JPH0349685A - Novel dna chain and its use - Google Patents

Abstract

PURPOSE:To obtain the subject DNA chain capable of extracellular secretion of an useful biologically active substance, etc., by a host bacterium by making the DNA chain a base sequence capable of coding a polypeptide having a specified amino acid sequence. CONSTITUTION:A DNA chain containing a base sequence capable of coding a polypeptide having an amino acid sequence from A to B shown in the figure. The above-mentioned DNA chain is obtained, e.g. by chemical synthesis of a part or more of the chain according to the nucleic acid synthetic method. The extracellular secretion of a desired protein or polypeptide is carried out by transforming a host bacterium using a DNA chain containing a fragment capable of coding a signal sequence for permeation through the endomembrane of the host bacterium and another fragment capable of coding an amino acid sequence of the desired protein or polypeptide and a DNA chain having the above-mentioned base sequence and culturing the resultant transformant in order to make the transformant secrete the desired protein or polypeptide into the culture medium.

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] Technical Field The present invention relates to a novel DNA strand that enables extracellular secretion production of useful physiologically active substances by a bacterial host, and
This paper relates to secretory production methods of useful physiologically active substances using chains.

Prior Art At present, various methods have been devised for producing genetically useful physiologically active substances using recombinant DNA technology, by causing host microorganisms to secrete and produce the physiologically active substances.

In particular, when using Escherichia coli as a host, attempts have been made to use various signal sequences to allow the gene product to penetrate the inner membrane of Escherichia coli (Japanese Patent Laid-Open No. 61-37099).
, JP 61-135599, JP 61-14908
9, JP 61-282084, JP 62-5508
7 and Japanese Patent Application Laid-Open No. 62-151193 jj40
In most cases, the gene product remains in the periplasmic fraction, and complete extracellular secretion is rarely achieved. In order to allow the gene products of the periplasmic fraction to permeate the outer membrane, a method has been considered that utilizes the kil gene, which is one of the colicin genes of Escherichia coli (Japanese Patent Application Laid-Open No. 158-1581).
Publication No. 790, Journal of the Japanese Society of Agricultural Chemistry, Volume 62, No. 11, 16
19-1628, 1988). It is known that complete expression of the kil gene has a lethal effect on the host, and attempts have been made to obtain resistant mutant strains (Japanese Patent Laid-Open No. 63-2
No. 02375).

[Summary of the invention]

Abstract: The present invention provides a novel DNA that provides the ability to secrete proteins or polypeptides produced by a bacterial host to the outside of the bacterial cell.
The purpose is to provide A chain and to provide a method for secretory production of a desired protein or polypeptide using this DNA #jl to secrete the desired protein or polypeptide outside the bacterial cell (into the medium). We have discovered that a DNA fragment derived from a factor that promotes the exocrine production of gene products in bacterial hosts such as Escherichia coli (NCI8 11038) can achieve this purpose, and based on this knowledge, we have developed the present invention. has been completed.

That is, the DNA chain according to the present invention is characterized in that it has a base sequence encoding a polypeptide whose amino acid sequence is substantially from A to B shown in FIG.

In addition, the method for secretory production of a desired protein or polypeptide according to the present invention provides a D
A method characterized in that a desired protein or polypeptide is distributed into a medium by transforming a host cell with N A fl and the DNAvi according to claim 1 and culturing the transformant. It is.

<Effects> By expressing the DNA chain of the present invention in a bacterium, it is possible to secrete and produce veliplasm proteins of the host bacterium outside the bacterium.

Therefore, by expressing the DNA strand of the present invention and the gene of a useful physiologically active substance (comprising a portion encoding a signal sequence for permeation of the inner membrane of the host bacterial cell) in a bacterial host, it is possible to Various desired and useful physiologically active substances, which were secreted only in small amounts or in small amounts outside the bacterial cells, are now secreted and produced outside the bacterial cells, and these physiological activities It becomes possible to obtain substances in the culture medium.

[Specific description of the invention]

DNA strand of the present invention> The DNA strand of the present invention has an amino acid sequence of ml in length.
It has a base sequence encoding a polypeptide from A to B shown in the figure, and the present invention D
The gene expression product of NAfi has the effect of promoting the secretion of proteins secreted to the veliplasm from the host bacterial cell to the outside of the bacterial cell (hereinafter also referred to as the effect of promoting extracellular secretion).

Here, the term "rDNA chain" refers to a polydeoxyribonucleic acid chain having a certain length. In the present invention, this rDNA chain is specified by the amino acid sequence of the polypeptide it encodes, and since this polypeptide has a finite length as described above, this DNA chain also It is of finite length. However, this D
The NA chain contains a gene encoding a polypeptide having the above-mentioned effect of promoting extracellular secretion, and the expression of this gene is useful for promoting extracellular secretion. This gene is not expressed by its 5'-upstream and/or
This gene can be expressed in a state in which a DNA chain of an appropriate length is bound to the 3' and downstream sides. Therefore, when referring to a "DNA chain" in the present invention, in addition to a DNA chain of this specific length (in terms of the corresponding amino acid sequence in FIG. 1, the length is A-8), a DNA chain of this specific length is also used. It shall include those in the form of a chain or cyclic DNA containing A as a member.

Typical forms of the DNA strand according to the present invention include a plasmid in which the DNA strand is a part of its structure, and a plasmid existing in bacteria, particularly Escherichia coli. A plasmid, which is one of the preferable forms of existence of the DNA strand according to the present invention, is a combination of the DNA strand of the present invention as a passenger or foreign gene, a plasmid vector and a promoter that stably exists and can be replicated in bacteria. This is what I did. Known plasmid vectors and promoters can be used in appropriate combinations. See below for suitable plasmid vectors and promoters.

Polypeptide Encoded by the Cucumber Gene> As mentioned above, the DNA jfl according to the present invention is specified by the amino acid sequence encoded by it. This polypeptide is responsible for promoting the extracellular secretion of veliplasm proteins in host bacterial cells, and its amino acid sequence is substantially the same as those from A to B among the amino acid sequences shown in Figure 1. be. Here, "the amino acid sequence is substantially the one from A to B among the amino acid sequences shown in Figure 1" means that as long as this peptide has the secretion-promoting effect, some of the amino acids are deleted, This indicates that substitution, addition, etc. may occur.

The polypeptide of the present invention that exhibits a typical extracellular secretion-promoting action consists of the amino acid sequences A to B in FIG. 1, and consists of 76 amino acids. The arrangement was unknown.

Although the mechanism by which the promotion of extracellular secretion occurs in the present invention is not clear, it can be said that at least the promotion of extracellular secretion occurs when the polypeptide having the amino acid sequence from A to B is expressed (see reference experimental examples below).

<Base sequence of DNA strand> The DNA strand of the present invention has the nucleotide sequence A to H in FIG. These are those with a base sequence that corresponds to the change, or their degenerate isomers. As used herein, "degenerate isomer" refers to DNA strands capable of encoding the same polypeptide that differ only in degenerate codons. For example, A-8 in Figure 1
For a DNA chain with a base sequence of
In the present invention, those in which the codon corresponding to (GAG) is changed to (GAA), which has a degenerate relationship with this, are referred to as degenerate isomers.

A preferred embodiment of the DNA strand according to the invention is one having at least one stop codon (eg TAA) adjacent to its 3'-end. Furthermore, the 5' side upstream and/or 3' side downstream of the present invention includes a DNA strand as an untranslated region (the first part of the 3' side downstream is a stop IL codon such as TAA). ) may continue for a certain length.

The base sequence of the DNA strand shown in Figure 1 is Achr
omobacter lophagus (NCIB
11038) was determined by the dideoxy method for a gene encoding a polypeptide having an effect of promoting extracellular secretion.

<Obtaining a DNA chain> One means of obtaining a DNA chain according to the present invention, that is, a DNA chain having a base sequence encoding the amino acid sequence of the polypeptide having the above-mentioned extracellular secretion-promoting effect, is by nucleic acid synthesis. The aim is to chemically contain at least a part of its growth and development according to its direction.

^chrosobaetor Iophagus (N
CIB 11038) from the eastern body gene library, U-1'-Kamigaku's part! It can also be obtained by a method commonly used for 1lf, such as hybridization with an appropriate probe, but if the bound amino acid is 7
Considering that there are six elements, it is easier to synthesize the total by chemical synthesis only.

Uses of the DNA strand of the present invention The DNA strand of the present invention can be used to express this gene in bacteria together with a gene for a useful physiologically active substance (having a portion encoding a signal sequence for permeation of the western membrane of host bacterial cells). This enables the exocrine production of the physiologically active substance by cultivating the bacteria, and is suitable for the exocrine production of various useful physiologically active substances.

Outline of secretory production method for desired protein or polypeptide
Essential> The method for secretory production of a desired protein or polypeptide according to the present invention uses a DNA containing a portion encoding a signal sequence for permeation through the inner membrane of a host bacterial cell and a portion encoding the amino acid sequence of the desired protein or polypeptide. transforming a host bacterium with the DNA chain and the DNA chain according to claim 1, and culturing the transformant to secrete the desired protein or polypeptide into the medium;
As mentioned above, it is characterized by the following.

Specifically, for example, (1) a DNA containing a portion encoding the above-mentioned signal sequence and a portion encoding a desired protein or polypeptide;
By creating a recombinant DNA in which both the A chain and the DNA strand of the present invention are incorporated into one expression vector, transforming a bacterial host with this, and culturing this transformant under appropriate conditions, the desired result can be obtained. The protein or polypeptide can be secreted into the culture solution. In addition, the above DNA strand (■) and the present invention DNA (■)
The A chain may be inserted into separate expression vectors that are compatible in the host bacterium, and both vectors may be used to transform the bacterial host. Furthermore, the above DNA strand (■), the present invention DNA (■)
One or both of the A chains may be integrated into the chromosome of the host bacterial cell and expressed.

Construction of an expression vector (1) A DNA chain containing a portion encoding a signal sequence for permeation of the inner membrane of the host bacterial cell and a desired protein, or a portion encoding the amino acid sequence of voripeptide It is generally known that a signal sequence necessary for cell membrane permeation exists in the N-terminal region of sexual proteins. It is known that the signal sequence is cleaved by a specific enzyme during cell membrane permeation to produce a mature protein.

In the present invention, the aforementioned DNA #J'l is a DNA strand that encodes a signal sequence that enables membrane permeation of a protein in a host bacterial cell and a DNA strand that encodes a desired protein or polypeptide. DN containing
It is the A chain. The part encoding the signal sequence and the part encoding the desired protein must be read at the same time. Preferably, the signal peptide is cleaved between the C-terminus of the signal sequence and the N-terminus of the desired protein, so that the two parts are linked so as not to contain unnecessary base sequences. Known methods in this case include methods for removing excess nucleotides by site-directed mutagenesis techniques using synthetic oligonucleotides.

Furthermore, as DNA synthesis technology has advanced in recent years, it is now possible to include everything from the signal sequence portion to the structural gene.

Any signal sequence can be used as long as it functions in the bacterial host used. When the host is Escherichia coli, for example, Om, which is a large intestine outer membrane protein,
pA (JP-A-63-237790), OmpF (
Japanese Patent Publication No. 61-282084), periplasmic protein alkaline phosph-7ase (Japanese Patent Publication No. 61-37099)
) and other signal sequences can be used.

The desired protein or polypeptide is not particularly limited, but one that is naturally secreted by the cell from which it is derived is preferred. Examples include human-derived growth hormone, human-derived GM-CSF, and the like.

(2) Construction of expression vectors Procedures or methods for constructing expression vectors may be those commonly used in the fields of molecular biology, bioengineering, or genetic engineering, such as those described by sT. Manl
atls, E. F. ['rlLsch. J. Saa
brookrMolecular Clonlng A
Laboratory Manual J, Col.
d Spring Laboraio
ry. (19g2), h( can be referred to.

Examples of vectors used include p for the colonic flexure.
There are plasmid vectors such as BR322 and pBR328, and pZA22 (see Japanese Patent Application Laid-Open No. 62-228278) for bacteria of the genus Zymomonas.

Examples of promoters include flac, Tc'CAT,
Trp, tac (for E. coli), Tc rSCA
T (for Zymomonas flexor), etc.

In addition, when constructing an expression vector into which both the DNA strand (1) and the DNA strand of the present invention (2) described above are inserted, the order and direction in which both DNA strands are arranged should be such that the genetic information can be expressed, i.e. As long as each gene is appropriately transcribed and translated within the host, it does not matter in what order or in what direction. In addition, regarding the promoter, one nophlomotor may be used so that it acts on both DNA strands, or both DNA fl
A promoter may be attached to each fi. The sequence necessary for liposomes to bind to mRNA (in the large intestine, SD
sequence) must be attached to both DNA strands.

Transformation> Bacteria, especially Durum-negative bacteria, are used as hosts. A specific example of such a preferred bacterial host is, for example, E. coli.

Transformation methods that are commonly used in this field may be used; for example, the above-mentioned Molecular Clo
ning A t, laboratory manual
etc. can be referred to.

As an example of transformation with an expression vector containing the DNA strand of the present invention and a DNA strand containing a portion encoding a signal sequence and a portion encoding a desired protein,
Eschcrich1a co11 e(ioO(pA
S23) PIEL?M BP-
2505) has been deposited.

Secretory production of protein> Using the transformant obtained as above, that is, a DNA strand containing a gene for a polypeptide having an extracellular secretion promoting effect and a gene for a desired protein having a portion encoding a signal sequence. If the 115-transformed bacteria are cultured, they will produce and accumulate the desired protein outside the body (that is, in the culture solution).

The culture or culture conditions of the transformant are essentially the same as those for the host bacteria used. Furthermore, recovery and purification of the desired protein from the culture solution can also be carried out according to conventional methods.

Experimental Examples> In the following experimental examples, DNA was handled using conventional methods (Mania mouth, molecular, molecular, etc.) unless otherwise specified.
eu-lar Clonlng. Cold Spring
g llarbor Laboratory (19g2
)).

Experimental example 1: DNA derived from Achromobaetor
Obtaining fragments of Aehrosobacter 1opha
gus (NCIB 1101g) on a slant medium (
Liquid medium (Bacto T
ryptonc (Dirco) 2. 0%, Baet
oYeast Extract (Dirco)0.
5%, NaCl2. 4%, pH 7.0), 3
After culturing with shaking at 0°C for 20 hours, the bacterial cells were collected,
Miura's method
gy. l2, pp. 543-545. Acadeslc
Chromosomal DNA was extracted and purified using (Press). Restriction enzyme EcoR1 (manufactured by Blind Shuzo Co., Ltd.) was added to this chromosomal DNA IOμK, and the mixture was incubated at 37°C for 5 minutes, 10 minutes, 20 minutes, 30 minutes, and 6 minutes.
Partial decomposition was carried out by reacting for 0 minutes. On the other hand, cloning vector p AT 3 2 5 (Nakasu
ra at al. J. Blotch'. 3.23
9-248 (1988)) 1 ug of ophthalmic enzyme E
After complete Mation with coRI, 1 μl of Escherichia coli alkaline phosphatase (1! manufactured by Shuzo) was added, and the mixture was incubated at 65°C.
After the treatment, the alcoholic acid was deactivated by phenol extraction. This vector DNA was mixed with the above-mentioned partial chromosomal DNA digest, and a reaction was carried out at 12° C. for 18 hours using T4 phage-derived DNA ligase (manufactured by Boehringer Mannheim Pharmaceutical Co., Ltd.). ? 1) The obtained recombinant DNA mixture was used to transform Otomo strain C600. The obtained transformants were grown on an agar medium (Bacto Tryptone) containing 50 μg/ml ampicillin.
D1fco) 1. 0%, Bac t O Yes
t Extract (Dirco)0. 5%, Na
Cl1. 096, Soluble College
n(SERVA) 0.5%, Sirius l? cd
(BDII) 150■/l), and colonies with visible loss of pigment around the colonies were selected. This colony was added to L containing 50 μg/ml ambicillin.
B medium (BacLo TrypLone (Dil'co
) 1%, BaetoYeast ExtracL (
Dlfco) 0. 5%, NaCl 19 (; )
After culturing overnight at 37°C, plasmid DNA was grown using a standard method.
was isolated to obtain plasmid pAs20.

pAs20 has the structure shown in FIG.
A 6.6 kb EcoRI fragment derived from r was inserted. Next, we created deletion mutants of pAs20 using various fermentation inhibitors, cultured them, and examined the protein levels in the culture supernatant. It was identified that it was located between Mlul and Pstl in Figure 2. Mlul-Pstl fragment into M13mpl
9 vector (manufactured by Boehringer Mannheim 11), and the base sequence was determined by the dideoxy method.

FIG. 1 shows the obtained nucleotide sequence and the open reading frame indicated by A to B in the figure.

Experimental Example 2: Secretory production of β-lactamase by Escherichia coli The DNA fragment shown in Figure 1 was treated with Klenow enzyme to make both ends blunt, and the plasmid vector pAT325
was inserted into the EcoRV site of the tetracycline resistance region of . The obtained plasmid was named pAS23 (Figure 3)
. Escherichia m strain C600 was transformed using plasmid pAS23 and pAT325 as a control, and the resulting transformant was inoculated into LB medium containing 30 μg/ml chloramphenicol and incubated at 37°C for 24 hours. After culturing, the growth of the bacteria and the amount of protein in the culture solution were measured by conventional methods. Crush the bacterial cells using ultrasound to obtain a bacterial cell extract.
Enzyme activities (β-lactamase, β-galactosidase) of the culture supernatant and bacterial cell extract were measured. β-lactamase was obtained using the method of Sawai et al. (Ant1s1crob.Agen
Ls ChesoLher. ．． 13.910-913(
(1978) ), β-galactosidase activity is
The method of Iler et al.
MolecularGeneties. p 352-3
55, Cold Sprlng HarborLab
I1-1 was determined according to the Oratory (f972)). The results are shown in Table 1. After 24 hours of culture, E. coli harboring pAS23 showed no difference in growth compared to the control. released into the supernatant. On the other hand, β
- Most of the galactosidase activity was found in the cell extract even in the large intestine containing pAS23, indicating that the extracellular secretion of β-lactamase in the previous series was +11
It can be seen that this is not the result of bacteriolysis. The above Yuri is
This figure shows the six uses of the DNA strand of the present invention regarding the extracellular secretion production of useful physiologically active substances in Escherichia coli.

Table 1: Extracellular secretion of β-lactamase by pAs23 (Reference experiment example: Loss of extracellular secretion-promoting effect due to linker Shindai) In order to confirm whether this was caused by the expression of the peptide, the following experiment was performed. The sequence CAGCTG from the 201st to 206th positions west of the base sequence in FIG. 1 is a site recognized by the restriction enzyme Pvu[I. PsL summer linker GC at PvuI1 site in 228bp of plasmid pAS23
Insert TGCAGC and create plasmid pAS23-Ps
I created t. In pAS23Pst, the above Pv
The amino acid sequence after the uI1 site is the original one (Figure 1A
-B's amino acid sequence). pAS23-Ps t into E. coli C60
After culturing at 37°C for 24 hours in LB medium containing 100 μg/ml ampicillin, the protein amount of the soil serum was determined by n1, and E. coli harboring pA323, pAT3
A comparison was made with the case of E. coli harboring 25. The result is second
As shown in the table, protein secretion did not occur in pA323-Pst, as in the case of pAT325. From the above, it has been revealed that this DNA fragment has an effect of promoting extracellular secretion by expressing the polypeptide shown in FIG. 1 AB.

Table 2 Plasmid with loss of secretion function due to linker insertion Secretion of protein into culture medium pAT32
5 pAS23-Ps t pAS23 Deposit of ten microorganisms The microorganisms related to the present invention have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as follows.

Microorganism Accession number Accession date E
schericl11a cot I Microtechnical Research Institute 7/16 0800 (pAS23)
No. 2505 (1'ERM BP-2505)

[Brief explanation of drawings]

FIG. 1 shows the sequence of the DNA chain and the amino acid sequence of the encoded polypeptide according to the present invention. Each amino acid is represented by a single letter, 7'k, and is represented by the first do of the corresponding codon. FIG. 2 shows a reduced structure of plasmid pAs20. Figure 3 shows the behavior of plasmid pAS23.

Claims (1)

[Scope of Claims] 1. A DNA strand having a base sequence encoding a polypeptide whose amino acid sequence is substantially from A to B shown in FIG. 2. A DNA strand comprising a portion encoding a signal sequence for permeation of the inner membrane of a host bacterial cell and a portion encoding an amino acid sequence of a desired protein or polypeptide, and the DNA strand according to claim 1 to transform a host bacterium. A method for secretory production of a desired protein or polypeptide, which comprises secreting the desired protein or polypeptide into a medium by transforming the transformant and culturing the transformant.