JP3398427B2 - How growth hormone is produced - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing human growth hormone, more specifically a molecular weight of 200.
A human growth hormone secretory plasmid that enables the secretion and accumulation of 00 dalton human growth hormone (hereinafter referred to as 20KhGH) in the periplasm of Escherichia coli, and a method for producing human growth hormone using an Escherichia coli transformant into which the plasmid is introduced. It is about.

[0002]

2. Description of the Related Art Human growth hormone having a molecular weight of 22,000 daltons (hereinafter referred to as 22KhGH) is currently used for treating pituitary dwarfism. In addition, human growth hormone is expected to expand its application for treatment of diseases such as children with chronic renal failure, bone fracture and burns (90/9)
1 Nikkei Bio Yearbook). However, 22K hGH is known to have side effects. Regarding the side effects of 22KhGH, it has been pointed out from the results of animal experiments that there is a risk of leukemia development when it is used for treating humans (R
ogers et al. , The Lancet,
p. 434, August. 27.1977). In fact, by 1987 it was confirmed that human growth hormone users had a 10-fold increase in the incidence of leukemia than expected, which is not negligible (Fisher et a.
l. , The Lancet, p. 1159, May.
21.1988, and Watanabe et al.
The Lancet, p. 1159, May. 21.
1988). Regarding diabetogenic activity, which is another side effect of 22KhGH, it has been reported that a decrease in glucose tolerance was observed in 30% of recipients during a clinical trial of Turner syndrome (hormone and clinical, Vol. 38. , Pp.
155-159, 1990). In order to develop such human growth hormone for a new indication, human growth hormone which is less likely to induce side effects is desired in clinical practice.

In addition to 22KhGH currently used as human growth hormone, 20KhGH is known. This 20K hGH has a lower proliferative activity against leukemia than 22K hGH (Endocrinol. Ja.
pon, Vol. 36 (1), pp. 9-13,198
9) and the absence of diabetogenic activity (protein nucleic acid enzyme, Vol. 30 (9), pp. 1044-1050,
1985) is known.

In recent years, recombinant DNA technology has made it possible to express the human growth hormone gene using a microorganism as a host and to produce human growth hormone in the cells, in the cells, or in the periplasm. Regarding the extracellular production of 22KhGH, a secretory production method of 22KhGH using Bacillus subtilis has been reported (JP-A-1-273591). This 22
In the production of 20KhGH using the extracellular production method of KhGH, the produced protein is the secretory signal of the neutral protease of Bacillus amyloliquefaciens and 20KhGH.
It is a GH-bound form, which is different from the native 20K hGH in this respect. Further, in this case, as a result of the examination of the culture method, it has succeeded in secreting and accumulating about 1 mg / L · A660 of about 20 KhGH in the culture solution. However, it is difficult to say that the accumulated concentration is industrially sufficient, and it is necessary to further improve the accumulated concentration. Further, in this case, since a rich medium is used for culturing, it is not possible to directly subject the culture supernatant to column chromatography by an ordinary method in the separation and purification of 20K hGH from the culture solution, that is, an operation such as column chromatography. It is possible, and column chromatography can be applied only after diluting the culture supernatant 5-fold to 10-fold in advance. This leads to an increase in the size of the column chromatography device, which is a negative factor in terms of manufacturing cost. 20K like this
An industrially satisfactory method has not yet been established for the extracellular production of hGH.

On the other hand, as for intracellular production of human growth hormone, various intracellular productions of 22KhGH and 20KhGH using Escherichia coli have been studied by MIYAMOTO et al. (MIYAMOTO et al., INTE).
RNATINAL SYMPOSIUM ON GR
F, GROWTH HORMONONE AND SOMA
TOMEDIN PROGRAM AND ABSTR
ACTS Vol. 28, NOV. 1-2,198
6). MIYAMOTO et al. Aimed at high expression of 20KhGH and attempted high expression of 20KhGH by using the already established 22KhGH production method. But 20
It is reported that the expression level of KhGH was only 1/20 of 22KhGH. These are two things that simply follow the already established 22KhGH production method.
0KhGH could not be expressed efficiently in E. coli and
It suggests that there is an inherent problem with hGH expression.
That is, no industrially satisfactory method has been established for the intracellular production of 20K hGH using Escherichia coli, which is technically easier than secretory production.

Regarding the secretory production into the E. coli periplasm, Gray et al. Reported the secretory production of 22KhGH (Gray et al., GEN).
E, Vol. 39, pp. 247-254, 198
5). Periplasm refers to the space between the inner and outer membranes of Gram-negative bacteria including E. coli. In Gram-negative bacteria,
When a precursor protein having a signal sequence passes through the inner membrane, the secretory signal required for membrane penetration is cleaved. Then, a protein that is cleaved from a secretory signal and becomes a mature form generally does not pass through the outer membrane and accumulates in the periplasm. Gray et al. Have investigated the alkaline phosphatase gene (p) of E. coli in order to secrete and accumulate 22KhGH into the E. coli periplasm.
A plasmid in which the 22KhGH gene was ligated immediately after the region encoding the promoter and secretion signal of hoA) was constructed and introduced into E. coli. The report of Gray et al. Relates to 22KhGH and 20KhHG.
Is not described at all. 20KhHG is 2
It is a deletion of 15 amino acid residues from 2KhHG. Therefore, the higher-order structure of 20KhHG is changed as compared with 22KhHG, and even if it is attempted to directly produce 20KhHG using a system for intracellular expression of 22KhHG, the production amount is significantly higher than that of 22KhHG as shown in the above example. It will be low. In the intracellular expression method, the produced protein is a non-natural type in which methionine is added to the N-terminal of the target protein, and the higher-order structure of the protein is generally not a natural type. Therefore, a refolding step is required for protein production by the intracellular expression method. On the other hand, the secretory production method is superior to the intracellular production method in that a naturally-occurring target protein can be obtained. However, the secretory production method is technically more difficult, and the target protein is
It is susceptible to lysis, and its secretion is largely inhibited by the higher-order structure of the target protein. Therefore, some (eg 2
With the exception of 2KhGH), efficient secretory production of animal-derived proteins using prokaryotic cells has not been industrially achieved. Regarding secretory production of native 20K hGH, very low levels of production have been reported so far, but they are not industrially satisfactory. In addition, as shown in the embodiments of the present specification, the
Even if the secretory signal used for the industrial production of KhGH was simply used for the secretory production of 20 KhGH, a satisfactory secretory production amount was not obtained.

[0007]

The object of the present invention is to construct a 20KhGH secretion plasmid, transform Escherichia coli with the plasmid, culture the transformed strain, and secrete and accumulate 20KhGH in the periplasm. It is to provide an efficient production method of 20K hGH.

[0008]

As a result of various studies in view of the above points, Bacillus amyloliquefaciens ( Ba
cillus amyloliquefaciens )
A DNA fragment in which a human growth hormone gene encoding 20KhGH is bound immediately after a promoter, a ribosome binding site, and a region encoding a secretory signal involved in the expression of a neutral protease gene of origin is bound to vector DNA. It was found that 20 KhGH is secreted and accumulated in the periplasm of transformed Escherichia coli when E. coli is transformed with a human growth hormone secreting plasmid and the obtained transformant is cultured, and the present invention was completed based on this finding. did. That is, the present invention is a Bacillus
Immediately after the promoter, the ribosome binding site and the region encoding the secretion signal involved in the expression of the neutral protease gene derived from amyloliquefaciens, 20KhG
A DNA fragment in which the mature human growth hormone gene encoding H is bound is transformed into Escherichia coli with a human growth hormone secretory plasmid in which vector DNA is bound, and the resulting transformant is cultivated. Molecular weight 2
It is a production method of 20K hGH that secretes and accumulates 0000 Dalton of human growth hormone.

The present invention will be described in detail below. The promoter refers to a region recognized and bound by RNA polymerase, and in the present invention, a promoter involved in the expression of a neutral protease gene derived from Bacillus amyloliquefaciens is preferably used. Also, the ribosome binding site is mR synthesized by RNA polymerase.
NA refers to the region that binds to the ribosome, and in the present invention, a neutral protease gene derived from Bacillus amyloliquefaciens is particularly used. These regions play an important role in gene expression. Further, it is widely known that the gene sequences of these regions are related to the expression level of the gene. It is known that when a gene encoding a desired protein is expressed in Escherichia coli as a host, promoters and ribosome binding sites of various microorganisms other than E. coli can be recognized. The secretory signal refers to a polypeptide existing in the upstream portion of the N-terminal of the extracellular protein precursor. Since this polypeptide is removed during the process of secretion, it is considered to play an important role in passage of extracellular protein precursors through the cell membrane (GB.
robel et al. Cell. Biol. ，
Vol. 67, p. 835, (1975)). The region encoding a secretory signal refers to the region of the polypeptide gene, and in the present invention, a neutral protease gene derived from Bacillus amyloliquefaciens is particularly used. Periplasm refers to the space between the inner and outer membranes of Gram-negative bacteria including E. coli. In Escherichia coli, the precursor protein that has passed through the inner membrane cannot pass through the outer membrane because the secretory signal required for membrane penetration is cleaved. Therefore, it accumulates in the periplasm.
Unlike the extracellular production in Bacillus subtilis described above, the protein can be recovered in a concentrated liquid without being released into the medium, and the types and amounts of other proteins contained in the periplasm are small. The subsequent purification operation becomes easy. The human growth hormone gene encoding 20K hGH includes those constructed by chemical synthesis, and 20K
Any cDNA such as cDNA based on hGH mRNA may be used, and known ones can be used. As the vector DNA for constructing the hGH secretion plasmid of the present invention, any vector can be used as long as it has a replication original that enables replication in E. coli, and examples thereof include pBR322 and pUC19. Can be done.

The promoter, the ribosome binding site, the region encoding the secretory signal and the gene encoding 20K hGH are sequence-ligated in this order in the 5'to 3'direction. As a result, a precursor in which a secretion signal and 20KhGH are bound is expressed by the instructions of the promoter and the ribosome binding site, and the obtained precursor is secreted into the periplasm of E. coli. From a practical point of view, when constructing a heterologous protein secretion plasmid capable of binding a heterologous protein gene downstream of a gene promoter, a ribosome binding site and a region encoding a secretory signal and capable of replicating in E. coli. Any gene derived from the extracellular enzyme gene secreted and produced outside the bacterial cell can be used, and various genes are known as such extracellular enzyme gene. However, simply binding a heterologous protein gene downstream of the promoter, ribosome binding site and secretory signal coding region of these genes does not always ensure that the desired heterologous protein is secreted and produced. This is also clear from Comparative Example 1. In the present invention, the promoter, the ribosome binding site, and the region encoding the secretion signal for secreting and producing the native 20K hGH in the E. coli periplasm include:
It was found that those derived from the Bacillus amyloliquefaciens neutral protease gene showed excellent results. Then, immediately after the promoter derived from the neutral protease gene, the ribosome binding site and the secretory signal, the DNA fragment binding the human growth hormone gene is ligated to the vector DNA to construct a human growth hormone secretory plasmid. The above-mentioned four units (promoter, ribosome binding site, region encoding secretory signal and gene encoding 20K hGH) are linked so as to achieve the above-mentioned object. The sequence between the promoter, the ribosome binding site and the region encoding the secretory signal is not particularly limited as long as the purpose can be achieved. Furthermore, regarding the 14th amino acid from the N-terminal of the amino acid sequence of 20K hGH, there are two types of reports, serine and methionine. That is, Masuda, N .; et a
l. , Biophysica Acta, Vol. 9
49, p. 125, (1988), the cDNA nucleotide sequence is AGT (encoding serine), and
l, J. A. et al Science, Vol. Two
05, p. 602, (1979), the RNA sequence encoding the 14th amino acid from the N-terminus of mRNA is AUG (encoding methionine). A method known per se can be used to construct the human growth hormone secretory plasmid. Examples of human growth hormone secretory plasmids include pSTY12 and pGHW3.

Using the human growth hormone secreting plasmid, Escherichia coli can be transformed by an ordinary method to obtain an E. coli transformant. The strain that can be used here may be any E. coli that can be transformed with the 20K hGH secretion plasmid of the present invention, but is preferably Escherichia coli which is not pathogenic and has abundant experience in use. Escherichia coli HB101 strain (Escherichia coli HB101) ( Escherich
ia coli HB101) or W3110 (ATC
C 27325) is preferably used. Thus, an Escherichia coli transformant carrying a human growth hormone secretory plasmid can be obtained. Examples of such strains include MT-012 (Microtechnical Laboratory Article 3966, FERM BP-3966) and MT-.
10712 (Ministry of Fine Arts, Article 4361, FERM BP
-4361) can be mentioned.

In order to culture the Escherichia coli transformant strain, it may be cultured by a known culture method using a medium composed of a carbon source, a nitrogen source and inorganic salts that can be assimilated by the strain, and a liquid culture method is used. It is preferably used. Of the medium components of the medium, those usually used as a carbon source may be used,
The inventors of the present invention have found that the following problems occur when glucose, which is the most commonly used carbon source, is used. That is, glucose was added to LB medium (polypeptone 20 g / L, yeast extract 10 g / L) at a double concentration at a low concentration (0.1 to 0.5%) to give an initial pH of 7.5 and a culture temperature of 30 ° C. Culture at 20KhG
The maximum concentration of H reaches 5 to 10 mg per liter of the culture solution, but since it rapidly decomposes, it is necessary to collect cells after culturing (1 to
During 2 hours, the accumulated amount of 20K hGH decreased. Generally, the cause of such a decrease in the accumulated amount after culturing is that when the glucose in the medium becomes low due to the consumption of the bacterial cells in the expression of the heterologous protein in the transformant, the accumulated amount of the target protein is increased by proteolysis. It is known to decrease rapidly (Trends in Biotechnology
Vol. 10, 0 pp. 310-315, 1992).
Further, in the culture in which a high concentration (0.5 to 2.0%) of glucose was added under the above-mentioned culture conditions, the higher the glucose concentration, the lower the concentration of glucose after the culture observed when the glucose was low. Although the decrease in the accumulated amount of 20KhGH was small, the growth was suppressed due to the decrease in pH over time during the culture.
The maximum cumulative concentration of GH decreased to about 2-8 mg / L.
With respect to such a problem caused by using glucose as a carbon source, it was considered that the influence of the suppression of expression by catabolite repression by glucose was possible. Therefore, the same study was performed using other substances that can be a carbon source. As a result, when the same culture was performed using glycerol as a carbon source, growth inhibition due to an extreme decrease in pH did not occur at a high concentration (1.0 to 2.0%), and 20K hGH was about 10 per 1 L of the culture solution. Accumulated up to -20 mg. Also,
In the same culture with a low concentration of glycerol (0.1 to 0.5%), no extreme decomposition after the culture took place for 3 hours or more,
The accumulated concentration was maintained at 20 mg or more per liter of culture solution. As described above, even when a carbon source such as glucose is used, it is possible to produce a sufficient amount of 20KhGH to achieve the object of the invention, but it is possible to suppress the growth of bacteria by degrading the produced 20KhGH or decreasing the pH. It is necessary to take measures to avoid this. Therefore, by adding glycerol as a carbon source to the medium, it is possible to overcome the two problems of growth inhibition due to pH decrease and rapid decomposition of the target substance after culturing. Glycerol can be preferably used as the carbon source. When the concentration of glycerol added to the medium is less than 0.1%, the accumulated substances are significantly decomposed after culturing as in the case of no addition of glycerol, and the concentration of microbial cells exceeds 2.0%. Since the growth slowed down and the accumulated amount of 20K hGH also decreased,
It is desirable to add in the range of 1 to 2.0%.

Among the medium components of the medium used for the culture of the present invention, polypeptone and yeast extract are preferably used as the nitrogen source, and the concentration of the component is the usual concentration (polypeptone 10 g / L, yeast extract 5 g / l). Although it can be cultured in L), a higher concentration is preferable because both the cell growth rate and the accumulated amount of 20K hGH are higher. However, when the added concentration of the component is extremely high, 20K hGH from the culture is
Since it may interfere with the purification of LB medium, LB medium (polypeptone 20 g / L, yeast extract 10
A concentration on the order of g / L) is used.

The culture temperature of the culture of the present invention is 20 ° C to 30 ° C.
It is preferable to carry out the treatment in the range described above because a large amount of 20K hGH can be stably obtained for the following reason. That is, in culturing the Escherichia coli transformant of the present invention, when culturing was carried out at various culturing temperatures using glycerol as a carbon source in order to investigate the influence of the culturing temperature, surprisingly, the culturing result at 25 ° C. was: The cell growth rate, the cell concentration reached, and the amount of accumulated 20KhGH were better than those at 30 ° C, and the accumulated concentration of 20KhGH was 3 per 1 L culture solution.
Reached 0 mg or more. At a culture temperature of less than 20 ° C, the growth of bacterial cells was extremely slow. Under the conditions of higher than 30 ° C., the bacterial growth was poor and 20KhGH was accumulated only in a small amount. The temperature normally used for the production of E. coli 3
At 7 ° C, a bacteriolysis phenomenon was often observed, and growth was sometimes impossible. The cause of such a phenomenon is not always clear, but it is considered that the cell growth is inhibited by the cytotoxicity due to the increase in the intracellular accumulation concentration of the product (20KhGH) due to the high temperature.

By culturing the Escherichia coli transformant of the present invention, 20 KhG was contained in the periplasm of the transformant.
H is secreted and accumulated. The 20K hGH can be prepared from the periplasm of the E. coli transformant strain by a conventional method for recovering and purifying the protein from the periplasm, for example, an osmotic shock method (Nossal G. et al.
N; J. Biol. Chem. Vol. 241 (1
3), pp. 3055-3062, 1966) and the like can be implemented.

As described above, according to the production method of the present invention, a significant amount of 20KhGH was added to the periplasm of an Escherichia coli transformant strain.
20 can be secreted and accumulated in the periplasm
KhGH can be finally recovered as a prepared periplasmic protein solution.

[0017]

EXAMPLES The present invention will now be described with reference to specific examples, but the present invention is not limited to these examples.

Example 1 Construction of 20KhGH Secretion Plasmid pSTY12 Immediately after the promoter, the ribosomal binding site and the secretory signal coding region involved in the expression of the neutral protease gene derived from Bacillus amyloliquefaciens, a region coding for 20KhGH was constructed. The containing DNA fragment was prepared from the plasmid phGH727 containing the region. The plasmid phGH727 is the deposited strain MT-727 (F
ERM-BP-3571), the method of Tabak et al. (Tabak et al., Nucleic Acid).
s Res. , Vol. 5, p. 2321, 1978)
Was prepared using. Next, a DNA fragment of about 2.3 Kb (hereinafter referred to as DNA fragment A) generated by digesting phGH727DNA with restriction enzymes EcoRI and SphI was prepared by gel electrophoresis using an agarose gel. This has the sequence of SEQ ID NO: 1 in the sequence listing.

Further, a vector DNA replicable in Escherichia coli
For this, pUC19 (purchased from Takara Shuzo) was used. p
UC19 was digested with restriction enzymes EcoRI and SphI to obtain a 2.64 Kb DNA fragment (hereinafter referred to as DNA fragment B).
Was prepared). The DNA fragments A and B prepared as described above were ligated using 0.01 μg of each DNA ligation kit (manufactured by Takara Shuzo) to give a molecular weight of about 4.
The 9 Kb plasmid pSTY12 was constructed. This pS
TY12 is a hybrid plasmid capable of replicating in Escherichia coli in a form in which a promoter of the neutral protease gene of Bacillus amyloliquefaciens, a ribosome binding site and a region coding for mature 20KhGH are linked immediately after the secretion signal.

Example 2 Construction of 20K hGH Secretion Plasmid pGHW3 Similar to Example 1, plasmid phGH727 was digested with restriction enzymes EcoRI and SphI, and the digested product was subjected to gel electrophoresis using agarose gel to give about 2.3 Kb. DNA fragment A was prepared. On the other hand, pBR322 (purchased from Takara Shuzo), which is a vector DNA replicable in E. coli, was digested with restriction enzymes EcoRI and ScaI, and the degradation product was subjected to gel electrophoresis using an agarose gel to obtain a 3.8 Kb DNA fragment ( Below DN
A fragment C) was prepared. The SphI end of DNA fragment A and the Sc of DNA fragment C prepared as described above
The aI ends were made blunt ends and ligated in the same manner as in Example 1, and the plasmid pGHW having a molecular weight of about 6.1 Kb was obtained.
3 was built. Similar to pSTY12 described in Example 1, this plasmid pGHW3 also matures immediately after the promoter, ribosome binding site and secretion signal of the neutral protease gene of Bacillus amyloliquefaciens 20.
It is a hybrid plasmid that can be replicated in E. coli in a form in which a region encoding KhGH is bound. Example 3 Production of native 20K hGH Using the plasmid pSTY12 constructed in Example 1,
Escherichia coli HB101 (Takara Shuzo) was transformed to obtain a transformant MT-012 (FERM BP-3966). Furthermore, E. coli W3110 (ATCC 273) was prepared by a known method using the plasmid pGHW3 constructed in Example 2.
25) and transformed strain MT-10712 (FE
RM BP-4361) was obtained. Each of the obtained two transformants was cultured in 20 ml of a double concentration of LB medium, and the concentration of 20KhGH contained in the culture supernatant fraction, the periplasm fraction, and the intracellular fraction was measured with an antibody against human growth hormone. Enzyme immunoassay used (Kato K et a
l. J. Immunol. , Vol. 116, p. 1
554, 1976). The method for preparing each fraction was generally according to the method of Heppel et al. (Nossal
G. N; J. Biol. Chem. Vol. 241
(13), pp. 3055-3062, 1966), and the periplasmic fraction was prepared as follows. That is, the culture solution is centrifuged to collect the bacterial cells, and
1/0 volume of isotonic solution (20% sucrose, 10m
10 mM Tris-HCl buffer containing MEDTA (pH
7.5)). After the suspension was left for 30 minutes, it was centrifuged to collect bacterial cells. Next, the recovered cells were suspended in cold water at 4 ° C. to extract the protein present in the periplasm of the cells into cold water. The extract was centrifuged to remove bacterial cell components, and the supernatant was collected as a periplasmic fraction. The results are shown in Table-1 (Table 1). Approximately 10 mg per liter of culture medium for the two types of cultured transformants
Accumulation of 20K hGH was observed in the periplasmic fraction. Since the volume of the solution was reduced to 1/10 in the process of preparing the periplasmic fraction, the concentration of 20KhGH was concentrated 10 times, resulting in the concentration of 20KhGH of the periplasmic fraction being 100 mg / L, respectively.

Next, 2 secreted and accumulated in the periplasm
To characterize 0KhGH, 20KhGH was purified from the periplasmic fraction. 10 mM periplasmic solution
After addition to a DE-52 column (manufactured by Whatman) equilibrated with Tris Buffer (pH 7.5), N
It was purified by eluting with a gradient of aCl (0-0.8M). Next, purified 20K hGH (1 nm
ole) was used to determine the N-terminal amino,
It was found to have an N-terminal amino acid sequence that corresponds to native 20K hGH. In addition, purified 20K hGH
As a result of analysis of the amino acid composition of the above, it was in good agreement with the theoretical value calculated from the natural type 20KhGH.

[0022]

[Table 1]

Comparative Example 1 Construction of 20K hGH secretion plasmid pSTY11 and production of 20 KhGH of Escherichia coli transformed with the plasmid pSTY11 is a promoter and a ribosome binding site involved in the expression of a neutral protease gene derived from Bacillus amyloliquefaciens. It is a hybrid plasmid that is replicable in Escherichia coli in a form in which the region encoding the secretion signal of the Escherichia coli outer membrane protein A gene (OmpA) is bound immediately thereafter, and further immediately thereafter the region encoding 20KhGH is bound. E. coli was transformed with pSTY11, and the resulting E. coli transformant strain E. Coli HB101 (pST
Y11) was cultured in the same manner as in Example 3 to prepare an extracellular fraction, a periplasmic fraction and a culture supernatant, and the amount of 20KhGH contained in each fraction was measured by an enzyme immunoassay method. The results are shown in Table 2 (Table 2). Comparing Table-1 (Table 1) and Table-2 (Table 2), it can be seen that the amount of 20K hGH contained in each fraction is larger in Table-1 (Table 1). In constructing a human growth hormone secretory plasmid for secretory accumulation of 20K hGH in the periplasm, it is not necessary to use any secretory signal, but it is necessary to select and use an optimal secretory signal. It is clearer than the comparison. The secretory signal used in this case encodes the secretory signal of the neutral protease gene derived from Bacillus amyloliquefaciens rather than the region encoding the secretory signal of the Escherichia coli outer membrane protein A gene (OmpA). It turns out that it is more appropriate to use the area to be filled.

[0024]

[Table 2]

Example 4 Achievement concentration of cultured cells and 20K hGH depending on the difference of carbon source
A solution containing 20 g / L of accumulated concentration of peptone, 10 g / L of yeast extract and 0.5% and 1.5% of the substances shown in Table 3 (Table 3) was prepared, and pH was adjusted to 7 using 5N KOH solution. Adjusted to 5. After high-pressure steam sterilization, ampicillin sodium was further added aseptically to a concentration of 100 mg / L. Three platinum loops of MT-012 strain were inoculated into this medium (30 ml), and the mixture was shake-cultured at a culture temperature of 30 ° C. Cell concentration over time in each medium and 20Kh in periplasm
Table 3 shows the results of measuring the change in GH concentration and measuring the time required for the bacterial cell concentration to reach a maximum, the maximum value of 20KhGH concentration and the concentration of 20KhGH 3 hours after reaching the maximum concentration.
It shows in (Table 3). The microbial cell concentration was determined by measuring the absorbance at 660 nm, and 20 KhGH in the periplasm was quantified by the enzyme immunoassay of the periplasmic solution prepared in the same manner as in Example 2. When glycerol is used, there is no growth inhibition due to extreme pH drop even at high concentration (1.5%), and there is rapid degradation of 20K hGH after reaching the maximum cell concentration at low concentration (0.2%). I can't.

[0026]

[Table 3]

Example 4 Culture temperature, concentration reached by cultured cells and accumulated concentration of 20K hGH Polypeptone 20 g / L, yeast extract 10 g / L and a solution containing 0.5% glycerol were prepared, and 5N K was defined.
The pH was adjusted to 7.5 with an OH solution. After high-pressure steam sterilization, ampicillin sodium was further added aseptically to a concentration of 100 mg / L. This medium (30 ml)
The MT-012 strain was inoculated with 3 platinum loops, and shake-cultured at the culture temperature shown in Table 4 (Table 4). Table 4 shows the results obtained by measuring the time-dependent change in the concentration of 20K hGH in the periplasm and the concentration of 20 KhGH in the periplasm, and the maximum value of the 20 KhGH concentration at each culture temperature.
It shows in (Table 4). The microbial cell concentration was determined by measuring the absorbance at 660 nm, and 20 KhGH in the periplasm was quantified by the enzyme immunoassay of the periplasmic solution prepared in the same manner as in Example 2. Culture temperature 20 ℃
The cells grew slowly in, and hardly grew at 37 ° C.

[0028]

[Table 4]

[0029]

EFFECTS OF THE INVENTION A mature human growth hormone gene encoding 20KhGH is bound immediately after a promoter, a ribosome binding site and a region encoding a secretory signal involved in the expression of a neutral protease gene derived from Bacillus amyloliquefaciens. It is possible to secrete and accumulate 20KhGH in the periplasm by constructing a human growth hormone secretory plasmid in which the DNA fragment containing the vector DNA is bound, introducing it into Escherichia coli, and culturing the obtained transformant strain. became. Then, by separating and collecting 20KhGH in the periplasm, 20KhGH
A large amount of GH can be produced. In addition, the method of the present invention differs from the case of extracellular production in which 2
20K without recovering 0K hGH from dilute solution
20K hGH can be recovered in a solution in which hGH is concentrated, and other proteins contained in the periplasm are less than those in the medium, and the amount thereof is also small, which facilitates the subsequent purification operation.

[0030]

[Sequence list]

[0031] SEQ ID NO: 1 Sequence length: 859 Sequence type: Nucleic acid Number of chains: double-stranded Topology: linear Sequence Type: Genomic DNA origin   Organism name: Bacillus amyloliquefaciens   Share Name: MT-272 Sequence features   179-208 S promoter   236-242 S RBS   251-331 S sig peptide   332-859 E mat peptide Array GATCTTAACA TTTTTCCCCT ATCATTTTTC CCGTCTTCAT TTGTCATTTT TTCCAGAAAA 60 AATCGTCATT CGACTCATGT CTAATCCAAC ACGTCTCTCT CGGCTTATCC CCTGACACCG 120 CCCGCCGACA GCCCGCATGG ACGAATCTAT CAATTCAGCC GCGGAGTCTA GTTTTATATT 180 GCAGAATGCG AGATTGCTGG TTTATTATAA CAATATAAGT TTTCATTATT TTCAAAAAGG 240 GGGATTTATT ATG GGT TTA GGT AAG AAA TTG TCT AGT GCT GTA GCC GCT TCC 292            Met Gly Leu Gly Lys Lys Leu Ser Ser Ala Val Ala Ala Ser                    -25 -20 -15 TTT ATG AGT TTA ACC ATC AGT CTG CCG GGT GTT CAG GCC TTC CCA ACT 340 Phe Met Ser Leu Thr Ile Ser Leu Pro Gly Val Gln Ala Phe Pro Thr             -10 -5 1 ATA CCA CTT TCG CGC CTA TTC GAT AAC GCA AGT CTA CGT GCT CAC CGA 388 Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Ser Leu Arg Ala His Arg       5 10 15 CTA CAT CAG CTG GCC TTT GAC ACC TAC CAG GAG TTT AAC CCC CAG ACC 436 Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Asn Pro Gln Thr  20 25 30 35 TCC CTC TGT TTC TCA GAG TCT ATT CCG ACA CCC TCC AAC AGG GAG GAA 484 Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu                  40 45 50 ACA CAA CAG AAA TCC AAC CTA GAG CTG CTC CGC ATC TCC CTG CTG CTC 532 Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu              55 60 65 ATC CAG TCG TGG CTG GAG CCC GTG CAG TTC CTC AGG AGT GTC TTC GCC 580 Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala          70 75 80 AAC AGC CTG GTG TAC GGC GCC TCT GAC AGC AAC GTC TAT GAC CTC CTA 628 Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu      85 90 95 AAG GAC CTA GAG GAA GGC ATC CAA ACG CTG ATG GGG AGG CTG GAA GAT 676 Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp 100 105 110 115 GGC AGC CCC CGG ACT GGG CAG ATC TTC AAG CAG ACC TAC AGC AAG TTC 724 Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe                 120 125 130 GAC ACA AAC TCA CAC AAC GAT GAC GCA CTA CTC AAG AAC TAC GGG CTG 772 Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu             135 140 145 CTC TAC TGC TTC AGG AAG GAC ATG GAC AAG GTC GAG ACA TTC CTG CGC 820 Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg         150 155 160 ATC GTG CAG TGC CGC TCT GTG GAG GGC AGC TGT GGC TTC 859 Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe     165 170 175

[0032] SEQ ID NO: 2 Sequence length: 859 Sequence type: Nucleic acid Number of chains: double-stranded Topology: linear Sequence type: Genomic DNA origin   Organism name: Bacillus amyloliquefaciens   Share Name: MT-272 Sequence features   179-208 S promoter   236-242 S RBS   251-331 S sig peptide   332-859 E mat peptide Array GATCTTAACA TTTTTCCCCT ATCATTTTTC CCGTCTTCAT TTGTCATTTT TTCCAGAAAA 60 AATCGTCATT CGACTCATGT CTAATCCAAC ACGTCTCTCT CGGCTTATCC CCTGACACCG 120 CCCGCCGACA GCCCGCATGG ACGAATCTAT CAATTCAGCC GCGGAGTCTA GTTTTATATT 180 GCAGAATGCG AGATTGCTGG TTTATTATAA CAATATAAGT TTTCATTATT TTCAAAAAGG 240 GGGATTTATT ATG GGT TTA GGT AAG AAA TTG TCT AGT GCT GTA GCC GCT TCC 292            Met Gly Leu Gly Lys Lys Leu Ser Ser Ala Val Ala Ala Ser -25 -20 -15 TTT ATG AGT TTA ACC ATC AGT CTG CCG GGT GTT CAG GCC TTC CCA ACT 340 Phe Met Ser Leu Thr Ile Ser Leu Pro Gly Val Gln Ala Phe Pro Thr -10 -5 1 ATA CCA CTT TCG CGC CTA TTC GAT AAC GCA ATG CTA CGT GCT CAC CGA 388 Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg       5 10 15 CTA CAT CAG CTG GCC TTT GAC ACC TAC CAG GAG TTT AAC CCC CAG ACC 436 Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Asn Pro Gln Thr  20 25 30 35 TCC CTC TGT TTC TCA GAG TCT ATT CCG ACA CCC TCC AAC AGG GAG GAA 484 Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu                  40 45 50 ACA CAA CAG AAA TCC AAC CTA GAG CTG CTC CGC ATC TCC CTG CTG CTC 532 Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu              55 60 65 ATC CAG TCG TGG CTG GAG CCC GTG CAG TTC CTC AGG AGT GTC TTC GCC 580 Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala          70 75 80 AAC AGC CTG GTG TAC GGC GCC TCT GAC AGC AAC GTC TAT GAC CTC CTA 628 Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu      85 90 95 AAG GAC CTA GAG GAA GGC ATC CAA ACG CTG ATG GGG AGG CTG GAA GAT 676 Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp 100 105 110 115 GGC AGC CCC CGG ACT GGG CAG ATC TTC AAG CAG ACC TAC AGC AAG TTC 724 Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe                 120 125 130 GAC ACA AAC TCA CAC AAC GAT GAC GCA CTA CTC AAG AAC TAC GGG CTG 772 Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu             135 140 145 CTC TAC TGC TTC AGG AAG GAC ATG GAC AAG GTC GAG ACA TTC CTG CGC 820 Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg         150 155 160 ATC GTG CAG TGC CGC TCT GTG GAG GGC AGC TGT GGC TTC 859 Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe     165 170 175

[Brief description of drawings]

FIG. 1 shows the construction method of 20K hGH secretion plasmid pSTY12.

[Explanation of symbols]

PM Promoter region required for expression of Bacillus amyloliquefaciens-derived neutral protease gene SD Ribosomal binding site of Bacillus amyloliquefaciens-derived neutral protease gene SS Bacillus amyloliquefaciens-derived neutral protease gene 20KhGH, a region encoding a secretory signal for humans. Human growth hormone gene encoding 20KhGH.

Continuation of the front page (56) Reference JP-A-4-316488 (JP, A) Science, 1979, Vol. 205, No. 4406, p. 602-607 Nucleic Acids Research, 1981, Vol. 9, No. 15, p. 3719-3730 Biochimica et Bio physica Acta, 1988, Vol. 949, p. 125-131 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/00-15/09 PubMed

Claims (6)

(57) [Claims] 1. A human growth hormone gene having a molecular weight of 20,000 daltons is bound immediately after a promoter, a ribosome binding site and a region encoding a secretory signal involved in the expression of a neutral protease gene derived from Bacillus amyloliquefaciens. DNA fragment is vector D
Escherichia coli was transformed with the human growth hormone secreting plasmid bound to NA, the E. coli transformant was cultured, and a molecular weight of 2000 was added to the periplasm of the E. coli transformant.
A method for producing human growth hormone having a molecular weight of 20,000 daltons, which comprises secretory accumulation of 0 dalton human growth hormone. 2. A promoter involved in gene expression,
The DNA fragment in which the human growth hormone gene having a molecular weight of 20,000 daltons is bound immediately after the region encoding the ribosome binding site and the secretory signal is the nucleotide sequence set forth in SEQ ID NO: 1 in the Sequence Listing. A method for producing human growth hormone having a molecular weight of 20,000 daltons. 3. A promoter involved in gene expression,
The DNA fragment in which the human growth hormone gene having a molecular weight of 20,000 daltons is bound immediately after the ribosome binding site and the region encoding the secretory signal is the nucleotide sequence of SEQ ID NO: 2 in the Sequence Listing. A method for producing human growth hormone having a molecular weight of 20,000 daltons. 4. The method for producing human growth hormone having a molecular weight of 20,000 daltons according to claim 1, wherein the vector DNA is a plasmid capable of replicating in Escherichia coli. 5. An Escherichia coli transformant is cultured at a culture temperature of 2.
The method for producing human growth hormone having a molecular weight of 20,000 daltons according to claim 1, which is performed at 0 ° C to 30 ° C. 6. The culture of the Escherichia coli transformant strain is performed at 0.1% to 0.1%.
The method for producing human growth hormone having a molecular weight of 20,000 daltons according to claim 1, which is carried out using a medium containing 2% glycerol.