JPS61254196A - Production of protein by secretion of outside of mold - Google Patents

Abstract

PURPOSE:To simplify production and recovery processes, to make purification easy and to obtain protein by secretion of outside of mold, by forming a transformant with a specific vector and cultivating it. CONSTITUTION:A vector (A) having a gene to code a promoter derived from an alkali phosphatase and a gene to code a signal sequence under the control of it, capable of multiplying in the cell of an expected host microorganism, is prepared. Then, a gene to code exotic protein is integrated into the component A to form a recombinant DNA, and the recombinant DNA is made to enter in the cell of a host microorganism, to give the transformant microorganism (B). The microorganism is cultivated in a medium containing an inorganic phosphorus substance in an amount required to synthesize protein from the latter period of the logarithmic phase of the growth process of the microorganism to the former period of stopping period. Then, the culture mixture is centrifuged, subjected to gel filtration and fraction separation, and exotic protein is recovered.

Description

BACKGROUND OF THE INVENTION Technical Field 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 zolomotor derived from alkaline phosphatase and a gene encoding a signal sequence under the control of the vector, which encodes the y'gT exogenous protein. A recombinant DNA is obtained by integrating the gene for the host microorganism (hereinafter, microorganisms are sometimes simply referred to as "bacteria"), thereby transforming the host and obtaining a transformant. The present invention relates to a method for producing proteins using genetic engineering techniques, which comprises secreting the desired protein outside the host cell (into a medium) by culturing it under certain conditions, 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 can be made to various publications, publications, published patent publications, and patent publications.

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.

By the way, when non-secretory proteins are produced by the method described above, the proteins produced within microbial cells are degraded by the protease of the host bacteria. Of the United Stake of America (P
roc, Natl, Acad.

Sci, USA), 79.1830-1833 (1
982))), so there was a problem that the desired protein could not be obtained stably and in large quantities. In addition, when recovering such non-secreted proteins, the host microorganism is physically destroyed and then the desired protein is isolated and purified, so such recovery operations are generally complex; 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 a signal sequence involved in the passage of proteins through membranes [Protein/Nucleic Acids/Enzyme Research, 26, 3].
86-394 (1981)], a method of secreting proteins produced within the host microbial cell to the outside of the cell or outside the cytoplasmic membrane has been proposed extensively (Public Patent Publication No. 19092/1981). No. 55-45395,
No. 56-137896, No. 56-1.45221,
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 Durham negative and has a cytoplasmic membrane and an outer membrane (the space between these membranes is called the periplasm), so recombinant DNA is introduced into such a bacterium to form a transformant. When this transformant is subjected to normal culture, the protein is accumulated in the veriplasm. The proteins accumulated in the periplasm are collected using the osmotic shock method and published in the Journal of Osmotic Biological Chemistry (
J, Biol, Cbem, ), 240, 36
85 (1965)], it cannot be recovered unless it is released from the periplasm to the outside of the bacterial cell. This osmotic shock method involves collecting bacterial cells by mass centrifugation, and then dipping them into high-concentration sucrose, 20% sucrose, 30mM) and Lith-HCl buffer (pH 8.0).
After suspending the cells in mM ethylenediaminetetraacetic acid (EDTA), collect the cells, suspend the cells in cold water, and leave them in a water bath to release both components containing the desired protein from the cells. In this method, the supernatant (containing the desired protein) is obtained by centrifugation. In order to recover the desired protein, this supernatant is further processed by chromatography or the like. In this way, it has traditionally been possible to use Durham-negative bacteria such as M. taichungen as a host, and to obtain a transformant by introducing recombinant DNA with a protein secretion function into the host, and by culturing the transformant, the desired protein can be produced. The production method requires extra processing operations (osmotic shock method) compared to when Durham-positive bacteria (Bacillus subtilis, yeast, etc.) are used as hosts.

However, when these Durham-positive boxes are used as hosts, there are problems with the stability of the recombinant DNA transferred into them, and no host-vector system has been developed for E. coli. Handling cups is also not easy due to E. coli.

Therefore, there is a need for the development of a method for efficiently (and in large quantities) producing the desired protein using a common bacterial strain such as E. coli.

SUMMARY OF THE INVENTION The present invention aims to solve the above-mentioned problems, and includes a gene encoding a promoter derived from alkaline phosphatase (hereinafter referred to as promoter gene), and under the control of which a gene encoding a signal sequence (signal sequence) is provided. A gene encoding a desired foreign protein (structural gene of the foreign protein) is inserted into a vector containing a gene) to create recombinant DNA, and this recombinant DNA is transferred into host microorganism cells. A method for producing proteins using genetic engineering techniques, in which a transformant is created, the transformant is cultured under certain conditions, the desired protein is secreted outside the bacterial cell (into the culture solution), and the protein is recovered. By providing this information, we aim to achieve this purpose. The present invention also provides that if Durham-negative bacteria (colon @) are transformed and cultured according to the method of the present invention, the desired protein is secreted outside the bacterial cells (into the medium) without lysis of the bacterial cells. This was done after confirming that the desired protein hardly remains within the bacterial body (inside the cytoplasm).

Therefore, the method for producing proteins by extracellular secretion according to the present invention is characterized by (1) comprising the following steps FA) to (D).

(d) To provide 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 cells.

(B) A gene that co-ISes a foreign rice 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. Obtaining transformants.

(C) The above transformant is cultured in a medium containing an amount of inorganic phosphorus necessary for protein synthesis to occur from the late logarithmic growth phase to the early arrest phase of the microorganism's growth process. thing.

(DJ: After the completion of the above-mentioned culture, collect the foreign 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) without retaining them in the periplasm as in the past. (a) Prepare a vector that is equipped with a promoter gene derived from alkaline phosphatase and also has a signal gene under its control, and (b) that can be propagated in the intended host cell, Next, a structural gene of a desired foreign protein is incorporated into this to obtain a recombinant DNA, and this is transferred into a host bacterium to obtain a transformant, which is incubated under certain conditions (b
The present invention relates to a method for producing proteins using genetic engineering techniques, in which a desired protein is secreted outside the bacterial cells (into a medium) by culturing (in step (C) of tJ), and the secreted protein is recovered.

A preferred embodiment of the present invention is a method in which the vector consisting of (a) and (o) above 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 the structural gene of a foreign protein immediately after the signal gene, it is recommended to use a restriction enzyme recognition site artificially created with a small number of base pairs (one of which is at least part of the member) of the signal gene. It is recommended to use one with

In creating this site, the fact that codons consisting of DNA base pairs are degenerate can be skillfully utilized. In other words, if the restriction enzyme recognition/cleavage site produced by d11 is cleaved with the restriction enzyme, the cleavage site or downstream of the signal gene ([if present adjacent to the end,
A foreign gene with an end complementary to the restriction enzyme cut end is prepared on the upstream side, and the exogenous gene is combined with the signal gene at the cut end to create a foreign gene downstream of the signal gene. It is possible to connect directly.

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. If the ligation is performed in the same manner as above, both strands of the signal gene or DNA which have been cut once are restored, and a structure in which the foreign gene is directly linked to the downstream side thereof is realized (details will be described later).

A structural gene of an undesired rice protein is inserted into such a vector to obtain a recombinant DNA, a host bacterium is transformed with this recombinant DNA to obtain a transformant, and this is transformed under certain conditions (
When cultured in step (C) of the present invention, even if the host bacterium is a Durham-negative bacterium such as Escherichia coli, the desired protein will not accumulate in the periplasm and the desired protein will not accumulate outside the bacterial cell (in the culture medium).
: Secreted in.

The object of the present invention is achieved by recovering the desired foreign protein from the culture solution.

Therefore, the present invention avoids the above problems and has the following advantages.

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

It should be noted that the method of the present invention is also applicable to Durum-positive bacteria such as Bacillus subtilis, which does not have a periplasm, so if the method of the present invention is followed, the desired protein will be secreted into the culture medium. Needless to say.

(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 membrane fermentation, so that a protein without a signal sequence can be obtained. If there is an extra gene (DNA 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 embodiment of the present invention, if the gene encoding the desired protein is linked immediately after the gene encoding the signal sequence, the desired protein can be completely produced without any extra amino acids attached. It is secreted into the culture medium as a mature protein. Therefore, if the culture of the transformant is terminated before the bacterial cells are lysed, substantially only the desired protein and the components of the culture solution 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.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing proteins by extracellular secretion. Alkaline phosphatase is affected by the amount of inorganic phosphorus [Biochem, Biophys.

Acta, ), 38.460 (1960), Nature, 1529 (1
959)], it is necessary to have a promoter gene derived from alkaline phosphatase. ) may be any vector that satisfies the requirements of ). Particularly preferable vectors are (a) and U'(1'
), and is designed so that the structural gene 'f: of the desired foreign protein can be linked immediately to the downstream end of the signal gene. A specific example of this preferred vector (plasmid) is the plasmid PTA52, which was previously proposed by a joint researcher of the present inventors.
9 (Patent Application No. 58-1.40748), pTA1529
(Patent Application No. 59-159703), pTA 253
9 (Japanese Patent Application No. 59-279585): 4. All of these plasmids have a promoter and signal sequence derived from alkaline phosphatase (the former two are derived from alkaline phosphatase, pTA2539 is β-
lactamase), and it is possible to attach the structural gene of the foreign protein immediately after the gene encoding the signal sequence. Here pTA 1
52.9 is pTA 529 (pYK 283[E,c
olt K] 2C600 (pYK 283) deposited with FIKEN (FEIKEN Article No. 556)], and by the method described in the specification of Japanese Patent Application No. 58-1.4.0748. pBR322[E, colt K12C600
Deposited as (pBR322) at FEIKEN (FEIKEN Article 2)
No. 35)] Total restriction enzymes coRI and Hind ll
and this EcoRI-Hlnd In portion was replaced with a synthetic linker shown by the following base sequence (1) (see JP-A-59-71692). For details of the procedure for constructing this plasmid, please refer to the specification of Japanese Patent Application No. 59-159703.

EcoRI Hpa I Sma I Hind
mHere, the broken line indicates the restriction enzyme cut @ site, EcoRj
etc. indicate the name of the restriction enzyme that performs the cleavage.

In addition, pTA 2539 converts the ampicillin resistance gene of pTA 1529 into a kanamycin resistance gene,
Furthermore, it is a plasmid in which the signal sequence has been converted to one derived from β-lactamase. For details of the preparation of alanine 1:'', please refer to the specification of Japanese Patent Application No. 59-279585.

Since PTA 1529 contains a DNA portion consisting of the following base sequence (II), 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 its downstream side, or in one embodiment of the present invention [immediately after the downstream end of the gene encoding the signal sequence]. "A gene encoding an undesired rice-based protein can be linked to the protein" refers to a product that contains an NA portion as described above. In addition, in the present invention, when referring to the "downstream side" with respect to DNA, 5'→3
It means the right side when the ' chain (■ chain) is displayed at the top and the 3'←5' chain (e chain) is displayed at the bottom.

The base sequence (If) indicates a part of a DNA gene consisting of double-stranded DNA, and A, G, C, and T each indicate adenine, guanine, cytosine, and thymine. Similarly), Lys%Ala and Trp represent lysine, alanine and tryptophan, respectively. Area (1) of this double-stranded DNA is the signal gene part, area (3) is the restriction enzyme f (ind I
II recognition site, and the dashed line is the HlndIn 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 base sequence (11) corresponds to the alanine codon GCC at the downstream end of the alkaline phosphatase-derived gene portion (1) modified to GCT, and the subsequent base C modified to T. Since co-12 of alanine is degenerate, the modified GCT is also an alanine codon,
Therefore, the DNA portion (1) in (l'l) above still corresponds to the signal gene derived from alkaline phosphatase.

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

Therefore, the alanine codon GCC at the downstream end of the signal gene for alkaline phosphatase
was changed to GC,T, and the base C following the alanine was changed to T.
By modifying this terminal base pair, 4 base pairs upstream, and 1 base pair downstream, ndn for restriction enzymes is created.
l recognition site (3) AAGCTT appears. That is, the signal gene has a small number of 611 restriction enzyme recognition sites, each of which includes at least a portion of the terminal base pairs.

In the specific example of (II) above for pTAL, 529, Hin
The cleavage site within the recognition site (3) of d In the sequence (]]), it exists between the already ligated region (2)) (the position of the cleavage site means that on the downstream side of the double-stranded DNA).

The presence of a restriction enzyme cleavage site at such a position means that
This is most preferred in vectors used in the present invention. This is because this cleavage site cannot be used to directly link the structural gene of a foreign protein to this signal gene, whereas the hybrid or fusion protein produced by the expression of this hybrid gene has a signal sequence and Since the following protein is cleaved by signal peptidase, if the restriction enzyme cleavage site and the signal peptidase cleavage site match like this, the above pTA1
In the case of sequence 529, Hin
This is because it is possible to bond with the sticky end of the signal gene after dIll digestion. F, it goes without saying that if you are willing to supplement the base on the :3'-side of the e chain of the foreign gene, it may be present on the θ1 side above the restriction fermentation site. However, vectors in which such a cleavage site exists are also within the scope of the vectors used in the present invention.

Genes that code for 1 signal sequence generally have various base sequences depending on the 81 types of signal sequences. Some specific examples of signal sequences include those of β-lactamase;
l, 'Acad, Sci, U, S.

A, ), Qian, 3737 (1978)), Riboproteins, Ibid., Dan, 1004 (19'77)), Alkaline phosphatase, European Journal of Biochemistry (Eur, J, Bio
chem, ), 96.49 (1979) 1st prize. Regarding signal sequences, see "Proteins, Nucleic Acids, and Enzymes" Special Issue ("Gene Manipulation"), Volume 26, No. 4, No. 3.
Reference may be made to pages 86-394. However, preferred signal sequences for use in the present invention include those of alkaline phosphatase and those derived from β-lactamase. The above plasmid having such a signal sequence may be used in step (C) of the present invention.
This is because the above-mentioned advantages can be obtained by using this culture method.

One specific example of the DNA gene included in the plasmid (vector) used in the present invention shown in (n) above is one made by modifying the DNA of alkaline phosphatase, so the signal gene DNA portion (1) A DNA portion (2) derived from alkaline phosphatase is bound immediately after the first stream end of the molecule. A specific example of the present invention is this DNA
Part (2) corresponds to the gene encoding foreign rice protein D
This is the NA part.

An example of a DNA gene included in the cilasmid used in the present invention, which falls within the scope of this specific example, has a signal gene portion consisting of 815 minutes derived from a natural product and a synthesized portion. That is, the part upstream of the restriction enzyme cleavage site is derived from a natural product, and the part downstream is synthetic. In this case, the downstream synthesized part is shown above (■)
In the case where the cleavage site is straight at position l', it may be the 4 bases (AGCT) of the Φ chain, or if the cleavage site is upstream from this, there may be a need for a synthetic part in the θ chain as well. Needless to say.

Transformation of microorganisms can be performed 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 plasmid to produce a recombinant DNA (chimeric plasmid),
This method uses known methods such as the Kushner method; Genetic Engineering (Genetic Engineering).
Engineering ) ) K978 ) 17 (
[1,978)], and then any suitable method for obtaining the desired transformant (usually using a cilasmid marker) can be used.

The microorganism that can be transformed with such a chimeric plasmid according to the present invention may be any microorganism as long as the above-mentioned cilasmid can proliferate within the microorganism, and specific examples thereof include Escherichia coli, Bacillus subtilis, and yeast. 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 bacteria. F;
, colt K12 C600 (Imitation Technology Research Institute No. 115), IC, coil K12 Yl (537 (Imitation Technology Research Institute No. 7941), E, coil RRI (ATCC
'31447) and E, coil HBIOI (AT
CC33694) etc. In a specific example of the present invention, E
, colt K12 YK537, E, colt RR
I and E, using coltHB 101.

A specific example of such a transformant used in the present invention is a chimeric cilasmid pTA1522' created by integrating an artificially synthesized structural gene of h-EGF (described below) into cilasmid pTA1529 (described above). , and then this chimera Shirasumi) 3 (recombinant DNA) was transferred to host bacteria E.
, colt K12 YK537, E, colt RR
I and I';, col+I (transformant E, colt K12 Y created by transferring to Blot
K537 (pTA1522), E, colt RRI
(pTA1522) Yohi E, colt HBIOI (
pTA1522) and pTA, 2539
pTA2532 was created by incorporating the structural gene of h-EGF artificially synthesized in the same manner as above, and this
, col'i K12 YK537; transformant P; , colt K12
YK537 (pTA2532).

The gene encoding the desired foreign protein to be incorporated into the plasmid may include hormones, immune-related substances, neuropeptides, enzymes, and the like. Possible methods for preparing these structural genes include obtaining them from natural chromosomal DNA and artificially synthesizing them.For actual methods of preparing structural genes, please refer to Yuzu's findings and literature. can do.

In one embodiment of the present invention, human epidermal growth factor (h-EGF) is used as a structural gene for such a foreign protein.
A chemically synthesized gene encoding A-UG0 (hereinafter referred to as h-F, GF) (see the specification of Japanese Patent Application No. 58-123520 for details of synthesis) was used.

Step (C)/Cultivation of microorganisms The culture according to step (C) of the present invention involves the incorporation of a structural gene of a desired foreign protein into a vector (Shirasumi P) having a promoter derived from alkaline phosphatase to produce a chimeric Shirasmi (organism). The target is a microorganism that has been transformed by transferring this DNA into a host microorganism. The culture method of this step is characterized by the nature of the vector (Shirasumi P) held by the transformed microorganism, that is, (depending on the inorganic phosphorus lid, protein synthesis ability is induced or not). By skillfully utilizing the properties of microorganisms (biochemistry, engineering, biophysics, acta, and natia), the growth of microorganisms and the subsequent induction of proteins by the microorganisms can be carried out in a single culture system.

Details of such a culture method are as follows.

■) Cultivation method The method for culturing microorganisms in this step involves culturing the transformed microorganisms in single-cell pure isolation, then subjecting them to preculture (the above is a well-known conventional method for culturing microorganisms; You can refer to literature and the bottom book.For example, bottom book ``Microbiological Experiment Method'' (published by Kodansha),
1 (published by Kyoritsu Shuppan), 1 Detailed Study Guide to Criminal Science (published by Maruzen), etc. ), which consists of carrying out the culture St in a single culture system.

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

Such cultivation is usually carried out using a jar fermentor, and it goes without saying that an appropriate scale amplifier is possible.
(See Volume 2 (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. MgSO4.7H20, antibiotics, etc.). Furthermore, it is composed of a material containing inorganic phosphorus with the light dispersion necessary to bring about the increase in protein synthesis ability from the late logarithmic growth phase to the early arrest phase during the growth process of the transformed microorganism. In addition, in the specific example of the present invention, as an antibiotic to be added as necessary, E, col
t K12 YK537 (pTA1522) E, co
lt HBlol (pTA1522) and E, col
When culturing tRRI (pTA1522), ampicillin is added, and E, colt K12 YK
When culturing 537 (pTA2532), kanamycin was added. In these specific examples of the present invention, the plasmids held by each transformant are each equipped with genes for anpicillin resistance and kanamycin resistance, and each transformant becomes ampicillin resistant or kanamycin resistant. Therefore, bacteria whose plasmids have fallen off during culture (non-resistant) can be inhibited from growing by ampicillin or kanamycin in the culture 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 any temperature that allows the microorganisms used to grow or grow and at which the product is stable. In the case of commonly used E. coli and E. coli used in the present invention, 37
°C is preferred.

(2) Culture time The culture time in this step is preferably the time at which the production amount of the desired substance secreted into the culture solution is maximized.

A more preferable culture time is a time during which the desired protein is secreted into the culture solution in large quantities. In one embodiment of the present invention, approximately 14 hours are employed when producing h-EGF. 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 the culture time can be performed using as indicators the quantification of glucose and inorganic phosphorus in the medium, the measurement of 0D66o, the measurement of the number of viable bacteria, and the quantification of produced substances in the periplasm and in the culture solution. Glucose was quantified using the glucose oxidase method using the euglucostat ``Fujisawa'' kit. ), the number of viable bacteria is E, c61
1 In the case of K12 YK537 (pTA1522), ampicillin [E, coli K12Y
Kanamycin for K537 (pTA2532)]
The amount of inorganic phosphorus was determined by measuring the number of colonies growing on the agar of L-medium (described above) containing -EGF Owase Hamasu culture solution (10 rng) was taken, centrifuged, and the supernatant was analyzed according to the RRA method (described below). For h-EGF in the periplasm, the bacterial cells were subjected to osmotic shock method and Biol, Chem], the synerte was centrifuged and the supernatant was subjected to radioreceptor assay (RRA) [Journal of Biological Chemistry = (J, Biol, Chem, ).

μ57.3053 (1982):1.
It was also investigated whether F was present within the bacterial body (cytoplasm).

That is, after the above-mentioned Osmochiink shock treatment, the resulting precipitate (bacterial cells) was suspended in pure water VLC, and then subjected to ultrasonic treatment to remove the bacterial cells, and then centrifuged to obtain a supernatant. By analyzing this supernatant according to the RRA method described above, h-gGF within the bacterial cells (inside the cytoplasm) was quantified.

(3) pH of culture medium The pH may be within a range in which microorganisms can grow, and a suitable 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, and a pH of 7.0 to 8.0 is particularly preferred.

(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 it does not contain inorganic phosphorus. A specific example of such an antifoaming agent is ``Antiform-AF-Emulsion'' (Gyudon Kagaku).

(5) #Oxygen present (Do) Oxygen present in the bath refers to molecular oxygen commonly understood in the liquid phase.

It is generally known that in aerated agitation culture, if there is too much DO, the growth of microorganisms will be inhibited, and if the DO is less than 1 ppm, the growth will be similarly inhibited. Therefore, it is preferable to control the amount of DO so that it does not become an inhibitor of microbial growth. In the case of one specific example of the present invention, the Do control device [Oriental Electric ■] FC-
DOi is maintained at 4 ppm by 4W1.

(Process)/Recovery of Protein Recovery of the produced protein can be carried out according to known conventional methods.

For example, ion exchange chromatography, affinity chromatography, electrophoresis, high performance liquid chromatography, or various combinations of these methods
[Refer to Biochemistry Central Research Institute 1 Tan A Refining Chemistry 11 Japanese Biochemistry Gold Line, Tokyo Kagaku Dojin Publishing (1982)], select the appropriate one according to the properties of the protein to be purified or PezotiP. It's fine to use.

In one embodiment of the present invention, the culture solution after completion of culture is centrifuged,
Next, the obtained supernatant is passed through a reverse phase column to perform gel filtration to collect a fraction containing h-EGF, and then this fraction is passed to DEAE-'I"0YOPEAR (Ion Father). Separate the h-EGF fraction by
The desired fractions were collected. In addition, in order to recover a larger amount (and more efficiently) of the target protein produced according to the method of the present invention, the amount of target protein secreted into the culture solution must be increased (and at a time when the bacterial cells are not lysed). After completing the culture,
After collecting the target protein from the culture solution and collecting the bacterial cells, the target protein in the periplasm can be recovered using the osmotiink shock method. According to the method of the present invention, the target protein produced within the host bacterial body does not remain within the bacterial body (in the cytoplasm) but is secreted into either the periplasm or the culture medium. Most of the proteins produced in the body will be recovered. The recovery of proteins in the periplasm can be carried out, for example, by the patent application Shoin-Shoin, which was previously proposed by the present inventors and co-researchers.
The method of No. 22630 may be used.

Experimental Examples Example 1 (1) Creation of transformants Transformants E and colt K12 were prepared according to the following method.
YK537 (pTA1522) was constructed.

pTA1529 (Creation details: 1987-1597)
03 specification)) was added to 50 μl of buffer solution [1
0mM Tris-HCl buffer (hereinafter referred to as Tris-HCI)
) (pH 7,5), H,) m M MgC+2.5
4 units of restriction enzyme Hi in 0 mM NaC]
nd ■C Takara] (hereinafter referred to as Hind m) 3
Hydrolysis was carried out at 7°C for 1 hour. Next, ethanol precipitation was performed, and the resulting precipitate was added to 30 μl of the reaction solution [67 m
M Tri a-HCI, (pH 8,8) 16.6
mM ammonium sulfate [hereinafter (NH4)2SO41,6
, 7mM ethylenediaminetetraacetic acid (EDTA),
0.66 mM each of dATP, dCTP,
The cells were treated with 1 unit of T4-DNA polymerase in dGTP, TTP for 15 minutes at 37°C. Next, the ethanol precipitate was added to 50 μl of the reaction solution [6 m
M Tris-HCI (pH 8,0) 6 m M M
Hydrolysis was carried out at 37° C. for 1 hour using 4 units of the restriction enzyme Sal I [Takara] (hereinafter referred to as Sal I) in 150 mM NaCl]. After the reaction is complete,
A 3900 bp DNA fragment (■ in Figure 1) was obtained by agarose gel electrophoresis.

Plasmid pBR322-hUG [pBR322(E,
colt K12C600<pBR322> [Feikoken Article No. 235]) was deposited as EcoRl and S
h-EGF artificially synthesized from that digested with al I
5 μg of a fragment obtained by digesting the structural gene with EcoRl and Sal I was added to 50 μl of the reaction solution [
100mM Tris-HCI (pH 7,5), 50
37 mM NaCl, 50 mM MgCl2) using 4 units of the restriction enzyme EcoRI [Takara].
After hydrolyzing at ℃ for 1 hour, T4 DNA was added as above.
After polymerase treatment and further Sal I treatment, a 160 bp DNA fragment (■ in Figure 1) was obtained by agarose gel electrophoresis.

The two DNA fragments prepared above (■ and ■ in Figure 1) were added to the reaction solution [20 m M Tris
-HCl (pH 7,5), 10 mM MgC12
,10m M D T T.

300 units of T4 in 0.5 mM ATP]
The reaction was carried out at 14° C. for 16 hours using DNA ligase (Takara).

After the reaction is completed, E. coli Kl 2YK537 is transformed with this, and the desired plasmid is transformed into the following pTA1522.
) (■ in Figure 1).
K12YK537 (pTA1522)) was obtained.

(2) Cultivation of transformants Microorganism E, colt K12 YK537 (pT
A1522) was cultured as follows. Single cell pure fraction 1j7 [t E, colt K12 YK537
(pTA1522) - Medium 1 consisting of tryptone 10g/liter, yeast extract 5g/rindl, NaC 15g/liter, ampicillin 201η/liter
00mQC inoculation, 500me volume of Sakalokolben 37
A shaking culture was performed overnight at °C.

Next, take 1.0 ml of this culture solution and add 1.0 ml of glucose.
Og/liter, tryptone 10 g/liter, yeast extract 5 g/liter, MgSO4.7H200.5
g/liter and ampicillin 201η/liter, and cultured in a 3-liter mini-jar fermenter. The culture temperature was 37°C.
The ventilation amount is 0.5 vvm (1 vvm is l volume
E-volume-minote means that 1 liter of air is introduced per 1 liter of culture per minute. ), pH is 7.2 (4
The dissolved enzyme (Do) concentration was maintained at around 4 ppm by changing the stirring speed using a DO control device. During the culture, the amount of h-EGF in the medium is determined by quantitatively determining glucose and inorganic phosphorus, measuring the number of viable bacteria, and quantifying h-11EGF in the periplasm inside the cells (inside the cells) and in the culture medium. h-EGF in the periplasm. Glutose was quantified according to the glucose oxidase method (using a kit from Fujisawa Kostat). The number of viable bacteria is ampicillin 20+i
#/! This was carried out by measuring the number of colonies appearing on an agar plate of L-medium containing J. 0D66o was measured as an indicator of bacterial mass, and inorganic phosphorus was determined using molybdenum sol-
It was carried out by the method (supra). Quantification of h-EGF in the bacterial body (in the cytoplasm), in the culture solution, and in the periplasm was performed by the following method. That is, the culture solution was 10 m. After centrifugation, h-EGF in the culture solution was quantified by analyzing the supernatant using the RRA method (described below). , 0.02 M Tris-HCI (pH 8,0), E
I'lTA O,00], M Gara'1[ru soaking liquid 2 (
1++ε and left at room temperature for 10 minutes. Then,
Collect the bacteria again by centrifugation, suspend the cells in 20ml of cold water (0°C to 4°C), and leave to stand in water for 10 minutes (treated by osmotic shock method).

Then, this suspension was centrifuged to obtain a supernatant, and h-EGF in the periplasm was quantified by analyzing this supernatant by radioresor assay (RRA). The obtained precipitate (l) was resuspended in 10 w of pure water, and then subjected to ultrasonic processing (1-Kuzeta Insonator Model 200M).
1) Perform step 1 (1 minute) to destroy the bacterial cells, then centrifuge (
270oog for 3o minutes) and RR
By analyzing according to Method A (described below), h-EGF in the bacterial cells (cell titer) was quantified.

The RRA method was performed as follows. KB cells derived from human nasopharyngeal carcinoma cells (ATCC CCL 1.7
) Monolayer culture was carried out in Dulbecco's modified Eagle (DIvI E) medium [Hinaga] in an 800 ml flask. The medium was then removed and the cells were scraped using phosphate balanced salt solution (PBS) containing 0.05% EDTA to create a cell suspension. Cells were then washed twice with Hanks Balanced Salt Solution (HBSS) containing 20 m M Hepes (pH 7,4). Binding trusion (Bi)
nding 5olution) CD IVi E
Medium 20 mM Hepes (pH 7,
4) ・0.35 g/liter Na) ICO3・1
After suspending in 0.00 μg/ml streptomycin], the number of cells was calculated and adjusted to yield 0.000 μg/ml/0.2 trd; binding/trusion.
The mixture was dispensed in 2 ml portions. Next, 0.2 ml of a sample solution containing each of the above three types of supernatant and 1251-mEGF (mouse epidermal growth factor (hereinafter referred to as mEGF)) was added to the tube and incubated at 37°C for 1 hour. After washing the cells three times with water-cooled HBSS, the cells are suspended in 10% trichlorochain 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. The amount of h-EGF was then converted to mEGF. The culture time, 0D66o1 viable cell count, amount of inorganic phosphorus, amount of glucose, and h-EGF (in the bacterial body (cytoplasm), periplasm, and culture solution) over time are shown in Table 1. As shown by the coating, the amount of h-EGF in the periplasm increases with the passage of culture time, but the amount of h-EGF O in the culture solution also begins to increase, and 12.
After 5 hours, the amount of h-EGFH in the culture solution was greater than the amount of h-EGF in the periplasm. Therefore, under such conditions, it is sufficient to incubate for at least 12.5 hours. Note that most of the h-EFF remains in the bacterial cells (cytoplasm) (Table 1). According to the method of the present invention, it can be said that h-EGF does not remain in the bacterial cells.
According to the ratio of h-EGF in the table (added with *** in the table), 63 of the proteins expressed within the bacterial cells were secreted into the culture solution 12.5 hours after the start of culture. .

(3) Protein recovery Centrifuge 1 liter of the above culture solution (12,000g).

10 minutes) to obtain a supernatant, which was subjected to reverse layer chromatography (column size: diameter 4.1 cm x length 8 cm).

Resin: Prep PAK 500/c18 reverse phase resin (Waters) to separate the desired protein fraction. Next, the desired fraction obtained by the above chromatography was applied to a DEAE-TOYOPEAR column (column size: diameter 1,56711 x length 25 (:In)), and the desired protein fraction was separated. When polyacrylamide electrophoresis was performed on both parts of the desired protein, a band (B, C1D, and E in Figure 2) was found at the same position as the h-EGF preparation (A in Figure 2). was observed, indicating that the desired protein h-EGF was recovered. In addition, B, C, D, and E in Figure 2 represent four different images that are thought to contain the desired protein. This figure shows the results of electrophoresis of separated white rice.

Example 2 (1) Construction of transformants Transformants E and colt K12 were prepared according to the following method.
YK537 (PTA2532) was constructed.

pTA2539 ←■ in Figure 3) 5 μg to 50 μm
buffer [, 6mM Tris-HCI pH 8,6
4 units of restriction enzyme Nael (N) in m M MgC + 2.20 m M NaCl].

E, Company B) for 1 hour at 37°C. Then,
Ethanol precipitation was carried out, and the resulting precipitate was collected (43) and treated with ndl114 units in the same manner as above, and a DNA fragment of approximately soo bp was recovered in the same manner as in Example 1 (Fig. 3). Medium ■).

On the other hand, pTA25395μg was treated with Hin
d III [DN of about 3800 bp after Takara 1 digestion
It was recovered as Fragment A (■ in Figure 3).

Furthermore, pBR322-hUG [above] (■ in Figure 3)
) 5 μg was digested with EcoRI in the same manner as above, and then
T4 DNA polymerase treatment and further Sal I
After digestion, a DNA fragment of approximately 160 bp was recovered (■ in Figure 3).

Then, in the same manner as in Example 1, 12YK537 (pTA2
532) was obtained.

(2) Transformants E, colt
K12 YK537 (pTA2532) was cultured.

The culture time, OD, number of viable bacteria, amount of inorganic phosphorus,
The changes over time in the amount of glucose and h-gGpz were as shown in Table 2.
25419 G (14) From the results in Table 2, the same thing as analyzed in Table 1 can be said. In other words, the amount of h-EGFO secreted into the culture solution increased over the amount of h-EGF in the periplasm within 2.5 hours after the start of culture, and, as in Table 1, the amount of h-EGFO secreted into the culture medium increased to 64% of the total, as shown in Table 1. % was secreted. Therefore, even under such conditions, it is sufficient to culture for at least 12.5 hours.

Furthermore, since h-EGF in the bacterial cells has hardly been determined, it can be seen that most of the proteins expressed within the bacterial cells are secreted into the periplasm or into the culture solution.

Example 3 Transformant E, colt K12 YK537 (pTA
1522) glucose 30 g/') liter, triftone 20 g/IJ liter, yeast extract], O
g / liter, MgSO4・7H201, Og /
') Culture was carried out under the same conditions and in the same manner as in Example 1 above, except for this point. The culture time at that time,
Changes over time in the number of 0D66o1 viable bacteria, inorganic phosphorus, glucose amount, and h-EGF amount (in the medium and periplasm) are shown in Table K3.

Incidentally, here, changes over time in β-lactamase (bla) are also shown. This is because bla, like alkaline phosphatase, is a verinolasm enzyme and accumulates in the periplasm. Therefore, whether the desired protein according to the present invention is secreted into the culture solution or due to bacterial lysis, bla is also thought to leak into the culture solution with the passage of culture time. In this example, the desired protein (e.g. h-EGr)
The bla was quantified as an indicator that the secretion of the secretion into the medium was not due to lysis of the bacterial cells but was caused by culturing according to the method of the present invention.

The quantification of bla in the periplasm of bacterial cells and in the culture solution is as follows. That is, to quantify bla in the culture solution, take the culture solution 1 (Di/I), centrifuge it, and dilute the upper groove with 0.I
Appropriately dilute with M sodium-phosphoric acid mild solution (pH 7.0) and prepare the sample bath solution (0.5 μg).
(Formula ■rnl below) is a substrate of PADAC (bla, which is decomposed by bla and changes from purple to yellow. When used, dissolve in the above-mentioned mild solution and 0D56゜S3゜0
(Calbiochem Pering Co., Ltd.)
CALB IOCHEM-BEHRING ) ) 0
．． After adding 5 ml of the mixture and carrying out the reaction at 37°C for about 10 minutes (formula t below), it was boiled for 5 minutes, immediately cooled in water, and then 0D56o was measured. From the difference (Δ0D56o) between this measured value and the measured value of 0D56o of the control (measured in the same manner as above using the above buffer as a sample solution),
The activity of bla was measured using the following formula. In addition, to measure bla in the periplasm, after treating the bacterial cells obtained above according to the osmotiink shock method (described above),
This solution was diluted with 0.1 M sodium-phosphate buffer (pH
7.0), and the following procedure was carried out in the same manner as the quantification of bla in the culture solution.

Here, one unit is the enzyme unit that hydrolyzes the β-lactam ring of 1 μmol of the substrate PADAC in 1 minute, t is the reaction time (minutes), and V is the amount of sample bath solution (α). [Proteins, Nucleic Acids, Enzymes, Dan, 391-400 (1978) 1゜The number of 0D660' viable bacteria at each culture time at that time,
Inorganic # amount, glucose amount, h-EGF amount and blaO
The results of measuring the amounts are shown in Table 3. From the results in Tables 1, 2, and 3, the secretion of the desired protein (h-EGF) into the medium increases with culture time; It is almost 100% 18 hours after the start. However, looking at the blaO value in the culture medium, it shows almost the same value up to 14 hours after the start of culture, so the h-EG secreted into the medium by this time is
F indicates that the bacterial cells do not become gonococcal. Therefore,
The culture time is preferably 14 hours (that is, if Neisseria gonorrhoeae grows, various substances inside the bacteria will be mixed into the culture solution, making it difficult to extract and purify the target product).

Example 4 E, colt RRI (ATCC31447
) by transferring recombinant DNA pTA1522 into transformant E, colt RRI (p
TA1522) was constructed and cultured under the same conditions as in Example 3 above. The culture results at that time are shown in Table 4. In addition, the meanings of *, ** and ne** in the table are as described above.

Example 5 E, colt HBlol (ATCC336
Transformant E, colt HBIO
I (pTA1522) was constructed and cultured under the same conditions as in Examples 3 and 4 above.

The results are shown in Table 5. In addition, in the table *, **Example 6 beta-galactosidase (hereinafter referred to as β~gal) is
An enzyme that exists within cells. Similar to the purpose of quantifying bla in Example 3, β-gal was quantified (the quantification was carried out using the equipment
in Molecu-1ar Genetics)
, 355, (1972) (according to the method of Miller, published by Cold Spring Harbor Laboratory) to determine whether the bacteria were lytic or not. Biology and Biotechnology (E
european Journal of Applied
Microbiolngy and Blotech
-nology), 16.1.4.6-150(1
982), 19.5-12 (1984)].

That is, transformant E, colt 1 (12YK53
7 (pTA1522) was added to the medium used in Example 3 with IPTG (Iaopropylβ-D-Thio-g
alactspyranoside) f 1 m M
Culture was carried out under the same conditions as in Example 3, with the addition of the following ingredients. OD at each culture time at that time
, number of viable bacteria, h-E in the periplasm and culture medium
The quantitative results of GF Xbla and β-gal are shown in Table 6. * and ** in the table have the same meanings as in Tables 1 to 5 above, and h-EGF.

Chi (Hayashi*) of each item of bla and β-gal is h-
For EGF, it shows the ratio of the quantitative value in the medium and in the periplasm to the sum of the quantitative values in the medium and in the periplasm, and in the case of bla and β-gal, and osmotic
It shows the ratio of the quantitative value of each item to the sum of the quantitative values in the bacterial cells after treatment by the shock method. From this result, even after 18 hours from the start of culture, almost no β-gal exists in the cell plasm or medium, and assuming that β-gal is stable in the medium, most of the bacterial cells will be lysed. It is thought that it is not.

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, 1988 (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 Kimoto ATCC (American Type
Culture Co11ec-tron)')
”i 9W 5 Mycological Properties (1) E, Colt K12C600 This bacterium is a Durham-negative bacillus and has the general attributes of Escherichia coli, such as producing spores and being facultatively anaerobic, and also contains F factor. , lacks the function of suppressor gene E, and has a defect in the rec B C gene that encodes a nuclease involved in genetic recombination. Also,
Taxonomically, it belongs to the family Enterobacteriaceae and the genus Escherichia coli.

The literature related to Honkan is as follows.

(a) Genetics, sadness,
440 (1,954) (b) Nature, 217. 11
40(2) E, colt K12C600 (pYK
283) This bacterium produces spores in Durham-negative rod cocoons.
In addition to having general attributes of the genus Escherichia coli such as facultative anaerobic properties, rB BC contains F factor, lacks the function of suppressor gene E, and encodes a nuclease involved in genetic recombination.
It has a genetic defect. As an auxotroph, it requires threonine and leucine for growth on its minimal medium. It contains plasmid pYK283, which is composed of the promoter-operator tiU region of the pho A gene, the replication initiation region derived from pAcyC177, and the bla gene of pBR322, and exhibits resistance to ampicillin. Furthermore, taxonomically, it belongs to the family Enterobacteriaceae and the genus Escherichia coli.

In addition, except for the traits derived from the plasmid pYK283, the Dutch properties of this strain are similar to that of its parent strain E, colt K12C.
It is the same as that of 600.

(3) E, col, i K12C600 (pBR3
22) This bacterium is a Durham-negative bacillus, does not produce spores, has general attributes of the genus Escherichia coli such as facultative anaerobic properties, contains E factor, lacks the function of suppressor gene E, and is involved in genetic recombination. rec B encoding nuclease
It has a defect in the C gene. As an auxotroph, it requires threonine and leucine for growth on its minimal medium. It also contains drug resistance plasmid pBR322. Regarding plasmid pBR322, Gen
e, 2.95 (1977), 12C6'00 to E. coli
Regarding this, reference may be made to the above-mentioned Nature. Except for the traits derived from pBR322, E, col
The orchidological properties of tK12C600(pBR322) are the same as those of the parent strain.

(4) E. coli K12YK537 large intestine m Kl
2YK537 is injected into E. coli using a known method.
Strain CM Microbiologtcal Reviews
12RR1 [Gene.

2.95 (1977), Biochem, Bioph
ys, Acta, .

655.243 (1981)), it exhibits the following properties, and other properties are in accordance with 1.
This strain is no different from that of 2RRI.

rec Al, pho A8, pro] (5
) E, colt HBIOI A bacterium created by hybridizing Escherichia coli K colon, 2 strains (above) and colon iB, and has the following genotype.

References regarding this strain include the Journal of Molecular Biology (J.

Mol, Biol, ) 41.459 (1969)
and Metho in Enzymology
ds Enzymol, ), 68.245 (1979)
There is.

F-1hsd S20 (rn 1mB-), rec
A13, ara-14, p r oA2.1acY1,
galK2, rpsL20 (Smr), xyl-5, m
tl-1,5upE44,λ-(6)E,colt
RRI large intestine (4Hni-ol modified to rccA+, E, co
It is a line conductor of ltHBIOI. Therefore, the genotype is [
Except for F-1recA, it is the same as HBOI].

However, the literature regarding this strain is Gene (Gene).
) 2.95 (1977) and Biochlmica et Byo
physica Acta), 655.243 (198
1) There is.

[Brief explanation of the drawing]

FIG. 1 is a flowchart for the construction of plasmid ppTA1522 used to transform E. colt K12Y537. FIG. 2 is a reproduction of an electropherogram obtained when polyacrylamide gel electrophoresis was performed. FIG. 3 is a flow chart of the construction of cilasmid pTA2532 used to transform E. colt K12Y537. FIG. 4 is a flowchart for construction of cilasmid pTA]524 used for total transformation of E. coli K12C600. Applicant's agent Kiyoshi Inomata■

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 D
Transforming a host by transferring NA into host microorganism cells to obtain a transformant. (C) Cultivating the above transformant 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. (D) After the completion of the above culture, the foreign protein is recovered from the medium. 2. 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 is capable of proliferating within the intended host microorganism cell, encodes the signal sequence. 2. The method for producing a protein by extracellular secretion according to claim 1, wherein the gene encoding a desired exogenous protein is linked immediately after the downstream end of the gene encoding the desired foreign protein. 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.