AU716408B2 - Novel HS gene, expression plasmid in which HS gene is ligated downstream of structural gene encoding foreign gene PR product, and process for producing foreign gene product using transformant containing the expression of plasmid - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

PATENTS ACT 1990 COMPLETE

SPECIFICATION

FOR A STANDARD PATENT

ORIGINAL

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TO BE COMPLETED BY APPLICANT 'Name of Applicant: Actual Inventor(s): Address for Service: Invention Title: HIGETA SHOYU CO., LTD.

Akimitsu Tanaka and Hiroaki Takagi CALLINAN LAWRIE, 278 High Street, Kew, 3101, Victoria, Australia "NOVEL HS GENE, EXPRESSION PLASMID IN WHICH HS GENE IS LIGATED DOWNSTREAM OF STRUCTURAL GENE ENCODING FOREIGN GENE PR PRODUCT, AND PROCESS FOR PRODUCING FOREIGN GENE PRODUCT USING TRANSFORMANT CONTAINING THE EXPRESSION OF PLASMID" The following statement is a full description of this invention, including the best method of performing it known to me:-

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NOVEL HS GENE, EXPRESSION PLASMID IN WHICH HS GENE IS LIGATED DOWNSTREAM OF STRUCTURAL GENE ENCODING FOREIGN GENE PRODUCT, AND PROCESS FOR PRODUCING FOREIGN GENE PRODUCT USING TRANSFORMANT CONTAINING THE EXPRESSION PLASMID DETAILED DESCRIPTION OF THE INVENTION: Field of the Invention The present invention relates to a biotechnology.

More specifically, the present invention relates to a novel gene, an expression plasmid in which the gene is ligated downstream of a structural gene encoding a foreign gene product, and a process for producing a foreign gene product, which comprises incubating a bacterium of the genus Bacillus which has been transformed with the plasmid to form and accumulate the foreign gene product in the culture, and collecting the same.

Prior Art The production of foreign gene products using recombinants has been widely used in industries of foods, chemicals, toiletries and the like. Bacteria such as Escherichia coli, Bacillus subtilis and the like, yeasts and molds have been used as hosts of gene recombination.

Since a gene recombination technology has been developed using mainly Escherichia coli, Escherichia coli has been utilized well as a host microorganism. However, in a system using Escherichia coli as a host, foreign gene products such as peptides or proteins are retained in a -2cytoplasm or in a periplasmic space between a cell outer membrane and a cytoplasmic membrane, and are hardly secreted and produced in a culture medium.

The accumulation of foreign gene products in cells is quantitatively limited. Besides, it is necessary to pulverize the cells and recover the foreign gene products, and also to separate the desired foreign gene products from co-existent intracellular substances such as nucleic acids and the like and purify the same. Further, peptides or .eo.

proteins produced sometimes form inclusion bodies. When the S inclusion bodies are formed, they have to be converted into active substances through regeneration.

The bacteria of the genus Bacillus secrete and produce large amounts of enzymatic proteins in many cases, and host S vectors have been actively developed utilizing this property.

Among the bacteria of the genus Bacillus, Bacillus subtilis has been studied well genetically and biochemically, and various studies on the secretory production of foreign gene products have been conducted. However, a system using Bacillus subtilis as a host has suffered problems in which foreign gene products such as peptides or proteins are decomposed by strong proteases inside or outside cells.

Assiduous studies have been conducted to eliminate these defects. Consequently, Udaka et al. found that most of strains belonging to Bacillus brevis do not produce proteases.

They produced a secretory vector by using the promoter 3 of the gene of the main extracellular protein [which is described as "outer wall protein and middle wall protein" and as "main extracellular protein" in J. Bacteriol., by H. Yamagata et al., 169, 1239 (1987) and Journal of Nippon Nogeikagaku Kaishi, by N. Tsukagoshi, 61, 68 (1987), respectively] of Bacillus brevis 47 [Methods in Enzymology, by S. Udaka and H.

Yamagata, 217, 23 33 (1993)] and the region encoding a signal peptide of MW protein (middle wall protein) which is one of the main extracellular proteins, and they succeeded in secretory production of -amylase [JP-A 62-201583 (1987) and SJ. Bacteriol., by H. Yamagata et al., 169, 1239 (1987)] or swine pepsinogen [Preprint of 1987 Convention of Agricultural Chemical Sciety of Japan, by S. Udaka, pp. 837 838, and Nippon Nogeikagaku Kaishi, by N. Tsukagoshi, 61, 68 (1987)] using this strain as a host.

Takagi et al. separated Bacillus brevis HPD31 [this strain is the same as Bacillus brevis H102 (FERM BP-1087)1 which does not produce protease outside the cell. They succeeded in the secretory production of heatresistant (-amylase at a high level using Bacillus brevis HPD31 as a host [Agric. Biol. Chem., by H. Takagi et al., 53, 2279 2280 (1989)] and Yamagata et al. succeeded in the secretory production of human epidermal growth factor hEGF at a high level using Bacillus brevis HPD31 as a host [Proc. Nati. Acad. Sci., by 4 H. Yamagata et al., USA, 86, 3589 3593 (1989)].

Problems To Be Solved by the Invention The productivity of foreign gene products using the bacteria belonging to the genus Bacillus, especially Bacillus brevis as host microorganisms has been rapidly improved, as mentioned above, in comparison with that using other host microorganisms. However, to produce foreign gene products in the system using the bacteria belonging to the genus Bacillus, it is required that an expression vector is *e used which is replicable in the bacteria of the genus Bacillus, and that a corresponding promoter is used and an SD sequence and a secretory signal sequence starting with a translation initiation codon are bound downstream thereof and ~a foreign gene is bound thereafter.

However, in this prior technology, the amount of foreign gene product is sometimes very small. Accordingly, a still higher level of the technology has been in demand for industrial usage.

Means Taken For Solving the Problems •o Under these technological circumstances, the present inventors have focussed on the excellent property that the bacteria of the genus Bacillus secrete and produce proteins extracellularly. They have conducted assiduous studies with respect to the presence of genes that improve the secretory production of Bacillus brevis for expediting 5 the production of proteins and increasing the production level from a laboratory scale to an industrial scale.

During the study of the secretory production of a human salivary gland amylase using Bacillus brevis, the present inventors have discovered strains that produce the human salivary gland amylase in quite a large amount from among 50,000 strains of Bacillus brevis transformed with the plasmid prepared by the method in which plasmid pTS7 having a human salivary gland amylase gene [Appln. Microbiol.

Biotechnol., by H. Konishi, 34, 297 302 (1990)j was cut with a restriction enzyme capable of cutting the plasmid at only one site and then ligated with a fragment prepared by cutting the chromosomal DNA of Bacillus brevis with the same restriction ~enzyme.

The plasmid was extracted from this transformant, and the gene analysis thereof was conducted. Consequently, it

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was found that the gene of approximately 150 bp was inserted downstream of the structural gene encoding the human salivary gland amylase. Therefore, this gene was taken out, the base sequence thereof was determined, and the structural analysis thereof was conducted. As a result, since this gene had sequences which are complementary to each other through the sequence of 15 bp; the 15 bp sequence interposed between the sequences, it was considered that when the gene is transcribed to a mRNA it takes a stem loop structure.

Then, this gene was ligated downstream of the structural gene encoding the foreign gene product other than the human 6 salivary gland amylase, and was introduced into the host microorganism to produce the foreign gene product. As a result, it was found that the amount of the foreign gene product was increased. According to the more detailed study, it was newly found that the sequences represented by Sequence Nos. 1 and 2 which are complementary to each other through any sequence of from 3 to 20 bp may be used to increase the amount of the foreign gene product.

The present inventors have conducted further studies on the basis of these useful findings, and have finally completed the present invention.

By the way, the gene having the sequences of Sequence Nos. 1 and 2 which are complementary to each other through any sequence of from 3 to 20 bp is hereinafter referred to as "HS gene".

Table 1 Sequence Listing: Sequence No. 1 Length of sequence: 16 Type of sequence: nucleic acid Type of strand: double strand Topology: linear Type of sequence: genomic DNA Sequence: GGACACTAAA TGGTGT 16 7 Table 2 Sequence Listing: Sequence No. 2 Length of sequence: 16 Type of sequence: nucleic acid Type of strand: double strand Topology: linear Type of sequence: genomic DNA Sequence: ACACCATTTG GTGTCC 16 That is, the present invention relates to novel HS gene, an expression plasmid in which HS gene is ligated downstream of a structural gene encoding a foreign gene product, and a process for producing a foreign gene product, which comprises incubating a bacterium of the genus Bacillus which has been transformed with the expression plasmid to 9 form and accumulate the foreign gene product in the culture, medium and/or the cells, and collecting the same therefrom.

The present invention will be described in detail below.

HS gene of the present invention is a gene having the sequence of Sequence Nos. 1 and 2 which are complementary to each other through any sequence of from 3 to 20 bp. The sequence of from 3 to 20 bp interposing between the sequences of Sequence Nos. 1 and 2 complementary to each other may be any sequence composed of bases selected optionally from 4 kinds of bases, adenine, 8 guanine, cytosine and thymine. Exemplarily, the gene represented by Sequence No. 3 (Table 3 below) in which the sequences of Sequence Nos. 1 and 2 are ligated through the sequence of 15 bp is shown. Further, in the production of a foreign gene product, a gene in which base pairs have been added to the 5'-terminus and/or the 3'-terminus of HS gene may be used. Exemplarily, the gene represented by Sequence No. 4 (Table 4 below) in which the sequence of 86 bp has been added to the 5'-terminus of S* the HS gene represented by Sequence No. 3 and the sequence of 24 bp to the 3'-terminus thereof respectively may be used.

oTable 3 Sequence Listing Sequence No. 3 Length of sequence: 47 Type of sequence: nucleic acid Type of strand: double strand Topology: linear Type of sequence: genomic DNA Sequence: GGACACTAAA TGGTGTCGTA TTCTCAAAGT AACACCATTT GGTGTCC 47 Table 4 Sequence Listing Sequence No. 4 9 Length of sequence: 157 Type of sequence: nucleic acid Type of strand: double strand Topology: linear Type of sequence: genomic DNA Sequence: AAGCTTCGGC ATTATAGTGC GGAGGCTTTT TCGC ATG CAG GTA GGG AAC AAT TAC Met Gin Val Gly Asn Asn Tyr ATT GTC TTT GAT TGT AAA AAT GCT GTT GAC AGG ACA CTA AAT GGT GTC 103 Ile Val Phe Asp Cys Lys Asn Ala Val Asp Arg Thr Leu Asn Gly Val 10 15 GTA TTC TCA AAG TAACACCATT TGGTGTCCAA TTGCAAGTCA TTTGGTAAGC TT 157 Val Phe Ser Lys HS gene is ligated downstream of the structural gene encoding a desired foreign gene product. In this case, HS gene may be directly ligated with the structural gene.

Alternatively, these genes may be ligated through the sequence of from several base pairs or scores of base pairs as shown in the gene represented by Sequence No. 4.

Foreign gene products which are secreted and produced in the present invention may be gene products derived from eucaryotes or procaryotes. Thus, the present invention can be applied to the production of gene products derived from 10 human, animal, bird, fish, microorganism, virus and the like (enzymes, hormones, interferons, immunoglobulins, physiologically active peptides, proteins and the like). For example, the present invention 'can be applied to the production of gene products such as human epidermal growth factor (hEGF) and the like.

In the present invention, a plasmid which is replicable in a host can be used as the vector by which the structural gene encoding a foreign gene product to be 9** secreted and produced and HS gene bound downstream thereof are introduced and retained in the host microorganism. For example, in the system using Bacillus subtilis as a host, pUB110 and derivatives thereof can be used, and in the system using Bacillus brevis as a host, 9* pNU200 ([Nippon Nogeikagaku Kaishi, by S. Udaka, 61, 669 (1987)], pHY700 [Biosci. Biotech. Biochem., by S. Ebisu et al., 56, 812 813 (1992)], pHT110 [JP-A 6-133782 (1994)] and derivatives thereof can be used.

These plasmids may be constructed by a known method, for example, the method described in Molecular Cloning, 2nd ed., A Laboratory Manual, Cold Spring Harbor Laboratory, 1989.

The bacteria which are used as host microorganisms in the present invention may be bacteria belonging to the genus Bacillus. Bacillus subtilis, Bacillus brevis and Bacillus 11 choshinensis are preferable.

The host microorganism may be transformed by a known method. Examples of this known method include the method of Takahashi et al. Bacteriol., by Takahashi et al., 156, 1130 (1983)] and the method of Takagi et al. [Agric. Biol.

Chem., by H. Takagi et al., 53, 3099 3100 (1989)].

Any culture media in which the resulting transformants can grow and produce desired foreign gene products may be used to culture the transformatns.

Examples of the carbon source which is contained in the culture medium include glucose, sucrose, glycerol, starch, dextrin, molasses, urea and organic acids. Examples of the nitrogen source include organic nitrogen sources such as casein, peptone, meat extract, yeast extract, casamino acid and glycine, and inorganic nitrogen sources such as ammonium sulfate. Inorganic salts such as potassium chloride, potassium phosphate monobasic, potassium phosphate dibasic, sodium chloride and magnesium sulfate may be added to the culture medium, as required. In the case of auxotroph, nutrient required for growth thereof may be added to the culture medium. Examples of the nutrient include amino acids, vitamins and nucleic acid bases.

Antibiotics such as penicillin, erythromycin, chloramphenicol, bacitracin, D-cycloserine, ampicillin and neomycin may be added to the culture medium, as required, in the 12 incubation. Further, an antifoamer such as soybean oil, lard, surfactants and the like may be added to the culture medium, as required.

The initial pH of the culture medium is between 5.0 and preferably between 6.5 and 7.5. The incubation temperature is usually between 15 0 C and 42 0 C, preferably between 24 0 C and 37 0 C. The incubation time is usually between 16 and 166 hours, preferably between 24 and 96 hours.

In the present invention, the transformant is incubated under the above-mentioned conditions to form and accumulate a foreign gene product. The thus-obtained foreign gene product may be purified by a known method such as membrane treatment, ammonium sulfate fractionation, chromatography Sand the like Tanpakushitsu Kakusan no Kisojikkenhou, Nankodo Publishing Co. (1985) The present invention has enabled various foreign gene products to be produced stably at a high level by ligating HS gene downstream of the structural genes encoding the desired foreign gene products.

The present invention will be illustrated more specifically by referring to the following Examples. However, the present invention is not limited to these examples.

Example 1 Cloning of a gene (HS' gene) containing HS gene and analysis thereof 13 The chromosomal DNA of Bacillus brevis HPD31 (FERM BP- 1087) was extracted by the method of Saito and Miura [Biochem. Biophys. Acta., by H. Saito and K. Miura, 72, 639 (1964)], and was digested with restriction endonuclease HindIII to form a DNA fragment. Then, plasmid pTS7 [Appl.

Microbiol. Biotechnol., by H. Konishi, 34, 297 302 (1990)] having the structural gene encoding human salivary gland amylase was treated with HindIII, and the 5'-terminus was further dephosphorylated with alkaline phosphatase.

.ot Thereafter, the thus-treated substance was subjected to 0.8 high agarose gel electrophoresis. A DNA fragment of 6.1 kb was recovered using a Gene clean (Bio 101, USA). This DNA fragment was ligated with the above-formed chromosomal DNA fragment through T4 ligase to obtain plasmid pTS7HS.

Bacillus brevis HPD31 was transformed with the thusobtained plasmid pTS7HS through the electroporation [Agric.

Biol. Chem., by H. Takagi et al., 53, 3099 3100 (1989)].

The selection of the strain that highly secreted 2 and produced the human salivary gland amylase from the resultant transformants was conducted by incubating the transformants in a T2 plate medium (containing 1 starch, 1 peptone, 0.5 meat extract, 0.2 yeast extract, 1 glucose and 1.5 agar, pH then spraying a 0.2 I 2 -KI solution thereon, and identifying whether or not each surrounding portion of the resultant colonies became transparent (identifying halo which is generated when starch is 14 decomposed).

Consequently, the colonies of approximately 50,000 forming halos were obtained. From among these colonies, the strain was found that formed the halo having the size of at least twice the size of the halo of Bacillus brevis HPD31 (pTS7 strain) containing plasmid pTS7. This strain is designated HS strain.

HS strain and pTS7 strain were inoculated in a T2Em liquid medium, and were incubated at 30 0 C for 2 days while being shaken. The amount of the human salivary gland amylase contained in the culture supernatant was measured by the method of Saito [Agric. Biol. Chem., by Saito et al., 155, 290 (1973)] using a soluble starch as a substrate.

Consequently, it was found that HS strain produced the human salivary gland amylase of approximately 8 times

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the amount by pTS7 strain as shonwn in Table 13.

Table 13 Amount of the human salivary gland amylase oo•• Strain Amount of human salivary gland amylase (mg/liter) pTS7 strain HS strain 160 Plasmid pTS7HS was extracted from HS strain through the alkali extraction [Nucleic Acids Res., by Birnoboim. H.

15 C. and Doly 2, 1513 (1979)], cleaved with HindIII and then subjected to 5 acrylamide gel electrophoresis.

Consequently, it was identified that in pTS7HS, a gene of approximately 150 bp was inserted in the HindIII site of plasmid pTS7.

The base sequence of the thus-obtained gene was analyzed, and it was found that the gene was composed of 157 bp as represented by Sequence No. 4.

The structural analysis was conducted on the basis of this sequence. As a result, two regions of between the 87th and 102nd bases and between the 118th and 133rd bases as counted from the 5'-terminus have complementary sequences (palindrome structure) which are considered to take a stem structure when these are transcribed to an RNA. In the sequences, an open reading frame encoding the protein composed of the amino-acid sequence represented by Sequence No. 4 was present.

The gene of 157 bp is designated HS' gene.

Secretory production of hEGF using HS' gene The HS' gene in plasmid pTS7HS was amplified by PCR using primer HSM1 represented by Sequence No. 5 shown in Table 5 below and primer HSRV represented by Sequence No. 6 16 shown in Table 6 below (HindIII site in the 5'-terminus of HS' gene was converted into BamHI site by using primer HSM1; this resultant gene is designated HS'B). The amplified HS'B gene was treated with BamHI and HindIII, and subjected to 5 acrylamide gel electrophoresis. The HS'B gene fragment was recovered by the electric elution (Molecular Cloning, 2nd ed., A Laboratory Manual, Cold Spring Harbor Laboratory, 1989).

oTable Sequence Listing: to* Sequence No. Length of sequence: 28 Type of sequence: nucleic acid Type of strand: single strand Topology: linear Type of sequence: other nucleic acid, synthetic DNA Sequence: TTTGGATCCC GGCATTATAG TGCGGAGG 28 Table 6 Sequence Listing: Sequence No. 6 Length of sequence: 28 17 Type of sequence: nucleic acid Type of strand: single strand Topology: linear Type of sequence: other nucleic acid, synthetic DNA Sequence: TTTAAGCTTA CCAAATGACT TGCAATTG 28 Then, plasmid pHT11OEGF [JP-A 6-133782 (1994)] prepared from plasmid pHT926 that Bacillus brevis HP926 (FERM BP-5382) contains was treated with BamHI and HindIII, and subjected to 0.8 agarose gel electrophoresis. A fragment of 3.5 kb was S recovered by using a Gene clean (Bio 101, USA), and then *e ligated with the above-obtained HS'B gene fragment by using T4 ligase to form plasmid pHT11OEGF-HS'B. This plasmid was introduced into Bacillus brevis HPD31 in the same manner as in to obtain Bacillus brevis HPD31/pHT110EGF-HS'B containing pHT11OEGF-HS'B.

The thus-obtained Bacillus brevis HPD31/pHT110EGF-HS'B was inoculated in test tubes containing a 2SLE liquid medium (containing 4 peptone, 0.5 yeast extract, 2 glucose, 0.01 MgSO 4 0.001 FeSO 4 0.001 MnS0 4 0.0001 ZnS04 and I-g/ml of erythromycin, pH 7.2) of 3 ml each.

18 The mixture was incubated at 300C for 3 days while being shaken. The amount of hEGF in the culture supernatant was measured by HPLC [column: C18-100 A, 4 mm (diameter) x 250 mm (length), buffer containing 0.1 TFA/H 2 0 and 0.1 TFA/50 acetonitrile, linear gradient, detection: UV 276 nmI and the measurement was effected in comparison with the peak area given when commercial EGF (made by Funakoshi used as a standard product was subjected to HPLC under the same conditions.

Consequently, Bacillus brevis HPD31/pHT110EGF-HS'B produced hEGF of approximately 1.4 times the amount by Bacillus brevis e HPD31/pHT110EGF.

Table 14 a.

Strain Amount of hEGF (g/liter) B. brevis HPD31/pHT11OEGF 0.8 B. brevis HPD31/pHT110EGF-HS'B 1.1 B. brevis HPD31/pHT110EGF-HSS 1.2 B. brevis HPD31/pHT110EGF-HSFS 1.1 B. brevis HPD31/pHT110EGF-HS3 1.2 B. brevis HPD31/pHT110EGF-HS20 B. brevis HPD31/pHT110EGF-HS 1.1 Example 2 Influence of the structure of HS' gene on the production of the foreign gene product A variant HS' gene HSS was prepared by PCR using primer HSRV of Sequence No. 6 and primer HSS of Sequence No. 7 shown 19 in Table 7.

Likewise, a variant HS' gene HSFS was prepared by using primer HSRV of Sequence No. 6 and primer HSFS of Sequence No.

8 shown in Table 8, a variant HS' gene HS3 by using primer HSRV of Sequence No. 6 and primer HS3 of Sequence No. 9 shown in Table 9, and a variant HS' gene HS20 by using primer HSRV of Sequence No. 6 and primer HS20 of Sequence No. 10 shown in Table 10, respectively.

Table 7 Sequence Listing: Sequence No. 7 Length of sequence: Type of sequence: nucleic acid Type of strand: single strand *Topology: linear Type of sequence: other nucleic acid, synthetic DNA Sequence: TTTGGATCCC GGCATTATAG TGCGGAGGCT TTTTCGCATG CAGGCTTAGG GAACAATTAC Table 8 Sequence Listing: Sequence No. 8 20 Length of sequence: 26 Type of sequence: nucleic acid Type of strand: single strand Topology: linear Type of sequence: other nucleic acid, synthetic DNA Sequence: TTTGGATCCG ACAGGACACT AAAATG 26 Table 9 Sequence Listing: Sequence No. 9 :Length of sequence: 48 Type of sequence: nucleic acid Type of strand: single strand Topology: linear Type of sequence: other nucleic acid, synthetic DNA Sequence: TTTGGATCCG ACAGGACACT AAATGGTGTG TAACACCATT TGGTGTCC 48 Table Sequence Listing: Sequence No. Length of sequence: 21 Type of sequence: nucleic acid Type of strand: single strand Topology: linear Type of sequence: other nucleic acid, synthetic DNA Sequence: TTTGGATCCG ACAGGACACT AAATGGTGTA TGCCCGTATT CTCAAAGTAA CACCATTTGG TGTCC **o In HSS, the length of from the 5'-terminus to HS of u Sequence No. 1 was 10 bp, and HSS was shorter by 76 bp than HS' gene of Sequence No. 4. In HSFS, the protein composed of 27 amino acids that HS' gene encoded was not translated because of the frame shift. When HS3 and were transcribed to the respective RNAs, loop portions of stem loops had sizes of 3 bp and 20 bp (15 bp of HS' gene is present in Sequence No. 4) respectively.

Each of the thus-prepared 4 variant HS' genes was cleaved with HindIII and BamHI, and subjected to 5 acrylamide gel electrophoresis, and each of the resulting fragments was recovered by the electric elution. Each of the fragments was inserted into plasmid pHT11OEGF in the same manner as in Example 1 to form pHT11OEGF-HSS, pHT11OEGF-HSFS, pHT11OEGF- 22 HS3 and pHT110EGF-HS20.

Bacillus brevis HPD31 was transformed with each of the plasmids, and the resulting transformant was incubated in the same manner as in Example 1. The amount of hEGF in the culture supernatant was measured. The results are shown in Table 14. The thus-prepared 4 variant HS' genes produced hEGF in approximately the same amount as the amount by the HS' gene of Sequence No. 4. Accordingly, it was considered that the Simprovement of the secretory production of the foreign gene product was not influenced by the peptide composed of 27 0. ~amino acid residues, and that the palindrome structure in the HS' gene was important to this production. Further, it was found that the loop portion of the stem loop may have a size of from 3 to 20 bp in transcribing HS' gene to a mRNA.

Example 3 Influence of HS gene (palindrome structure region of HS' gene) on the production of the foreign gene product From the results in Example 2, it was considered that the palindrome structure of HS' gene was important to the improvement of the secretory production of the foreign gene product.

23 Accordingly, only the palindrome structure region (Sequence No. 3) of HS' gene was inserted downstream of the hEGF gene of pHT11OEGF, and the productivity of hEGF was examined.

The palindrome structure region (HS gene) in HS' gene was amplified by using primer STEM of Sequence No. 11 shown in Table 11 and primer STEMRV of Sequence No. 12 shown in Table 12. The thus-obtained HS gene was treated with PstI, and then reacted with T4DNA polymerase to blunt the HS gene fragment. This fragment was subjected to 10 I* acrylamide gel electrophoresis to recover a fragment of approximately 60 bp. Subsequently, pHT11OEGF was treated with BamHI and HindIII, then blunted a with T4DNA polymerase, treated with alkaline phosphatase, and subjected to agarose gel electrophoresis. A fragment of kb was recovered by using a Gene clean, and ligated with the above-obtained HS gene through T4 ligase to construct plasmid pHT11OEGF-HS. This plasmid was introduced into Bacillus brevis in the same manner as in Example 1, and the direction of the HS gene inserted was identified by DNA sequencing.

Table 11 Sequence Listing: 24 Sequence No. 11 Length of sequence: 28 Type of sequence: nucleic acid Type of strand: single strand Topology: linear Type of sequence: other nucleic acid, synthetic DNA Sequence: TTTCTGCAGG ACACTAAATG GTGTCGTA 28 .be* Table 12 Sequence Listing: Sequence No. 12 Length of sequence: 27 Type of sequence: nucleic acid Type of strand: single strand Topology: linear Type of sequence: other nucleic acid, synthetic DNA Sequence: TTTCTGCAGG ACACCAAATG GTGTTAC 27 The resulting transformant was incubated in the same manner as in Example 1, and the amount of hEGF produced was determined. As a result, pHT11OEGF-HS produced hEGF in approximately the same amount as that by pHT11OEGF-HS' and in an amount which was 1.3 times that by pHT110EGF.

Effects of the Invention The present invention has developed the novel HS gene. This novel HS gene can be ligated downstream of the structural gene encoding a foreign gene product. The novel expression plasmids thus prepared can secrete and produce various foreign gene products extracellularly in large amounts by transforming bacteria of the genus Bacillus through these plasmids, and incubating the thus-obtained transformants.

Bacillus brevis H 102 was deposited with the Fermentation Research Institute Agency of Industrial Science and Technology on 24 June 1986 and assigned accession/deposit number FERM BP-1087.

Bacillus brevis HP 926 was originally deposited with the Fermentation Research Institute Agency of Industrial Science and Technology on 18 December 1991 and assigned accession/deposit number FERM P-12664.

This was subsequently transferred on 8 February 1996 with the National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology Ministry of International Trade and Industry and assigned accession/deposit number FERM BP-5382.

Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component or group thereof.

o* o• *e 22/12/99,document4,2

Claims (8)

1. A gene comprising an HS gene which is composed of Sequence No. 1, Sequence No.2, and a base sequence of 3 to 20 bases on the same nucleic acid strand; said Sequence Nos. 1 and 2 being separated by said base sequence of 3 to 20 bases.

2. A gene according to claim 1, wherein the base sequence of 3 to 20 bases is composed of bases selected from the group consisting of adenine, guanine, cytosine and thymine.

3. A gene having the base sequence of Sequence No. 3.

4. A gene having the base sequence of Sequence No. 4. An expression plasmid comprising a gene according to any one of claims 1 to 4 ligated downstream of a structural gene encoding a foreign gene product.

6. A process for producing a foreign gene product, which comprises incubating a bacterium of the genus Bacillus containing an expression plasmid according to claim 5 to form and accumulate the foreign gene product in a medium and/or in the i bacterium and collecting the foreign gene product.

7. The process of claim 6, wherein the foreign gene product is a human epidermal 25 growth factor.

8. A gene comprising an HS gene which is composed of Sequence No. 1, Sequence S° No.2, and a base sequence of 3 to 20 bases on the same nucleic acid strand; said Sequence Nos. 1 and 2 being separated by said base sequence of 3 to 20 bases, 30 which gene is substantially as herein described with reference to at least one of the 9 accompanying Examples.

9. An expression plasmid comprising a gene comprising an HS gene which is composed of Sequence No. 1, Sequence No.2, and a base sequence of 3 to 20 bases 35 on the same nucleic acid strand; said Sequence Nos. 1 and 2 being separated by S' said base sequence of3 to 20 bases and said expression plasmid being substantially as herein described with reference to at least one of the accompanying Examples. A process for producing a foreign gene product, which comprises incubating a bacterium of the genus Bacillus containing an expression plasmid according to claim 5 to form and accumulate the foreign gene product in a medium and/or in the bacterium and collecting the foreign gene product said process being substantially as herein described with reference to at least one of the accompanying Examples. 22/12/99,mg9022 spe,2