, the above-mentioned fragments (2) and (3) and the fragment (4) are mixed, ligated, and transformed into Escherichia coli (ie, the fragment (4) is used instead of the fragment (1) shown in FIG. 1). As a result, a secretory plasmid pGHR521 was obtained. Similarly, fragment (4)
The fragment (5) is used instead of the fragment (5), and the secretory plasmid pGHR525 is used instead of the fragment (1), and the fragment (7) is used instead of the fragment (1). By using the fragment (8) instead of the fragment (1), the secretory plasmid pGHR527 can be used.
The secretion plasmid pGH was prepared by using GHR529 instead of fragment (1) with fragment (9).
R524 was replaced with fragment (10) in place of fragment (1) to allow secretion plasmid pGHR
526 were obtained (FIG. 2).

[3] 20K containing OppA secretion signal
Evaluation of Escherichia coli Transformant Transformed with hGH Secretory Plasmid The Escherichia coli W3110 strain (ATCC 27325) was transformed with the secretory plasmid pGHR52 constructed in [1] to obtain an Escherichia coli transformant W3110 / pGHR52. This transformant was treated with tetracycline 10 μg / ml.
And cultured at 30 ° C. for 18 hours on an LB agar medium containing The cultured cells were added to a liquid medium (polypeptone 20 g / L, yeast extract 10 g / L, succinic acid 10 g / L, glycerol 1
0 g / L, tetracycline 30 mg / L).
C., and cultured for 24 hours. The periplasm fraction containing 20 KhGH secreted and accumulated by the osmotic shock method was recovered from the cultured cells, and the periplasm fraction in the periplasm fraction extraction solution was recovered.
The concentration of 0KhGH was measured by an enzyme immunoassay using an antibody against hGH. From the measured value, 1 L of culture solution
Per 20 KhGH secretory production was converted. For comparison of secretory production, MT-10765 (FERM BP-
5020) was similarly cultured. Table 1 shows the results.

[0063]

[Table 1] From the results shown in Table 1, the E. coli transformant transformed with the plasmid using the OppA secretion signal was different from the E. coli transformant transformed with the plasmid using the conventional Bacillus amyloliquefaciens neutral protease-derived modified secretion signal. Was also found to have excellent secretory productivity of 20KhGH.

[4] Evaluation of Escherichia coli Transformants Transformed with Plasmids Using Modified OppA Secretion Signal
An E. coli transformant obtained by transforming 10 strains (ATCC 27325) was obtained. These transformants were cultured in the same manner as described in [3], and the amount of secretion was measured in the same manner. Table 2 shows the results.

[0065]

[Table 2] From the results in Table 2, the E. coli transformants transformed with the recombinant plasmid using the modified OppA secretion signal were:
20KhGH secretion production ability was superior to that of Escherichia coli transformants transformed with a plasmid using a modified secretion signal derived from a conventional Bacillus amyloliquefaciens neutral protease. In particular, E. coli transformant MT-10853 (FER) transformed with pGHR525 containing the modified OppA secretion signal of the sequence shown in SEQ ID NO: 3
MBP-6291) had the best secretory production ability.

Example 2 Usefulness of Signal Peptidase 1

[1] 20KhG co-expressing signal peptidase 1 using OppA modified secretion signal
Preparation of H secretion plasmid PCR of a DNA fragment encoding signal peptidase 1 from the chromosomal gene of Escherichia coli W3110 using the oligonucleotides shown in SEQ ID NO: 40 and SEQ ID NO: 41
This PCR product was amplified by the restriction enzyme MunI,
Cleavage with PstI yielded a DNA fragment (11) encoding signal peptidase 1. This fragment (11) and pGHR1 described in JP-A-8-322586 were used.
0 was cut with restriction enzymes EcoRI and PstI, the fragment (12) on the vector side was mixed, ligated by a conventional method, and then mixed with E. coli W3110 (ATCC 273).
25) to form the secreted plasmid pGH.
S15 (FIG. 3) was obtained. The obtained pGHS15 was digested with restriction enzymes SacI and XbaI to obtain a vector fragment (13). Further, the recombinant plasmid pGHR525 obtained in Example 1 was replaced with restriction enzymes SacI and XbaI.
To obtain a 20 KhGH expression cassette side fragment (14). Thereafter, the fragment (13) and the fragment (14) were mixed and ligated to obtain a secretion plasmid pGHS525 (FIG. 3). By transforming Escherichia coli W3110 using this plasmid, the transformant W3110 / pGHS525 (M
T-10852 (FERM BP-6290) was obtained.

[2] Evaluation of E. coli transformant transformed with plasmid using signal peptidase 1 Transformant W3110 / pGHS52 constructed in [1]
5 was cultured according to the method described in Example 1 [3].
The secretion amount of hGH was measured. Further, W3110 / pGHR10 and W3 recovered by the method described in Example 1 [3]
110 / pGHR525, W3110 / pGHS52
5 was determined according to the method of Groves et al. (Groves, WE et al., Anal. Bioc).
hem. 20Kh in total protein in the periplasmic fraction
The ratio of GH was compared with W3110 / pGHR10 as 1. Table 3 shows the results.

[0069]

[Table 3] From the results in Table 3, in the E. coli transformant obtained by transforming with the recombinant plasmid co-expressing signal peptidase 1, the secretory production of 20KhGH was obtained by transforming with the conventional plasmid co-expressing glutathione reductase. It was found that the ratio was 20 KhGH, which was equal to or higher than that of the obtained Escherichia coli transformant, and occupied 20 KhGH in the total protein of the periplasmic extraction solution.

[Example 3] 20K by changing culture conditions
Evaluation of hGH productivity E. coli transformant w311 prepared in [4] of Example 1
0 / pGHR525 (MT-10853 (FERM B
P-6291)), w3110 / pGHS525 (MT
-10852 (FERM BP-6290)) and, for comparison, a known strain w3110 / pGHR10 (MT-10765 (FE-10BP) described in JP-A-8-322586 (or European Patent Publication No. 0735140).
RM BP-5020) was subjected to the following evaluation by changing the culture method. 10 μg of tetracycline was used for each transformant.
/ Ml of LB agar medium at 30 ° C for 18 hours. The cultured cells were added to a liquid medium (20 g / L polypeptone, 10 g / L yeast extract, 15 g / L succinic acid, 36 g / L).
g / L glycerol, 30 mg / L tetracycline)
And cultured at 30 ° C. for 42 hours. From the cells after culture,
2 secreted and accumulated in the periplasm by osmotic shock
0KhGH was collected, and the concentration in the periplasmic fraction solution was measured by an enzyme immunoassay using an antibody against hGH. From the measured value, the amount of hGH secreted per 1 L of the culture solution was converted. The total amount of protein in each periplasmic fraction was determined by the method of Groves et al. (Groves, WE.
Et al., Anal. Biochem. , 22, 195-21
0,1968). The ratio of 20KhGH in the total protein in the periplasm fraction was determined according to [Example 1].
W3110 / pGHR10 from which the culture and the periplasmic fraction were extracted by the method described in [3] above was designated as 1. Table 4 shows the results.

[0071]

[Table 4]

[0072]

According to the present invention, there is provided a method for secreting and producing 20KhGH using Escherichia coli, which can secrete and produce 20KhGH in a larger amount than the conventional method.

Further, in the method of the present invention, the amount of contaminating proteins in the obtained periplasmic extraction solution is small, so that purification using the extraction solution is easy, and high-purity 20KhG can be mass-produced inexpensively. Become. Furthermore, by using a medium containing at least one of glycerol 0.01 to 10% and succinic acid 0.01 to 5%, 20KhG
Can be further increased.

[0074]

[Sequence List] SEQUENCE LISTING <110> Mitsui Chemicals, Incorporated <120> Method of Secretory Production of Human Growth Hormone <130> 31990032 <150> JP 193003/1998 <151> 1998-07-08 <160> 41 <210> 1 <211> 26 <212> PRT <213> E. Coli <220> <221> SIGNAL <222> (1) ... (26) <223> OppA secretion signal <400> 1 Met Thr Asn lle Thr Lys Arg Ser Leu Val Ala Ala Gly Val 1 5 10 Leu Ala Ala Leu Met Ala Gly Asn Val Ala Leu Ala 15 20 25 <210> 2 <211> 27 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (27) <223> Modified OppA secretion signal <400> 2 Met Thr Asn lle Thr Lys Arg Ser Leu Leu Val Ala Ala Gly Val Leu 1 5 10 15 Ala Ala Leu Met Ala Gly Asn Val Ala Leu Ala 20 25 <210> 3 <211> 28 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <220> <221> (1) ... (28) <223> Modified OppA secretion signal <400> 3 Met Thr Asn lle Thr Lys Arg Ser Leu Leu Leu Val Ala Ala Gly Val 1 5 10 15 Leu Ala Ala Leu Met Ala Gly Asn Val Ala Leu Ala 20 25 <210> 4 <211> 29 <212 > PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (29) <223> Modified OppA secretion signal <400> 4 Met Thr Asn lle Thr Lys Arg Ser Leu Leu Leu Leu Val Ala Ala Gly 1 5 10 15 Val Leu Ala Ala Leu Met Ala Gly Asn Val Ala Leu Ala 20 25 < 210> 5 <211> 30 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (30) <223> Modified OppA secretion signal <400> 5 Met Thr Asn lle Thr Lys Arg Ser Leu Leu Leu Leu Leu Val Ala Ala 1 5 10 15 Gly Val Leu Ala Ala Leu Met Ala Gly Asn Val Ala Leu Ala 20 25 30 <210> 6 <211> 31 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (31) <223> Modified OppA secretion signal <400> 6 Met Thr Asn lle Thr Lys Arg Ser Leu Leu Leu Leu Leu Leu Val Ala 1 5 10 15 Ala Gly Val Leu Ala Ala Leu Met Ala Gly Asn Val Ala Leu Ala 20 25 30 <210> 7 <211> 32 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) .. . (32) <223> Modified OppA secretion signal <400> 7 Met Thr Asn lle Thr Lys Arg Ser Leu Leu Leu Leu Leu Leu Leu Val 1 5 10 15 Ala Ala Gly Val Leu Ala Ala Leu Met Ala Gly Asn Val Ala Leu Ala 20 25 30 <210> 8 <211> 3 3 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (33) <223> Modified OppA secretion signal <400> 8 Met Thr Asn lle Thr Lys Arg Ser Leu Leu Leu Leu Leu Leu Leu Leu 1 5 10 15 Val Ala Ala Gly Val Leu Ala Ala Leu Met Ala Gly Asn Val Ala Leu 20 25 30 Ala <210> 9 <211> 34 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (34) <223> Modified OppA secretion signal <400> 9 Met Thr Asn lle Thr Lys Arg Ser Leu Leu Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Val Ala Ala Gly Val Leu Ala Ala Leu Met Ala Gly Asn Val Ala 20 25 30 Leu Ala <210> 10 <211> 27 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1). . (27) <223> Modified OppA secretion signal <400> 10 Met Thr Asn lle Thr Lys Lys Arg Ser Leu Val Ala Ala Gly Val Leu 1 5 10 15 Ala Ala Leu Met Ala Gly Asn Val Ala Leu Ala 20 25 <210 > 11 <211> 28 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (28) <223> Modified OppA secretion signal <400> 11 Met Thr Asn lle Thr Lys Lys Arg Ser Leu Leu Val Ala Ala Gly Val 1 5 10 15 Leu Ala Ala Leu Met Ala Gly Asn Val Ala Leu Ala 20 25 <210> 12 <211> 29 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (29) <223> Modified OppA secretion signal <400> 12 Met Thr Asn lle Thr Lys Lys Arg Ser Leu Leu Leu Val Ala Ala Gly 1 5 10 15 Val Leu Ala Ala Leu Met Ala Gly Asn Val Ala Leu Ala 20 25 <210> 13 <211> 30 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (30) <223> Modified OppA secretion signal <400> 13 Met Thr Asn lle Thr Lys Lys Arg Ser Leu Leu Leu Leu Val Ala Ala 1 5 10 15 Gly Val Leu Ala Ala Leu Met Ala Gly Asn Val Ala Leu Ala 20 25 30 <210> 14 <211> 31 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (31) <223> Modified OppA secretion signal <400> 14 Met Thr Asn lle Thr Lys Lys Arg Ser Leu Leu Leu Leu Leu Val Ala 1 5 10 15 Ala Gly Val Leu Ala Ala Leu Met Ala Gly Asn Val Ala Leu Ala 20 25 30 <210> 15 <211> 32 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (32 ) <223> Modified OppA secretion signal <400> 15 Met Thr Asn lle Thr Lys Lys Arg Ser Leu Leu Leu Leu Leu Leu Val 1 5 10 15 Ala Ala Gly Val Leu Ala Ala Leu Met Ala Gly Asn Val Ala Leu Ala 20 25 30 <210> 16 <211> 33 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (33) <223> Modified OppA secretion signal <400> 16 Met Thr Asn lle Thr Lys Lys Arg Ser Leu Leu Leu Leu Leu Leu Leu 1 5 10 15 Val Ala Ala Gly Val Leu Ala Ala Leu Met Ala Gly Asn Val Ala Leu 20 25 30 Ala <210> 17 <211> 34 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1 ) ... (34) <223> Modified OppA secretion signal <400> 17 Met Thr Asn lle Thr Lys Lys Arg Ser Leu Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Val Ala Ala Gly Val Leu Ala Ala Leu Met Ala Gly Asn Val Ala 20 25 30 Leu Ala <210> 18 <211> 35 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (35) <223> Modified OppA secretion signal <400> 18 Met Thr Asn lle Thr Lys Lys Arg Ser Leu Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Val Ala Ala Gly Val Leu Ala Ala Leu Met Ala Gly Asn Val 20 25 30 Ala Leu Ala <210 > 19 <211> 28 <212> PR T <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (28) <223> Modified OppA secretion signal <400> 19 Met Thr Asn lle Thr Lys Lys Lys Arg Ser Leu Val Ala Ala Gly Val 1 5 10 15 Leu Ala Ala Leu Met Ala Gly Asn Val Ala Leu Ala 20 25 <210> 20 <211> 29 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (29) <223> Modified OppA secretion signal <400> 20 Met Thr Asn lle Thr Lys Lys Lys Arg Ser Leu Leu Val Ala Ala Gly 1 5 10 15 Val Leu Ala Ala Leu Met Ala Gly Asn Val Ala Leu Ala 20 25 <210> 21 <211> 30 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (30) <223> Modified OppA secretion signal <400> 21 Met Thr Asn lle Thr Lys Lys Lys Arg Ser Leu Leu Leu Val Ala Ala 1 5 10 15 Gly Val Leu Ala Ala Leu Met Ala Gly Asn Val Ala Leu Ala 20 25 30 <210> 22 <211> 31 <212> PRT <213 > Artificial Sequence <220> <221> SIGNAL <222> (1) ... (31) <223> Modified OppA secretion signal <400> 22 Met Thr Asn lle Thr Lys Lys Lys Arg Ser Leu Leu Leu Leu Val Ala 1 5 10 15 Ala Gly Val Leu Ala Ala Leu Met Ala Gl y Asn Val Ala Leu Ala 20 25 30 <210> 23 <211> 32 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (32) <223> Modified OppA secretion signal <400> 23 Met Thr Asn lle Thr Lys Lys Lys Arg Ser Leu Leu Leu Leu Leu Val 1 5 10 15 Ala Ala Gly Val Leu Ala Ala Leu Met Ala Gly Asn Val Ala Leu Ala 20 25 30 <210> 24 < 211> 33 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (33) <223> Modified OppA secretion signal <400> 24 Met Thr Asn lle Thr Lys Lys Lys Arg Ser Leu Leu Leu Leu Leu Leu 1 5 10 15 Val Ala Ala Gly Val Leu Ala Ala Leu Met Ala Gly Asn Val Ala Leu 20 25 30 Ala <210> 25 <211> 34 <212> PRT <213> Artificial Sequence < 220> <221> SIGNAL <222> (1) ... (34) <223> Modified OppA secretion signal <400> 25 Met Thr Asn lle Thr Lys Lys Lys Arg Ser Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Val Ala Ala Gly Val Leu Ala Ala Leu Met Ala Gly Asn Val Ala 20 25 30 Leu Ala <210> 26 <211> 35 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (35) <223> Modified OppA secretion signal <400> 26 Met Thr Asn lle Thr Lys Lys Lys Arg Ser Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Val Ala Ala Gly Val Leu Ala Ala Leu Met Ala Gly Asn Val 20 25 30 Ala Leu Ala 35 <210> 27 <211> 36 <212> PRT <213> Artificial Sequence <220> <221> SIGNAL <222> (1) ... (36) <223> Modified OppA secretion signal <400> 27 Met Thr Asn lle Thr Lys Lys Lys Arg Ser Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu Val Ala Ala Gly Val Leu Ala Ala Leu Met Ala Gly Asn 20 25 30 Val Ala Leu Ala 35 <210> 28 <211> 80 <212> DNA <213> Artificial < 220> <222> (1) ... (80) <223> DNA sequence for PCR to produce a DNA fragment having a promoter of B. amyloliquefaciens and the first half part of OppA secretion signal. <400> 28 ttcgagctcg gtacccggag tctagtttta tattgcagaa tgcgagattg ctggtttatt 60 atacaatata agttttcatt 80 <210> 29 <211> 83 <212> DNA <213> Artificial <220> <222> (1) ... (83) <223> DNA sequence for PCR to produce a DNA fragment having a promoter of B. amyloliquefaciens and the first half part of OppA secretion signal. <400> 29 cgcctgcagc tactaaactt ct cttggtga tgttggtcat atgaaatccc cctttttgaa 60 aataatgaaa acttatattg tat 83 <210> 30 <211> 14 <212> DNA <213> Artificial Sequence <220> <222> (1) ... (14) <223> Dsigned DNA sequence to act as a primer for PCR to produce the latter half of a OppA secretion signal. <400> 30 aatttaactg tgat 14 <210> 31 <211> 76 <212> DNA <213> Artificial Sequence <220> <222> (1). .. (76) <223> Dsigned DNA sequence to act as a primer for PCR to produce the latter half of a OppA secretion signal. <400> 31 gtagctgcag gcgttctggc tgcgctaatg gcagggaatg tcgcgctggc attcccaact 60 ataccacttt cgcgcc 76 <210> 32 > 15 <212> DNA <213> Artificial Sequence <220> <222> (1) ... (15) <223> Designed DNA sequence to act as a primer for PCR to produce a DNA fragment having a promoter of B amyloliquefaciens and a modified first h alf part of OppA secretion signal. <400> 32 tgcaacttta tccgc 15 <210> 33 <211> 45 <212> DNA <213> Artificial Sequence <220> <222> (1) ... (45) <223> Designed DNA sequence to act as a primer for PCR to produce a DNA fragment havin ga promoter of B. amyloliquefaciens and a modified first half part of OppA secretion signal. <400> 33 gcctgcagct accagacttc ttttcttttt ggtgatgttg gtcat 45 <210> 34 <211> 51 <212> DNA <213> Artificial Sequence <220> <222 > (1) ... (51) <223> Designed DNA sequence to act as a primer for PCR to produce a DNA fragment having a promoter of B. amyloliquefaciens and a modified first h alf part of OppA secretion signal. <400 > 34 gcctgcagct accaataaca gacttctttt ctttttggtg atgttggtcat 51 <210> 35 <211> 57 <212> DNA <213> Artificial Sequence <220> <222> (1) ... (57) <223> Designed DNA sequence to act as a primer for PCR to produce a DNA fragment having a promoter of B. amyloliquefaciens and a modified first h alf part of OppA secretion signal. > DNA <213> Artificial Sequence <220> <222> (1) ... (63) <223> Designed DNA sequence to act as a primer for PCR to produce a DNA f ragment having a promoter of B. amyloliquefaciens a <400> 36 gcctgcagct acgagcagca ggagcaataa cagacttctt ttctttttgg tgatgttggt 60 cat 63 <210> 37 <211> 69 <212> DNA <213> Artificial Sequence <220> <222> (1 ) ... (69) <223> Designed DNA sequence to act as a primer for PCR to produce a DNA fragment having a promoter of B. amyloliquefaciens and a modified first half part of OppA secretion signal. <400> 37 gcctgcagct accagcagga gcagcaggag caataacaga cttcttttct ttttggtgat 60 gttggtcat 69 <210> 38 <211> 54 <212> DNA <213> Artificial Sequence <220> <222> (1) ... (54) <223> Designed DNA sequence to act as a primer for PCR to produce a DNA fragment having a promoter of B. amyloliquefaciens and a modified first h alf part of OppA secretion signal. <400> 38 gcctgcagct acgagcaata acagacttct tttctttttg gtgatgttgg tcat 54 <210> 39 <211> 60 <212> DNA <213> Artificial Sequence <220> <222> (1) ... (60) <223> Designed DNA sequence to act as a primer for PCR to produce a DNA fragment having a promoter of B. amyloliquefacie ns and a modified first h alf part of OppA secretion signal. <400> 39 gcctgcagct accagcagga gcaataacag acttcttttc tttttggtga tgttggtcat 60 <210> 40 <211> 30 <212> DNA <213> Artificial Sequence <220> <222> (1) ... (30) <223> Dsigned DNA sequence to act as a primer for PCR to produce a DNA fragment encoding signal peptidase 1 <400> 40 ttccaattga tggcgaatat gtttgccctg 30 <210> 41 <211> 30 <212> DNA < 213> Artificial Sequence <220> <222> (1) ... (30) <223> Dsigned DNA sequence to act as a primer for PCR to produce a DNA fr agment encoding signal peptidase 1 <400> 41 tttctgcagt taatggatgc cgccaatgcg 30

[Brief description of the drawings]

FIG. 1 is a diagram showing a method for constructing a 20KhGH secretion plasmid pGHR52.

FIG. 2. 20KhGH secretion plasmid pGHR521-
529 is a diagram illustrating the construction method of H.529.

FIG. 3 shows a method for constructing a 20KhGH secretion plasmid pGHS525.

[Explanation of symbols]

NP promoter A promoter Bacillus amyloliquefaciens glutathione derived neutral protease promoter region 20KhGH molecular weight of about 20000 genes human growth hormone GR E. coli reductase gene tac tac promoter region lacIq lacI ^q gene tetr tetracycline resistance gene oppA Signal E.coli oppA protein secretion signal m of -oppA signal Modified secretion signal of Escherichia coli opppA protein SPI signal peptidase 1 rop pBR322-derived rop gene ori pBR322-derived replication origin

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) C12R 1:19) (C12P 21/02 C12R 1:19) (72) Inventor Hiroshi Uchida Togo, Mobara City, Chiba Prefecture 1144 Mitsui Chemicals Co., Ltd. (72) Inventor Daishi Mochizuki 1144 Togo, Mobara-shi, Chiba Mitsui Chemicals Co., Ltd. (72) Inventor Kazuya Matsumoto 1144 Togo, Mobara-shi, Chiba Mitsui Chemicals Co., Ltd.

Claims (21)

[Claims] 1. A secretion signal that differs from the OppA secretion signal of Escherichia coli or Salmonella but has at least 60% homology in amino acid sequence with the Escherichia coli OpppA secretion signal, and is a DNA encoding the secretion signal.
Ability to secrete and produce human growth hormone with a molecular weight of about 20,000 using a microorganism containing a recombinant DNA in which the 3 'end of A and the 5' end of the DNA encoding human growth hormone with a molecular weight of about 20,000 are directly linked. Secretion signal. 2. The following modifications in the amino acid sequence of SEQ ID NO: 1 in the sequence listing: (1) insertion of 1 to 3 basic amino acids into the 2nd to 7th portions of SEQ ID NO: 1; Substitution of 1 to 3 amino acids of the acidic amino acids and neutral amino acids at positions 2 to 7 of SEQ ID NO: 1 with basic amino acids, (3) To positions 9 to 23 of SEQ ID NO: 1 (4) substitution of the non-hydrophobic amino acid in the 9th to 23rd amino acids of SEQ ID NO: 1 with 1 to 9 amino acids by a hydrophobic amino acid, The secretion signal according to claim 1 having the following. 3. The basic amino acids inserted or substituted at positions 2 to 7 of SEQ ID NO: 1 in the sequence listing are Lys and A
rg, which is an amino acid selected from SEQ ID NO:
The secretion signal according to claim 2, wherein the hydrophobic amino acid inserted or substituted in the portion 9 to 23 of 1 is an amino acid selected from the group consisting of Leu, Ile, Val and Ala. 4. The secretion signal according to claim 3, which has the amino acid sequence of SEQ ID NOs: 2 to 27 in the sequence listing. A DNA encoding the secretion signal according to any one of claims 1 to 4. 6. A recombinant DNA comprising a DNA encoding a secretion signal having 60% or more homology in amino acid sequence with OppA secretion signal of Escherichia coli, and a DNA encoding human growth hormone having a molecular weight of about 20,000. A recombinant DNA, wherein the 5 'end of the DNA encoding human growth hormone having a molecular weight of about 20,000 is directly linked to the 3' end of the DNA encoding the secretion signal. 7. The recombinant D according to claim 6, wherein the secretion signal having 60% or more homology in amino acid sequence with the E. coli OppA secretion signal is a modified secretion signal of the Escherichia coli or Salmonella OppA secretion signal.
NA. 8. The secretion signal according to claims 2 to 4, wherein the secretion signal has 60% or more homology in amino acid sequence with the E. coli OpppA secretion signal.
3. The recombinant DNA according to item 1. 9. A recombinant comprising a promoter and the recombinant DNA according to any one of claims 6 to 8, wherein the recombinant DNA is incorporated at a position capable of being expressed by the promoter. Plasmid. 10. The recombinant plasmid according to claim 9, wherein the promoter enables expression of the recombinant DNA in E. coli, and further has a replication origin in E. coli. An Escherichia coli transformant transformed with the recombinant plasmid according to claim 9 or 10. 12. The Escherichia coli transformant is FERM BP.
The E. coli transformant according to claim 11, which is -6291. 13. A human having a molecular weight of about 20,000 by culturing the transformant of Escherichia coli according to claim 11 or 12 and extracting a periplasmic fraction solution obtained by extracting a periplasmic fraction from the obtained cells. A method for producing human growth hormone having a molecular weight of about 20,000, comprising obtaining growth hormone. 14. When culturing an E. coli transformant, glycerol 0.01 to 10% and succinic acid 0.0
14. The method according to claim 13, wherein a medium containing at least one of 1 to 5% is used. 15. A recombinant DNA wherein the 5 'end of DNA encoding human growth hormone having a molecular weight of about 20,000 is directly linked to the 3' end of DNA encoding a secretory signal.
And DN encoding E. coli signal peptidase 1
A. A recombinant plasmid comprising A. 16. The DNA encoding a secretion signal,
A DNA encoding an E. coli or Salmonella OppA secretion signal, or the DN of claim 5.
The recombinant plasmid according to claim 15, which is A. 17. A promoter that enables expression of the recombinant DNA in Escherichia coli, a promoter that enables expression of the DNA encoding the signal peptidase 1 of Escherichia coli in Escherichia coli, and a replication origin in Escherichia coli. 17. The recombinant plasmid according to claim 15, further comprising: An Escherichia coli transformant transformed with the recombinant plasmid according to claim 16 or 17. 19. The Escherichia coli transformant is FERM BP.
The transformant of Escherichia coli according to claim 18, which is -6290. 20. The E. coli transformant according to claim 18 or 19, which is cultured, and a periplasmic fraction solution containing few contaminating proteins obtained by extracting a periplasmic fraction from the obtained cells is purified to obtain a molecular weight. A method for producing human growth hormone having a molecular weight of about 20,000, which comprises obtaining about 20,000 human growth hormone. 21. When culturing a transformant of Escherichia coli, glycerol 0.01 to 10% and succinic acid 0.0
The method according to claim 20, wherein a medium containing at least one of 1 to 5% is used.