JPS633799A - Production of protein by extracellular secretion - Google Patents

Abstract

PURPOSE:To obtain protein by extracellular secretion, useful in the field of drugs, etc., readily and efficiently, by cultivating a transformant transformed with a specific vector at different temperatures in low-temperature and in high- temperature ranges. CONSTITUTION:Plasmid pTA529 and pHSI are linked to give a vector (e.g. pTA1529) which contains both a gene to code a promoter derived from alkali phosphatase and a gene to code signal sequence and can multiply in a host cell. Then the vector is integrated with a gene to code artificially synthesized human epidermal growth factor (hEGF) to give pTA1522 (figure), recombinant DNA. Then the pTA1522 is transduced to Escherichia coli of host mold to give a transformant, which is cultivated in a medium containing inorganic phosphorus, etc., in a low-temperature range (about 30 deg.C) before protein synthesis ability is induced and in a high-temperature range (about 37 deg.C) after the protein synthesis ability is induced by making >=4 deg.C temperature difference at pH 4.6-8.8 for about 20.5hr. Then hEGF, protein, is recovered from the prepared culture mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION (Background of the Invention) [Prior Art] The present invention relates to a method for producing proteins by extracellular secretion using genetic engineering techniques.

More specifically, the present invention provides a vector that includes a gene encoding a promoter derived from alkaline phosphatase and a gene encoding a signal sequence under the control of the gene encoding a desired foreign protein. This is integrated into a recombinant DNA, which is then transferred to a host microorganism (
(Hereinafter, microorganisms may be simply referred to as "bacteria") The host is transformed by transferring it into cells to obtain a transformant, and by culturing this under certain conditions, the desired protein is transferred to the host cell. The present invention relates to a method for producing a protein using genetic engineering techniques, which comprises secreting the desired protein outside the body (into a medium) and then recovering the desired protein from the medium.

(Prior Art) Currently, methods for producing useful substances through genetic engineering using recombinant DNA technology are being established, and for specific methods, reference may be made to various publications, published patent publications, and patent publications. can.

A method for producing a substance using such a method usually involves transforming a host microorganism using a recombinant DNA created by incorporating a gene encoding a desired substance into a vector, culturing the resulting transformant, and then culturing the resulting transformant. , recovering the desired substance.

When non-secreted proteins are produced by such a method, the proteins produced in microbial cells are degraded by host bacterial protease (PrOC).

Natl, Acad, Sci, USA, 79.1
830-1833<1982))), there was a problem in that the desired protein could not be obtained stably and on a regular basis. In addition, in recovering such non-secreted proteins, the host microorganism is physically destroyed and then the desired protein is separated and purified; therefore, such recovery operations are generally complex; Therefore, there are problems in that the yield of the desired protein is low, and that the active protein may be deactivated during the recovery operation. Therefore,
In order to address the above-mentioned problems, we developed signal sequences involved in protein membrane passage (proteins, nucleic acids,
4 (1981)), various methods have been proposed for secreting proteins produced within host microorganism cells to the outside of the cell or the cytoplasmic membrane.
No. 2, No. 55-45395, No. 56-137896, No. 56-145221, No. 56-154999, etc.).

By the way, the host microorganism into which recombinant DNA can be transferred in the production of proteins by genetic engineering techniques using the above-mentioned signal sequences is usually Escherichia coli. This Escherichia coli is a Durham-negative bacterium, and such bacteria have a cytoplasmic membrane and an outer membrane (the space between these membranes is called the periplasm), so recombinant DNA is introduced into such bacteria to create a transformant. When this transformant is subjected to normal culture, the desired protein is accumulated in the elliplast. The proteins accumulated in the periplasm are absorbed by the osmotic shock method (
J, Riot, Chet 240.3865 (19
65)), it cannot be recovered unless it is released from the periplasm to the outside of the bacterial cell. This osmotic
In the shock method, cells are first collected by centrifugation, and then added to a high-concentration sucrose solution [20% sucrose, 30mM
The desired protein is collected by suspending the cells in Tris-HCl buffer (DH8,0), 1mM ethylenediaminetetraacetic acid (EDTA), suspending the cells in cold water, and leaving them in a water bath. The containing fraction is released outside the bacterial cells, and then a supernatant (containing the desired protein) is obtained by centrifugation.
This is the method. And to recover the desired protein,
This supernatant will be further processed by chromatography or the like. In this way, it has been conventional to use Durham-negative bacteria such as Escherichia coli as a host, and to obtain a transformant by introducing recombinant DNA with the ability to secrete a protein into the host, and to obtain a transformant, which is then cultured. The method of producing 100 ml requires an extra processing operation (osmotic shock method) compared to the case where Durham-positive bacteria (Bacillus subtilis, yeast, etc.) are used as hosts.

However, when Durham-positive bacteria are used as hosts, even though they do not have the above-mentioned problems observed with Durham-negative bacteria, there is a problem with the stability of the recombinant DNA transferred into the bacterial body, and E. coli No host-vector system has yet been developed, and handling of Escherichia coli is not easy.

One Solution Therefore, in order to efficiently and easily produce the desired protein using a common bacterial strain such as E. coli, the present inventors have conducted various studies and have developed the following method. Established. That is, the method involves injecting a desired exogenous gene into a vector that is equipped with a gene that encodes a promoter derived from alkaline phosphatase (hereinafter referred to as promoter gene) and also has a gene that encodes a signal sequence (signal gene) under its control. Recombinant DNA is created by incorporating a protein-encoding gene (structural gene of a foreign protein).
A, this recombinant DNA is transferred into host microorganism cells to create a transformant, and this transformant is cultured under certain conditions to secrete the desired protein outside the microbial cell (into the culture solution). This is a method for producing proteins using genetic engineering techniques, in which the protein is recovered (patent application 1986-951).
See specifications of No. 81 and No. 60-121249.

Hereinafter, these will be referred to as "prior inventions." ).

This method is useful as it solves the above-mentioned problems, but as is common in technology, this prior invention may also be subject to further improvement.

[Summary of the invention]

The present invention was completed based on the discovery that the production efficiency of a desired protein can be improved by shifting the temperature during the culture conditions in the culture process of the prior invention.

Therefore, the method for producing proteins by extracellular secretion according to the present invention is characterized by comprising the following steps (A) to (D).

(A> Prepare a vector that is equipped with a gene that encodes a promoter derived from alkaline phosphatase, and also has a gene that encodes a signal sequence under its control, and that can proliferate within the intended host microorganism cell.

(8) A gene encoding a foreign protein is inserted into the above vector to create a recombinant DNA, and then the host is transformed by transferring this recombinant DNA into host microorganism cells to produce a transformant. to get.

(C) The above transformant is cultured in a medium containing the amount of inorganic phosphorus necessary for induction of protein synthesis ability from the late logarithmic growth phase to the early arrest phase in the growth process of the microorganism; Before induction of protein synthesis ability occurs, culturing is carried out in a low temperature range, and after induction of protein synthesis ability, culture is carried out in a high temperature range,
The temperature difference between both cultures should be at least 4°C.

(D) After the completion of the above culture, collect the produced exogenous protein from the medium.

Effects As described above, the present invention enables the production of proteins using genetic engineering techniques using Durham-negative bacteria (Escherichia coli) by secreting the desired proteins outside the bacterial cells (into the medium) instead of retaining them in the periplasm as in the past. In order to perform this, (a) a promoter gene derived from alkaline phosphatase is provided,
A vector that also has a signal gene under its control and (b) is capable of proliferating in the intended host cell is prepared, and then the structural gene of the desired foreign protein is incorporated into it for recombination. A transformant is obtained by transferring the DNA into a host bacterium, and the desired protein is secreted outside the bacterium (into the medium) by culturing this under constant conditions (in step (C) above). The present invention relates to a method for producing proteins using genetic engineering techniques, which comprises the steps of:

A preferred embodiment of the present invention is a method in which the vector consisting of the above (a) and (b) is designed to be able to bind the structural gene of a desired foreign protein immediately after the downstream end of the signal gene. It is. In order to facilitate the introduction of a structural gene of a foreign protein immediately after the signal gene, it is necessary to recognize an artificially created restriction enzyme with at least one of the base pairs of the signal gene as a member. It is better to use one that has parts.

In creating this restriction enzyme WX recognition site, the DNA
The fact that codons, which are made up of base pairs, are degenerate can be cleverly exploited. That is, when the created restriction enzyme recognition/cleavage site is cleaved with the restriction enzyme, if the cleavage site is in contact with the downstream end of the signal gene, the end complementary to the restriction enzyme cleavage end is cut. By preparing an exogenous gene formed on the upstream side and linking it to the signal gene at the cut end, the exogenous gene can be directly linked to the downstream side of the signal gene. In addition, if the cleavage site of the signal gene is located upstream of the downstream end of the gene, a fragment that is linked to the upstream side of the foreign gene is prepared by synthesizing the downstream part of the gene from the cleavage site. When the same ligation as described above is performed, the previously cleaved signal gene is restored on both strands of the DNA, and a structure in which the foreign gene is directly linked to the downstream side is realized (details will be described later).

A structural gene of a desired exogenous protein is incorporated into such a vector to obtain a recombinant DNA, and a host bacterium is transformed with this recombinant NA to obtain a transformant, which is then transformed under constant conditions (in this case). When cultured according to step (C) of the invention), even if the host bacterium is a Durham-negative bacterium such as Escherichia coli, the desired protein is secreted to the outside of the bacterium (into the culture solution) without accumulating in the cell plasma. The object of the present invention is achieved by recovering the desired foreign protein from the culture.

Therefore, the present invention has the following advantages.

(1) The process of producing and collecting proteins using genetic engineering techniques will be simplified.

According to the method of the present invention, a host microorganism is transformed with a recombinant DNA obtained by incorporating a structural gene of a desired foreign protein into a vector that satisfies the requirements (a) and (b) above, and a transformant is obtained. When obtained and cultured, the desired protein is secreted into the culture medium as a mature protein (the signal sequence is cleaved when the protein is secreted into the periplasm). That is, it is sufficient to simply recover the protein from the culture solution without subjecting the transformed host microorganism to the osmotic shock method. Therefore, the process of producing and recovering proteins using genetic engineering techniques is simplified.

The method of the present invention can also be applied to Durum-positive bacteria, but since Durum-positive bacteria such as Bacillus subtilis do not have a plasmid, if the method of the present invention is followed, the desired protein will be secreted into the culture solution. That goes without saying.

(2) It is easy to purify the desired protein.

According to the method of the present invention, the desired protein is secreted into the culture medium, and during secretion, the signal sequence is cleaved by a membrane enzyme, so that a protein without a signal sequence attached can be obtained. If there is an extra gene (DNAI gene as a linker) between the gene encoding the signal sequence and the gene encoding the desired protein, the protein obtained here will be secreted with the extra amino acids attached. Ru. However, as shown in one example of the present invention, if the gene encoding the desired protein is linked immediately after the gene encoding the signal sequence, the desired protein will be a complete mature protein without any extra amino acids attached. It is secreted into the culture medium as Therefore, if the culture of the transformant is terminated at step II when the bacterial cells are not lysed, substantially only the desired protein and the constituent components of the culture medium will be present in the culture solution. Since the constituent components of the culture solution used are known, it is therefore easy to purify the target protein.

(3) Protein production efficiency is better than the prior invention.

Improving production efficiency by shifting the culture temperature during culturing in step (C) of the earlier invention (1.5 to 1.5% of the earlier invention)
2 times) was realized.

[Detailed description of the invention]

The present invention relates to a method for producing proteins by extracellular secretion, and this method consists of steps (A) to (D).

Step (A)/Preparation of Vector In the vector used in the present invention, alkaline phosphatase is affected by the amount of inorganic phosphorus present in the culture method in step (C) of the present invention (8iochem).

Biophys, Acta, 38.460 (1960
)), Nattlre, 183.

1529 (1959)), it is necessary to have a promoter gene derived from alkaline phosphatase, and
Furthermore, it is a vector that has a signal gene involved in protein secretion under its control and is capable of proliferating within the intended host cell (a vector that satisfies the requirements of (a) and (b) above). Good to have.

A particularly preferable vector is one that not only meets the essential requirements (a) and (b), but also has a structure that allows the structural gene of the desired exogenous protein to be linked immediately to the downstream end of the signal gene. It is something that was given.

A specific example of this preferred vector (plasmid) is the plasmid pTA529 (Japanese Patent Application No. 140748/1982), which was previously proposed by the present inventors' collaborators.
, oTA1529 (Patent Application No. 59-159703),
pTA2539 (Japanese Patent Application No. 59-279585), etc. All of these plasmids contain a promoter and signal sequence derived from alkaline phosphatase (the former two are derived from alkaline phosphatase, pTA2
539 is derived from β-lactamase), and allows for the binding of the structural gene of the foreign protein immediately after the gene encoding the signal sequence. Here, pTA1529 is pTA529[)YK283(E,
coli K12C600 (pYK283>, deposited with FIKEN (FeIKEN Article No. 556))], this was prepared using methods commonly used in recombinant DNA technology (in particular, Japanese Patent Application No. 140748/1983). pBR322 (E,co I 1K12C600(
DBR322), deposited with the FEIKEN (FEITEC Article No. 23)
No. 5)] with the v1 restriction enzyme EC0RI and Hin dl [
[] and this IEcoRI-Hindl [[] portion was replaced with a synthetic linker represented by the following base sequence (I) (see Japanese Patent Application Laid-open No. 71692/1983)]. For details of the procedure for constructing this plasmid, please refer to the specification of Japanese Patent Application No. 59-159703.

EcoRI 81)a I 5Ila I
Hind 11! (I> Here, the broken line indicates the restriction enzyme cleavage site, and ECOR etc. indicates the name of the restriction enzyme that performs the cleavage.

Furthermore, pTA2539 is a plasmid in which the ampicillin resistance gene of pTA1529 was converted to a kanamycin resistance gene, and the signal sequence was further converted to one derived from β-lactamase. For details of this plasmid construction, please refer to Japanese Patent Application No. 59-279585, Mei 18.

Since pTA1529 contains a DNA portion consisting of the following base sequence (It), it is a plasmid that allows the structural gene of a foreign protein to be ligated immediately after the signal gene.

Sequence (II) consists of a signal gene part (1) and a DNA part (2) linked to the downstream side thereof, and in one embodiment of the present invention, the "downstream end of the gene encoding the signal sequence" 1 that is designed to be able to immediately bind a gene encoding a desired foreign protein is one that contains an NA portion as described above.In addition, in the present invention, when referring to DNA as "downstream side", 5 ′→
It means the right side when the 3' chain (■ chain) is displayed at the top and the 3'←5' chain (O chain) at the bottom.

The m-base sequence (If) is DNA3! consisting of double-stranded DNA!
They show part of the genes, A and G.

C and T represent adenine, guanine, cytosine and thymine, respectively; the same applies to (I) above), LySSA
+a and Trp represent lysine, alanine and tryptophan, respectively. This area of double-stranded DNA (1
) is the signal gene part, area (3) is the recognition site of the υ1 critical element Hindlll, and the broken line is the Hindlll part.
l[[is the cleavage site. Area (2) is the DNA portion joined immediately downstream of the signal gene.

The vector used in the present invention has a signal gene derived from alkaline phosphatase.

In the base sequence of this gene, the alanine codon at the downstream end is GCC.

On the other hand, the above nucleotide sequence (I) corresponds to the alanine codon GCC at the downstream end of the alkaline phosphatase-derived gene portion (1) changed to GCT and the base sequence (base C' changed to T). Since the alanine codon is degenerate, the modified GCT is also an alanine codon, and therefore the DNA portion (1) in (II) above still corresponds to the signal gene derived from alkaline phosphatase.

Incidentally, the signal gene derived from alkaline phosphatase has a lysine codon AAA upstream of the alanine codon at its downstream end and an arginine codon CGG downstream.

Therefore, the alanine codon GCC at the downstream end of the signal gene derived from alkaline phosphatase
was modified with GCT4C, and the salt MC connected to alanine was modified with T.
By modifying this terminus, the restriction enzyme Hind
The nl recognition site (3) AAGCTT appears. In other words, a restriction enzyme recognition site is created in the signal gene, in which at least a portion of its members are at least the base pairs at the ends.

In the specific example of (II) above of pTA1529, )1in
The cleavage site within the recognition site (3) of dIII is as shown by the broken line, and its position is the DNA part that should be linked to the signal gene and immediately downstream of it.
In the base sequence (II) related to TA529, it exists between the already bound region (2)) (the position of the cleavage site means that on the downstream side of the double-stranded DNA). I
It is most preferable that the II restriction cleavage site be present at such a position in the vector used in the present invention. This is because this cleavage site is used to express the structural gene of a foreign protein and the resulting hybrid or fusion protein is cleaved by a signal peptidase between the signal sequence and the following protein. Therefore, if the restriction enzyme cleavage site and the signal peptidase cleavage site match in this way, the above DT
In the case of A1529, Hi
This is because it is possible to bond with the sticky end of the signal gene after ndl digestion. In addition, the 3' e chain of the foreign gene
It goes without saying that a restriction enzyme cleavage site may be present on the upstream side, as long as you do not mind supplementing the - side with a base, and vectors containing such a cleavage site can also be used in the present invention. It is within the range.

"Genes encoding signal sequences" generally have various base sequences depending on the type of signal sequence. Some specific examples of signal sequences include those of β-lactamase (Proc).

Acad, Sci, U, S, A, 75.3737
(1978)), riboproteins (ibid., 74.10)
04 (1977)), that of alkaline phosphatase (Eur, J., Biochet 96.49
(1979)) etc. Regarding the signal sequence,
Special issue of "Proteins, Nucleic Acids, and Enzymes"("GeneManipulation")
, Vol. 26, No. 4, pp. 386-394. However, preferred signal sequences for use in the present invention are those of alkaline phosphatase and those derived from β-lactamase. This is because the above-mentioned plasmid having such a signal sequence can provide the above-mentioned advantages by using the culture method of step (C) of the present invention.

The example of a DNA gene included in the plasmid (vector) used in the present invention shown in (IF) above is one made by modifying the DNA derived from alkaline phosphatase, so the signal gene DNA portion (1) is A DNA portion (2) derived from alkaline phosphatase is bound immediately after the downstream end. A specific example of the present invention is this DNA
Part (2) is a DNA part corresponding to a gene encoding a foreign protein.

An example of a DNA gene included in a plasmid used in the present invention that falls within the scope of this specific example is one in which the signal gene portion consists of a natural product-derived portion and a synthetic portion. That is, the part upstream of the restriction enzyme cleavage site is derived from a natural product, and the part downstream is a synthetic part. In this case, the synthesized part on the downstream side is the 4 bases of the ■ chain (AGC
T), but it goes without saying that if the cleavage site is located upstream from this, a synthetic portion is also required in the O chain.

Step (B)/Creation of transformants Transformation of microorganisms can be carried out according to conventional methods known in the field of recombinant DNA technology.

For example, a structural gene of a desired foreign protein is incorporated into the above-mentioned plasmid to obtain a recombinant DNA (chimera plasmid), and this is used to generate a microorganism by a known method [for example, the Schikschner method (GenetiCE Engineering, Volume 17, pp.
(1978))), and then any suitable method for obtaining the desired transformant (usually using a plasmid marker) can be used.

The microorganism that can be transformed with such a chimeric plasmid according to the present invention may be one in which the above-mentioned plasmid can proliferate within the microbial cell, and specifically, the microorganism that can be transformed with the chimeric plasmid according to the present invention is a microorganism that can be transformed with the chimeric plasmid of the present invention. There are bacteria such as Bacillus subtilis and Bacillus subtilis. Among them, Escherichia coli is relatively commonly used, and the present invention particularly has the above-mentioned effects when using Durham-negative bacteria such as Escherichia coli as a host bacterium. As E. coli, for example, E.
, coli K12 C600 (Feikoken Joyori No. 11
5), E, Co11 K12 YK537 (Feikoken Bacteria No. 7941), E, coliRRl (ATC
C31447) and E.

coli HBlol (ATCC33694). In embodiments of the invention, E. coli K12 YK
537 is used.

A specific example of such a transformant used in the present invention is plasmid E) TA1529. A chimeric plasmid pTA1522 was constructed by incorporating the structural gene of hEGF (described later) which was artificially synthesized into (above), and then this chimeric plasmid (recombinant DNA) was injected into host bacteria E, coli.
Transformant E, coli K12 YK537 (
pTA1522), E. coli RRI (pTA1
522) and E. coli HBIOI (DTA1
522).

In addition, the gene encoding the desired foreign protein to be incorporated into the above-mentioned plasmid may include hormones, immune-related substances, neuropeptides, enzymes, and the like. Methods for preparing these structural genes include methods for obtaining them from natural chromosomal DNA and methods for artificially synthesizing them.For actual methods for preparing structural genes, refer to various books and literature. be able to.

In one embodiment of the present invention, human epidermal growth factor (hEGF/
A chemically synthesized gene encoding hUG (hereinafter referred to as fiEGF) (details of the synthesis are provided in the patent application filed in 1983).
-123520) was used.

Step (C)/Cultivation of microorganisms Cultivation according to step (C) of the present invention is carried out by inserting a structural gene of a desired foreign protein into a vector (plasmid) having a promoter derived from alkaline phosphatase to generate a chimeric plasmid (recombinant DNA). ), which is then transferred into a host microorganism and used as the target of the transformed microorganism.

The characteristic of the culture method in this step is that the protein synthesis ability may or may not be induced depending on the nature of the vector (plasmid) held by the transformed microorganism, that is, the amount of inorganic phosphorus. Properties (the above B10Che1
．． B10E) hys, ACta, and Nattl
re), can be skillfully used to grow microorganisms and subsequently induce protein by the microorganisms in a single culture system, and even cultivate the microorganisms in the middle of the culture! ! By shifting the temperature from a low temperature region to a high temperature region, the production efficiency of the desired protein is improved.

Details of such a culture method are as follows.

1) Cultivation method The method for culturing microorganisms in this step typically involves culturing the transformed microorganism in single-cell pure isolation, and then subjecting it to preculture (the above is a known conventional method for culturing microorganisms). You can refer to a large number of documents and foundations.

For example, Seishan's ``Experimental Methods of Microbiology'' (published by Kodansha), ``Experimental Methods of Microorganisms'' (published by Kyoritsu Shuppan), and ``Cytology Training Presentation''.
(published by Maruzen), etc. ), which consists of culturing in a single culture system.

This single culture system belongs to the category of aerated agitation culture, and specifically, after inoculating bacteria in the culture system, sterile air (with pure oxygen mixed in as necessary) is added to the culture medium.
The microorganism is introduced into the microorganism, and the microorganism is physically stirred while the pH, temperature, dissolved oxygen concentration, etc. are maintained under suitable conditions to suit the growth conditions of the microorganism to be cultured.

Such culture is usually carried out using a jar fermenter, and it goes without saying that appropriate scale-up is possible (see ``Biochemical Engineering'' (1), (1)
(see below), University of Tokyo Press).

2) Medium The medium composition used in this step is LB medium (tryptone, yeast extract, NaCl) as the basic medium, with other components added as necessary (e.g. MaSO4, 7H20, antibiotics, etc.). Furthermore, it contains a sufficient amount of inorganic phosphorus to induce protein synthesis ability from the late logarithmic growth phase to the early arrest phase during the growth process of the transformed microorganism. Further, in the specific example of the present invention, ampicillin is added as an antibiotic added as necessary. In a specific example of the present invention, the plasmid held by the transformant contains an ampicillin resistance gene, making the transformant resistant to ampicillin. Therefore, since the growth of bacteria (not resistant) to which the plasmid has parented during culture is suppressed by ampicillin in the medium, only host bacteria that retain the plasmid will grow. In this way, the addition of antibiotics to the culture medium is a means to facilitate the growth of host bacteria.

3) Culture conditions (1) Culture temperature The culture temperature may be within a range where M≠ allows the microorganism (transformant) used to multiply or grow and the product is stable.

According to the present invention, within this temperature range, the culture temperature?
Shift from low temperature range to high temperature range. Culture in both temperature ranges'7
! The difference in temperature should be at least 4°C.

Here, in the case of mesophilic bacteria, the low temperature range is within the range of approximately 25°C to 35°C, and the high temperature range is within the range of approximately 32°C to 42°C. For example, when the transformant used in one example of the present invention is E. coli, the low temperature range is preferably around 30°C, and the high temperature range is preferably around 37°C.

Then, the shift of the PJ difference temperature from the low temperature range to the high temperature range is
This is done from the perspective of inducing protein synthesis ability. That is, before induction of protein synthesis ability occurs, the culture is carried out in a low temperature range, and after induction, the culture temperature is shifted to a high temperature range. It is desirable to shift the culture temperature from a low temperature range to a high temperature range as quickly as the heat capacity of the culture solution allows. The means for this purpose may be arbitrary, but one means is to rapidly increase the temperature of the external heating medium of the culture tank. Another option is to transfer the culture fluid from a low-temperature culture tank to a high-temperature culture tank.

The culture temperature in each temperature range is usually kept substantially constant according to conventional methods for culturing microorganisms.

It is convenient to determine the timing of culture temperature shift by monitoring the amount of inorganic phosphorus in the culture solution. That is, the amount of inorganic phosphorus is quantified over time from the start of culture until the amount reaches the minimum (in one embodiment of the present invention, at about 9:00 p.m.
! It is best to culture in a low temperature range and then suddenly shift the culture temperature to a high temperature range.

(2) Culture time The culture time in this step is the time after the culture temperature has been shifted from a low temperature range to a high temperature range, at which the production amount of the desired substance secreted into the culture solution is at its maximum. preferable. A more preferable culture time is a time during which the bacteria are not lysed and the desired protein is secreted into the culture solution.

In one embodiment of the present invention, when producing hEGF, approximately 2
0.5 hours is adopted. This is because if the culture is carried out any longer, the bacterial cells will be lysed and various miscellaneous substances in the bacterial cells will be mixed into the medium, making it difficult to recover the desired protein. Such examination of culture time can be performed using as indicators the determination of glucose and inorganic phosphorus in the medium, the measurement of OD 660, the measurement of the number of birthplaces, and the quantification of produced substances in the periplasm and in the culture solution. Glucose can be quantified by the glucose oxidase method (using the New Glucostat "Fujisawa" kit). The country of birth is E, coli K12 YK537 (pTAL
In the case of 522), the number of colonies growing on agar of L-medium (described above) and L-medium containing ampicillin may be measured. In one embodiment of the present invention, the plasmid retention rate is calculated as the ratio (%) of the number of colonies growing on one agar medium containing ampicillin to the number of colonies growing on agar one medium medium.

The amount of inorganic phosphorus was determined according to the molybdenum blue method (factory wastewater test method: JIS K 0102). Also,
To quantify the product hEGF in the culture solution, first quantitate the culture solution (10si
After centrifuging this, the supernatant was subjected to RRA method (see below).
For hEGF in the periplasm, bacterial cells were subjected to the osmotic shock method (J. Biol.
After treatment by the radioreceptor assay (RRA) method (J, Chew), the solution was centrifuged and the supernatant was purified by radioreceptor assay (RRA)
Biol, Chet, 257.3053 (1982
)) For details of the hEGF quantification carried out by analysis, see Reference Examples and Japanese Patent Application No. 159703/1983, which will be described later.

It should be noted that hEGF expressed within the bacterial cells is expressed within the bacterial cells (1
We also investigated whether it was present in the cytoplasm.

That is, after the above-mentioned osmotic shock treatment, the resulting precipitate (bacterial cells) is resuspended in pure water, and then subjected to ultrasonic treatment to destroy the bacterial cells, and then centrifuged to obtain a supernatant. By analyzing this supernatant according to the RRA method described above, hEGF within the bacterial cells (inside the cytoplasm) was determined.

(3) pH of culture medium The pH may be within a range in which microorganisms can grow, and a preferable pH may be set as appropriate depending on the microorganisms used.

When using Escherichia coli, the pH at which Escherichia coli can grow is usually 4.6 to 8.8, among which pH 7.0 to 8.0.
Particularly preferred is between.

Incidentally, although the pH during the culture normally fluctuates, it is preferable to keep it around a - constant pH.

(4) Antifoaming agent If foaming is significant during culturing, use an antifoaming agent (higher alcohol,
It is a common practice to add vegetable oil, etc.). Since the antifoaming agent used in the present invention controls the protein synthesis ability of microorganisms depending on the amount of inorganic phosphorus present, it is important that the antifoaming agent does not contain inorganic phosphorus. A specific example of such an antifoaming agent is "Antiform-AF-Emulsion" (Hani Chemical).

(5) Dissolved oxygen (Do) Dissolved oxygen refers to molecular oxygen dissolved in the liquid phase.

It is generally known that in aerated agitation culture, if Do is too large, the growth of microorganisms will be inhibited, and even if -11) 9m or less, the growth will be similarly inhibited. Therefore, it is preferable to control DOffi so that it does not become an inhibitor of microbial growth. In the case of one example of the present invention, the Do control device [Oriental Electric-F
C-4 type] holds the Do boar in 41) l) Im.

The protein produced by / can be recovered according to known conventional methods.

For example, ion-exchange chromatography, affinity chromatography, electrophoresis, high-performance liquid chromatography, or various combinations of these methods [Sokoguchi, "Biochemistry Experiment Course 1 Chemistry of Proteins", edited by the Japanese Biochemical Society,
(See Tokyo Kagaku Dojin Publishing (1982)), and an appropriate one can be selected and used according to the properties of the protein or peptide to be produced.

In one embodiment of the present invention, the culture solution after completion of culture is centrifuged,
The resulting supernatant was then gel-filtered through a reverse phase column, and the hEGF-containing fraction was purified by DEAE-TOY.
1E by being familiar with OPEARL■ (ion exchange)
The GF fraction was separated and the desired fractions were collected. In addition, if you want to recover more (and more efficiently) the target protein produced according to the method of the present invention, culture should be carried out at a time when the amount of target protein secreted into the culture solution is large and the bacterial cells are not lysed. After completing the process, the target protein is recovered from the culture solution, and the target protein is recovered from the periplasm after treatment by the osmotic shock method after collecting the bacterial cells.According to the method of the present invention, the target protein is recovered from the host bacterial body The target protein produced does not remain within the bacterial body (in the cytoplasm) but is secreted either into the periplasm (a small amount) or into the culture medium (mostly); therefore, with this recovery method, the target protein produced within the bacterial body is secreted either into the periplasm (a small amount) or into the culture medium (mostly). Most of the proteins in the periplasm will be recovered.Recovery of proteins in the periplasm is possible, for example, as described in Japanese Patent Application No.
The method of No. 30 may be used.

Tomo-1-1 (1) Creation of transformants Transformants E and coli K12 were prepared according to the following method.
YK537 (pTA1522) was constructed.

pTA1529 (details of construction are in patent application No. 59-15970)
5 μ9 (see the light Il of No. 3) in a 50 μm buffer solution (
10mM Tris-salt i! Buffer solution (hereinafter referred to as Tris-HCI
><pH 7,5), 10mM MgCl 50mM
Hydrolysis was carried out at 37°C for 1 hour using 4 units of the restriction enzyme Hindll (Takara) (hereinafter referred to as Hindll) in NaCI). Ethanol precipitation was then performed, and the resulting precipitate was diluted with 30 μm of the reaction solution (67 mM Tri
s-HCIIH8,8), 1 unit of T4-DNA in 16.6mM ammonium chloride [hereinafter (NH4)2S04], 6.7mM ethylenediaminetetraacetic acid (hereinafter EDTA), 0.66mM each of dATPS, dCTP, dGTP, TTP). 1 at 37°C using polymerase.
Processed for 5 minutes. Then, the ethanol precipitate was added to 50μ
m reaction solution (6mM Tris-HCI (p
H8,0), 6mM MgC
Hydrolysis was carried out at 37°C for 1 hour using 4 units of restriction enzyme 3al I (Takara) (hereinafter referred to as 3alJ) in 2.150 mM NaCl). After the reaction, a 3900 bp D
An NA fragment (■ in Figure 1) was obtained.

Plasmid pBR322-hLIG (pBRa22
(E, co l i Kl 2 C600 (pBR
The hEGF structure gene, which was artificially synthesized by digesting the hEGF structure deposited as 322) [Feikoken Article No. 235]) with EC0RI and SaII, was synthesized with EC0RI and 5.
5μ9 [incorporating the fragment digested with alI],
0μm reaction solution (100mM Tris-Hcl (p
H7,5), 50mM NaCl, 50mM Mg
4 units of restriction enzyme EcoRI in Cl2) (Takara)
YK537 (oTA1522) was cultured as follows. Single cell pure isolated E, Co Ii
After hydrolysis at 37℃ for 1 hour, T4
After DNA polymerase treatment and further 5alI treatment, 160b was isolated by agarose gel electrophoresis.
A DNA fragment of p (■ in Figure 1) was obtained.

The two DNA fragments prepared above (■ and ■ in Figure 1)
) to 30 μm reaction solution (20mM Tris-HCI
(pH 7,5), 10mM MgC12, 10mM D
300 units of T4 DNA in TT, 0.5mM ATP)
The reaction was carried out at 14° C. for 16 hours using ligase (Takara).

After the reaction was completed, E. coli was transformed with 12 YK537, and the target plasmid [hereinafter pTA1522] was transformed.
) (■ in Figure 1).
K12 YK537 (pTAL 522)) was obtained.

(2) Cultivation of transformants The microorganism E, coli K12YK537 (pTA1
522) was cultured as follows. Single cell pure isolated E. coli K12 YK537 (pTA15
22) - Platinum loops were inoculated into 100 m of a medium consisting of 0/l of Trypto, 59/l of NaCl, and 20 q/l of ampicillin, and
Shaking culture was carried out overnight at 37°C in a 00d volume of Sakarokorpen.

Next, this culture? Take 1k10d, glucose 109
/Little, Tryptone 109/Little, Yeast Extract 5g Liter, MaSO4.78200, 5SF/
1 liter of ampicillin and 20 η/liter of ampicillin, and cultured in a 3 liter mini jar fermenter. The culture temperature was kept at around 30°C until the inorganic phosphorus content was at its minimum (approximately 9 hours after the start of culture), and then rapidly changed to 37°C. The ventilation amount is 0,
5vvi (1 vvm is i VOIu+me
-VOIu+ae-minute means that 1 liter of air is introduced per 1 liter of culture solution per minute. ), pH is 7.2 (4
(adjusted with N NaOH or 4N HCI), and the dissolved enzyme (Do) concentration was maintained near 41) DI by changing the stirring speed using a DO control device. Cultivation involves quantifying glucose and inorganic phosphorus, measuring the number of viable bacteria, and measuring hEG within the bacterial body (inside cells), in the periplasm, and in the culture solution.
hEGF in the medium was determined by quantifying F over time.
The cultivation was continued until the amount of hEGF exceeded the amount of hEGF in the periplasm (approximately 20.5 hours from the start of culture). Glucose was quantified according to the glucose oxidase method using a kit for euglucostat "Fujisawa"). The number of viable bacteria is determined by one medium containing 20ttty/liter of ampicillin (
This was done by measuring the number of colonies appearing on an agar plate of Trypto 2 Go 0 NaCl 59/liter). 0D6
6. was measured as an indicator of bacterial mass, and inorganic phosphorus was determined by the molybdenum blue method (described above).

Note that this constant m is used as a guideline for the timing of culturing temperature shift. Quantification of hEGF in the bacterial cells (in the IT cytoplasm), in the culture solution, and in the Bribelladum was performed by the following method. That is, 10 d of the culture solution was taken, centrifuged, and the supernatant was analyzed by the RRA method (described below) to quantify hEGF in the culture solution.
% Sucrose, 0.03M Tris-HCI (
pH 8.0), EDTA O. The suspension was suspended in a solution of 0.001M and left at temperature V for 10 minutes. Then, centrifuge again to collect the bacteria, and soak the bacteria in 20 m of cold water (0°C).
~4°C) and left in water for 10 minutes (treatment by osmotic shock method). This suspension was centrifuged to obtain a supernatant, and hEGF in the periplasm was quantified by analyzing this supernatant by radioreceptor assay (RRA). In addition, the precipitate (bacterial cells) obtained in the Osmotic Shock Treatment II bond was resuspended in 10 d of pure water, and then subjected to ultrasonication (Kubota Insonator Model 200Mj) (10 minutes). The population of hEGF in the bacterial cells (cytoplasm) was determined by disrupting the bacterial cells and analyzing the supernatant obtained by centrifugation (27,000 g, 30 minutes) according to the RRA method (described below).

The RRA method was performed as follows. First, KB [1ffd (ATCCIIQCC
L17) was cultured in an 800 d flask in Dulbecco's modified Eagle (DME) medium [Hinaga]. The medium was then removed and the cells were detached using phosphate balanced salt solution (PBS) containing 0.05% EDTA to create a cell suspension.

Then 20mM Hepes (D
Cells were washed twice with Hanks Balanced Salt Solution (HBSS) containing H7,4). Binding solution
n) (DME medium 20 top 7, 4>-0.35 g/liter NaHCO3"100
μ9/d streptomycin] to li! ! After turbidity, the number of cells was calculated to be 300,000 to 400,000. 2al! Adjust so that it becomes a pin ding trusion, and add 0.2 to the tube.
d portions were dispensed. Next, each of the above three types of supernatant and a sample solution containing I-mEGF (71x epithelial cell growth captive (hereinafter referred to as mEGF)) 0, 2nd! was added to the tube and incubated at 37°C for 1 hour. After washing the cells 3 times with ice-cold HBSS, 10% Trichlor 0
The cells were suspended in acetic acid (hereinafter referred to as TCA) and fixed using a glass filter. After removing TCA with acetone,
Counts were performed using a liquid scintillation counter. Then, the amount of hEGF was converted to mEGF.

Note that β-galactosidase (β-gal) is an enzyme that exists within cells. Therefore, if the bacterial cells are lysed, it is inferred that this enzyme will also leak out of the bacterial cells. Therefore, by quantifying β-gal, it was determined whether the host bacteria (transformants) were lysed (European Journal of A
pplied Hicrobiolngyand Bi
otechnology, 16, 14G-150 (
1982), Around, 5-12 (1984)).

Note that β-gal was quantified using Miller's method [bottom Exp
erimental in Molecular Ge
netics.

355, (1972), Cold Spring Harbor Laboratory), 0D42. showed its activity.

In these experiments, the culture time, OD, number of viable bacteria, m of inorganic phosphorus, amount of glucose, suspension of hEGF (in the body (cytoplasm), periplasm and culture medium) and β-g
The results of the determination of al are shown in Table 1.

Furthermore, these results are illustrated in FIGS. 2 and 3 (a) and (b). Figure 2 plots the constant volume results for glucose, viable count, absorbance, and inorganic phosphorus, and Figure 3 plots the total hEGF (total), hEGF in the culture medium, hEGF in the periplasm, and intracellular The results of measuring hEGF (a) and the ratio of each fraction to the total hEGFffi (b) are shown.

From these tables and figures, it can be seen that the amount of hEGF in the culture solution reaches its maximum 4120.5 hours after the start of culture. In addition, β-ga, an enzyme that should be present in the I8 cell,
Since the ratio of 1 in the cells has been constant since the start of culture, it is suggested that the bacterial cells have not been destroyed. Therefore, in this culture process, the culture temperature is shifted 9 hours after the start of culture, and then further culture is performed. By continuing the culture until 20.5 hours after initiation, 54.71 Rg/liter of hEGF could be obtained, which was twice as much as the conventional method.

(3) Protein recovery One liter of the above culture solution was centrifuged (12,000g, 10 minutes) to obtain a supernatant, which was then subjected to reverse layer chromatography (column size: diameter 4.1a+
x length 8 an, resin: prep PAK500/
The desired protein fraction was separated using C18 reverse phase resin (Waters). Then, the desired fraction obtained by the chromatography described in F was purified by DEAE-TOYOPEA.
RL■ (Column size: diameter 1.5c IRX length 25
cta ) column, the desired protein fraction was separated. In addition, when a portion of both parts of the desired protein obtained here was subjected to polyacrylamide electrophoresis, h
Since a band was observed at the same position as the EGF standard, it was suggested that the desired protein hEGF was recovered.

Comparative Example In order to establish the culture method according to the present invention, culture was carried out while keeping the culture temperature constant, culture was shifted from a high temperature range to a low temperature range, or from a low temperature range to a high temperature range during the culture, etc. saw.

The results are shown below.

The experiment was conducted in the same manner as in the example except that the culture temperature was maintained at 30° C. and the culture was continued.

The results are shown in Table 2. From this result, the maximum amount of hEGF secreted into the culture solution was 26.4#+9/liter, which was obtained at 27 hours after the start of culture. Note that the meanings of *1 to *4 in the table are the same as in Table 1.

The experiment was conducted in the same manner as in the example except that the culture temperature was maintained at 37° C. and the culture was continued. The results are shown in Table 3. From this result, the h secreted into the culture medium
The maximum EGF was 28.61R9/liter, which was obtained 23 hours after the start of culture. Note that the meanings of *1 to *4 in the table are the same as in Table 1.

Until 9 hours after the start of culture, the culture temperature was 37°C.
Cultivate after that? & The experiment was conducted in the same manner as in the example except that the humidity was suddenly shifted to 25°C. The results are shown in Table 4.

The meanings of Ne1 to Ne4 in the table are the same as in Table 1.

From this result, hEGF reached its maximum level of 3.9019/liter 30.5 hours after the start of culture. Note that in addition to β-ga1, β-lactamase (b I a) was also quantified here.

The reason why bla was quantified is because bla, like alkaline phosphatase, is a periplasmic enzyme and accumulates in the periplasm. Therefore, if the secretion of the desired protein into the culture solution is due to bacterial lysis, bla
It is also thought that the microorganisms leak into the culture medium over time. The quantification of bla in the periplasm of bacterial cells and in the culture mouth fluid is as follows. That is, bla in the culture solution
The constant m is as follows: Take 10 - of the culture solution, centrifuge it, and dilute the supernatant to 0.1
Appropriately diluted with M sodium-phosphate buffer (9 days 7,0)! Prepare a nanbul solution, and add PADAC (b I a substrate, which changes from purple to yellow when it is decomposed by bla, to 0.5 d (formula vIR1 below). '?3
．． Adjust to 0 (CALBIOCHEH-BEIIRING) 0
, 5d was added thereto, and the reaction was carried out at 37°C for about 10 minutes (formula t below), then boiled for 5 minutes, immediately cooled in water, and then the oD was measured.

Based on the difference (ΔoD) between this measured value and the measured value at 0D56° of a control (measured using the above buffer as a sample solution in the same manner as above), the activity of bla was measured using the following formula. In addition, to measure bla in the periplasm, the bacterial cells obtained above were treated according to the osmotic shock method (described above), and then this solution was mixed with 0.1M sodium-phosphate buffer (p17. Appropriately dilute with O>
The following procedure was carried out in the same manner as the quantification of bla in the culture solution.

Here, the unit is the β-
The enzyme activity that hydrolyzes a lactam ring in 1 minute is defined as 1 unit, where t is the reaction time (minutes), and ■ is the fli! of the sample solution. (d) (“Protein・
Nucleic Acids and Enzymes” and 390-400 (1978)). From the emplacement results of β-gal and bla in this table, β-
The majority of gal remains within the cell, and b
The fact that most of the la remains in Belibraz suggests that the host bacteria are not destroyed.

Comparative Example 4 An experiment was conducted in the same manner as in Example except that the culture temperature was 37°C for about 9 hours from the start of culture, and thereafter the culture temperature was shifted to 30°C. The results are shown in Table 5. From this result, the E secreted into the culture medium
The maximum GF is 27.7 #/liter, which is
& It was obtained 29 hours after the start. The meanings of Books 1 to 3 in the table are the same as in Table 1.

Technique 1 The experiment was carried out in the same manner as in Example except that the culture temperature was 37°C for about 7 hours from the start of culture, and thereafter the culture temperature was shifted to 40°C.

The results are shown in Table 6. From this result, the maximum amount of EGF secreted into the culture solution was 28.5 IItg/liter, which was obtained in the mouth 21 hours after the start of culture. The meanings of Books 1 to 3 in the table are the same as in Table 1.

From the results of Examples and Comparative Examples in Table 7 of Table 7 of Le J. Ro, the shift range of culture temperature, shift time, culture time at which EGFm in the culture solution reached its maximum, and EGFm at that time <Rg/J)
are summarized in Table 7. From this, it can be seen that the culture method of shifting the culture from a low temperature range to a high temperature range, as in this example, increases the production (2) by about twice that of the normal culture method (constant at 37° C.).

Mycological properties and accession numbers of related microorganisms The mycological properties and accession numbers of the microorganisms disclosed in the present invention are as follows.

Entrustment date (1) June 9, 1981 (2) April 30, 1981 (3) June 9, 1981 (4) November 14, 1981 Japan Ministry of International Trade and Industry, Agency of Industrial Science and Technology Microbial Technology Research Institute accession number ** A T CC (American Type)
Cur1ture Collection) accession number 3" υ1 Wataru (1) E. coli K12C600 This bacterium is a Gram-space bacillus, does not produce spores, has general attributes of the genus Escherichia coli such as facultative anaerobic ability, and has the F factor. It lacks the function of suppressor gene E and has a defect in the recBcLf gene, which encodes a nuclease involved in genetic recombination. As an auxotroph, it requires threonine and leucine for growth on its minimal medium. Furthermore, taxonomically, it belongs to the family Enterobacteriaceae and the genus Escherichia coli. The literature regarding the J3 water bottle is as follows.

(b) Genetics, 39.440 (1954
) (mouth) Nature, 217.1110 (1
968) (2) E. coli K12C600 (
pYK This bacterium is a Durham-negative bacillus, does not produce spores, has general attributes of the genus Escherichia coli such as being facultatively anaerobic, does not contain F factor, lacks the function of suppressor gene E, and has no nuclease involved in genetic recombination. It has a defect in the recBG gene encoding. As for nutritional requirements,
Requires threonine and leucine for growth on its minimal medium.

Promoter-operator region pA of phOA gene
Plasmid pYK composed of the replication initiation region derived from CYC177 and the b I a31i gene of σpBR322
283 and is resistant to ampicillin. Furthermore, taxonomically, it belongs to the family Enterobacteriaceae and the genus Escherichia coli.

The mycological properties of this strain are those of its parent strain E, except for the traits derived from plasmid pYK283.

It is the same as that of coli K12C600.

(3) E. coli K12C600 (pBR) This bacterium is a Durham-negative bacillus, does not produce spores, exhibits the general attributes of the genus E. coli such as facultative anaerobic properties, and does not contain the F factor.
It lacks the function of suppressor gene E and has a defect in the recBC gene, which encodes a nuclease involved in genetic recombination. As auxotrophs, threonine and leucine are added to the i! ! Required for breeding. It also contains drug resistance plasmid pBR322. Regarding plasmid pBR322, see Gene, 2
．． 95 (1977), Nature, supra, regarding E. coli Kl 2C600. pBR3
Except for the traits derived from E. coli K12C6
The mycological properties of 00 (pBR322) are the same as those of the parent strain.

(4) E. coli K12YK537 12YK537 is a known strain of E. coli 12YK537.
Stock () licrobioloqica! IteVie
WS.

44.1-56 (1980)) derivative of Escherichia coli Kl 2
RR1 (Gene, 2.95 (1977), Bioc
hem, Biophys.

ACta, 655.243 (1981)), and exhibits the following properties, and is a strain that does not differ from that of 12RR1 in other properties. (recAl, phOA8, pro+) (5)
F: coli is a bacterium created by hybridizing 12 strains of E. coli HB101 (described above) and E. coli B, and has the following genotype. The literature related to this strain is J, t4o1. Biol, 41.459
(1969) 83 and) lcthodsEnzymo
1, 68.24B (1979).

F'', hsds20 (r-, mB-), rec
Al3, ara-14, prOA2, lacYl,
aa l K2, rpsL20 (3m), xy+-
5, mt l -1, 5uDE44, λ- (6) E, coli RRI large [E with IHBlol modified to recA+.

coli)-18101. Therefore,
The genotype is (F-, same as HBLol except reCA+). In addition, the literature regarding this strain is cane
2.95 (1977) and Biochimica'
et Biophysica ACta, 655
．． There are 243 (19&i).

[Brief explanation of drawings]

FIG. 1 is a flowchart for constructing plasmid pTA1522 used to transform E. coli K12Y537. FIG. 2 and FIG. 3 <a) and (b) are drawings in which the results of Table 1 are plotted.

Claims (1)

[Scope of Claims] 1. A method for producing proteins by extracellular secretion, which comprises the following steps (A) to (D). (A) To prepare a vector that is equipped with a gene encoding a promoter derived from alkaline phosphatase, and also has a gene encoding a signal sequence under its control, and is capable of propagating within the intended host microorganism cell. (B) A gene encoding a foreign protein is inserted into the above vector to create a recombinant DNA, and then this recombinant DNA is
Transforming a host by transferring A into host microorganism cells to obtain a transformant. (C) The above transformant is cultured in a medium containing an amount of inorganic phosphorus necessary to induce protein synthesis ability from the late logarithmic growth phase to the early arrest phase in the growth process of the microorganism. Culture should be carried out in a low temperature range before induction of protein synthesis ability occurs, and in a high temperature range after induction of protein synthesis ability, so that the difference in temperature between the two cultures is at least 4°C. (D) After the completion of the above culture, the produced exogenous protein is recovered from the medium. 2. A vector that is equipped with a gene encoding a promoter derived from alkaline phosphatase and also has a gene encoding a signal sequence under its control, and is capable of proliferating within the intended host microorganism cells, encodes the signal sequence. The method for producing a protein by extracellular secretion according to claim 1, which is designed to allow a gene encoding a desired foreign protein to be linked immediately after the downstream end of the gene. 3. The gene encoding the foreign protein to be incorporated into the vector in step (B) is human epidermal growth factor.
A method for producing a protein by extracellular secretion according to claim 1 or 2.