JPH062066B2 - Plasmid having DNA encoding a novel signal amino acid sequence - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel plasmid that promotes extracellular secretion of a gene product.

(Prior Art) In recent years, genetic engineering research has progressed, and it has become possible to produce useful substances such as insulin, interferon, and interleukin-2 by microorganisms. In the case of such genetic manipulation, Escherichia coli is generally used as a host, but since Escherichia coli belonging to Gram-negative bacteria has a double membrane structure of an inner membrane and an outer membrane, the produced useful substance is secreted outside the cells. However, there is a problem that the separation and purification becomes difficult.

As a method for solving this problem, a method of producing and accumulating in the periplasm using the signal sequence of the alkaline phosphatase gene which is a periplasmic enzyme of Escherichia coli (for example, JP-A-58-69897 and 59-39899). )
And the outer membrane protein of E. coli. Method of secreting through the outer membrane using ompF gene (Japanese Patent Laid-Open No. 59-88092)
Have been proposed.

However, such a method is still few in number, and the former method has a problem that it is only secreted into the periplasm, and development of a better method has been desired.

(Problems to be solved by the invention) Therefore, as a result of diligent studies to develop a method for imparting extracellular productivity to E. coli, the present inventors secreted a protein of the same gram-negative bacterium as E. coli. It has been surprisingly found that the extracellular amino acid sequence of the extracellular protein of Serratia bacterium having the above-mentioned property can be imparted to Escherichia coli to produce extracellular acid, and the present invention has been completed.

(Means for Solving Problems) Thus, according to the present invention, there is provided a hybrid plasmid containing a DNA encoding a signal amino acid sequence of an extracellular protein of the genus Serratia.

The hybrid plasmid of the present invention is one in which a DNA encoding a signal amino acid sequence of extracellular protein of Serratia bacteria is incorporated into a plasmid capable of replicating in a host. Extracellular protein of Serratia spp. Means Serratia spp. (Eg Serratia marcescens IFO3046)
Means a protein secreted extracellularly, and specific examples thereof include metalloprotease and serine protease SS.
Examples include P-1, thiol protease, deoxyribonucleic acid degrading enzyme and the like.

Of these, serine protease SSP-1 is a novel enzyme discovered by the present inventors and has the following physicochemical properties.

1. Action: A protein such as casein is used as a substrate to hydrolyze this.

2. Optimum pH: The action on casein is optimal at around pH 9.0.

3. pH stability: pH5.5 to pH11.0 when treated at 37 ℃ for 30 minutes
When casein is used as a substrate, the residual activity is 90% or more.

4. Optimal temperature: At pH 7.5, it is around 45 ° C when casein is used as a substrate.

5. Temperature stability: 95% at 40 ° C for 10 minutes at pH 7.5
It is stable as above and has about 90% residual activity after treatment at 45 ° C for 10 minutes.

6. Effect of inhibitor: Complete inactivation by pretreatment with 1 mM PMSF (phenylmethanesulfonyl fluoride) for 30 minutes at 37 ° C. No change in activity was observed after pretreatment with 10 mM EDTA at 37 ° C.

7. Molecular weight: about 65,000 (SDS polyacrylamide gel electrophoretic method) 8. Active center: It belongs to serine proteases that have serine in the active center.

The signal amino acid sequence of the present invention is a chromosome of a Serratia bacterium having the ability to produce such extracellular protein.
The gene portion encoding the extracellular protein from DNA is ligated to a plasmid vector, the obtained hybrid plasmid is transformed into Escherichia coli, and the extracellular protein-producing strain is selected and present in the bacterial cell. It can be determined by comparing the DNA sequence of the hybrid plasmid with the N-terminal amino acid sequence of the extracellular protein.

The raw material plasmid used for the preparation of the hybrid plasmid of the present invention may be any of those commonly used in the field of genetic engineering, and specific examples thereof include pB
R322, pBR325, PACYC184, pYEJ001 and the like can be mentioned.

The hybrid plasmid of the present invention can be obtained by inserting a DNA fragment containing a signal sequence as described above into an appropriate restriction site of a raw material plasmid according to a conventional method.

In this case, it is necessary to consider that a liposome binding site (SD sequence) and a promoter are present upstream of the DNA encoding the signal amino acid sequence.

The DNA fragment to be inserted may be chemically synthesized, or may be obtained by cleaving the chromosomal DNA of Serratia bacterium (for example, Serratia marcescens IFO 3046).

An example of a method for creating such a hybrid plasmid is shown below.

(Creation of hybrid plasmid having signal sequence of Serratia protease SSP-1) Serratia spp. Having ability to produce Latia protease SSP-1, for example, chromosome D of Serratia marcescens IFO3046
A serratia protease SSP-1 producing DNA fragment was isolated from NA and used as a raw material plasmid (for example, pBR32
The desired hybrid plasmid can be created by inserting into 2).

First, Serratia marcescens IFO 3046 was cultivated in an appropriate manner to obtain a large amount of cells, and the cells were collected.
Extract and separate the DNA. The obtained chromosomal DNA is partially decomposed by Sau 3 AI, and the decomposed product is subjected to agarose electrophoresis to obtain a DNA fragment having a molecular weight of 1 Kb or more.

On the other hand, the commercially available plasmid pBR322 was digested with Bam HI, the digested product was treated with BAPase (bacterial alkaline phosphatase), and then the above DNA fragment was added thereto to prepare T4 DNA.
Gauze is added to allow the ligation reaction to occur.

The resulting ligation reaction product was subjected to Escherichia coli according to a conventional method.
^{C600 (r -, m -)} ( Proceedings Day Packaging Of Natural
Academia of Science (Proceeding of Natur
al Academy of Science) 71 , 4579-4588, 1974).

The transformed cells were cultured in a plate medium containing skim milk, and the strain producing the cloudy ring (Turbit zone) was isolated to obtain the serratia protease SSP-1.
It is possible to obtain a strain which secretes and produces the bacterium.

One exemplary transformant obtained here is Escherichia coli
It was named C600 (pSP11). Also, Escherichia coli C60
The hybrid plasmid isolated from cultured cells of 0 (pSP11) was designated as plasmid pSP11. The restriction enzyme cleavage map of pSP11 is shown in FIG.

The base sequence of pSP11 thus obtained was analyzed according to the method of Messing et al.
It was confirmed that a part of 11 had the sequence as shown in FIG.

The part corresponding to the signal amino acid sequence in this sequence was determined to be 81 base pairs corresponding to 1 to 81 in FIG.
The basis is as follows.

1. There is a portion corresponding to the SD sequence 6 base pairs upstream of the start codon (ATG).

2. It has a tendency for typical signal sequences rich in hydrophobic amino acids.

3. N-terminal amino acid sequence 8 of Serratia protease SSP-1
And the DNA sequence of 82-105 is completely matched.

The hybrid plasmid of the present invention thus obtained is
It is possible to impart to Escherichia coli a desired useful protein or peptide extracellular production. For example, by inserting and constructing a structural gene for a protein or peptide so that the structural gene of the protein or peptide is located downstream of the signal sequence, the desired useful substance in the form of a fusion protein or a mature protein can be secreted out of the cells. .

Next, examples of the present invention will be described.

Example 1 (Creation of pSP11 and transformation) Serratia marcescens IFO 3046 was added to LB medium [(g /) bactotryptone 10.0, yeast extract 5.0, NaC
l 5.0, glucose 1.0 adjusted to pH 7.2 with Hcl]
After shaking culture at 26.5 ° C for 7 hours, the cells in the late logarithmic growth phase were collected and then subjected to the phenol method (Biochem Biofiz.
Acta) (Biochem. Biophys. Acta) 72 , 619〜, 1963
) And extracted and purified the chromosomal DNA.

Take 10 μg of the obtained chromosomal DNA, add restriction endonuclease Sau 3AI, and allow it to react at 37 ° C for 25 minutes to partially decompose it. Then, subject the reaction product to agarose gel electrophoresis for 1K.
A 30 Kb DNA fragment was recovered from b.

On the other hand, the commercially available plasmid pBR322 (Takara Shuzo) was completely digested with the restriction endonuclease BamHu and then treated with BAPase (bacterial alkaline phosphatase). This is the previous DN
The A fragment was mixed and the DNA strands were ligated with T4 DNA ligase at 16 ° C. for 14 hours.

Separately, a culture of Escherichia coli C600 (r ⁻ , m ⁻ ) was used as a permissive (competent) cell, and the ligation reaction product was added with the above T4 DNA gauze to transform the cell, and the cell was cultured in LB medium for 1 hour. , Ampicillin (Ap) 50 μg / ml, skim milk
Ampicillin resistant (Ap ^r ) strains were selected on LB agar containing 1%. The method of transformation is Mandel and Higa [Journal of Molecular Biology
(J. Mol. Biol.), 53 , 159 (1970)].
Of the 25,000 obtained Ap ^r strains, 2 strains forming a cloudy ring (Turbit Zone) around the colony were obtained. When the plasmids possessed by these two strains were isolated by a conventional method and the cleavage patterns by restriction enzymes were compared, they had exactly the same pattern. This plasmid was named pSP11, and its restriction map is shown in FIG. A bacterium containing pSP11 is designated as Escherichia coli C600 (pSP11).

(Determination of DNA base sequence) pSP11 was double-digested with two kinds of restriction enzymes (HindIII and Ecorl), and a fragment of 790 base pairs (bp) size containing the signal sequence of the Serratia protease SSP-1 gene (first In the figure (Messing) and the method of Messing et al. Using M13 phage (Messing et al.
ing J. et al) Gene 19 269-276 (1982), Sanger, F, Science 214 , 1205-1210 (1981)
The base sequence was determined from the EcoRI cleavage site side by. In this DNA fragment, there is an open reading frame starting at the start codon ATG immediately after the Shine-Dalgarno (SD) sequence, two lysines and one arginine near the N-terminus, and then a hydrophobic mino acid. There was a so-called signal sequence that followed. FIG. 2 shows the nucleotide sequence downstream of the SD sequence.

The N-terminal amino acid sequence of the Serratia protease SSP-1 obtained by the method described below was determined by the Edman degradation method. As a result, the 28th alanine to the 35th leucine of the amino acid sequence predicted from the nucleotide sequence were determined. 8
Mamino acids were the same on both sides.

(Expression of Serratia Protease SSP-1) The transformant having the above pSP11, Escherichia coli C600 (pSP11) was inoculated into an LB medium and cultured at 37 ° C. for 8 hours,
The supernatant was collected by centrifugation. Ammonium sulfate was added to this supernatant so as to be 60% saturated, and the mixture was stirred at 4 ° C. for 12 hours and then centrifuged to obtain a precipitate. This precipitate was dissolved in 10 mM trishydroxymethylamine pH 7.9 and dialyzed against the same solution three times. The dialyzed product was passed through arginine sepharose 4B (manufactured by Pharmacia) equilibrated with the same buffer to obtain purified Serratia protease SSP-1.

Example 2 Escherichia coli C 600 (pSP11) obtained in Example 1 was mixed with 50
The cells were inoculated into an LB medium containing μg / ml of ampicillin, cultured for 6 hours, and then collected by centrifugation to separate the supernatant (extracellular fraction) and cells. After washing the cells twice with physiological saline,
% Shoe cloth, 30mM Tris pH8.0)
mM EDTA was added, and the mixture was stirred at room temperature for 10 minutes. After collecting the cells, the cells were suspended in ice-cold water and stirred at 0 ° C. for 10 minutes. The supernatant (periplasmic fraction) and the precipitate were separated by centrifugation.
The precipitate was then suspended in 10 mM Tris-Bach pH 7.5,
The cells were disrupted by sonication and centrifuged to obtain a supernatant (cytoplasmic fraction).

The protease activity in each of the extracellular fraction, the periplasmic fraction and the cytoplasmic fraction thus obtained was measured, and the results are shown in Table 1. For comparison, pSP1
The transformed strain Escherichia coli transformed with pBR322 instead of 1.
The same experiment was conducted on Kori C600 (pB322), and the results are also shown in Table 1.

First, 5 ml of 0.6% casein solution (50 mM phosphate buffer pH
7.5) for 10 minutes at 37 ° C, add 1 ml of crude enzyme solution, and
React at 30 ° C for 30 minutes, add 5 ml of TCA solution (0.11M trichloroacetic acid, 0.22M acetic acid, 0.33M sodium acetate) to stop the reaction. After standing for 30 minutes, the precipitate is separated by filtration and the absorbance of the supernatant at 280 nm is measured. The activity is shown by the difference between this absorbance and the absorbance of the control (ΔA ₂₈₀ ).

As a control, a solution obtained by adding 5 ml of TCA solution to 1 ml of crude enzyme solution and then adding 0.6% casein solution and treating in the same manner is used.

From these results, it can be seen that Escherichia coli C600 (pBR322) does not secrete the protease outside the cells, whereas Escherichia coli C600 (pSP11) secretes a large amount of the protease outside the cells. The extracellular protease activity was attributed to a single protease, and its N-terminal sequence was confirmed to be Serratia protease SSP-1.

When cells were collected over time from the start of the culture and the state of protease secretion was observed, secretion was observed outside the cells from the beginning of the logarithmic growth phase, and retention in the periplasm was not observed.

On the other hand, the activity of β-lactamase, which is a periplasmic enzyme of Escherichia coli, was determined by the macroiodine method (Method in Enzymology P. 69-85, 1975).
Yearly), Escherichia coli C6
It was confirmed that most of both 00 (pSP11) and Escherichia coli C600 (pBR322) were accumulated in the periplasm.

[Brief description of drawings]

FIG. 1 shows the restriction enzyme cleavage map of the hybrid plasmid pS11, and FIG. 2 shows the map of pSP11 shown in the upper part of FIG. 2 shows the base sequence of the SD sequence, signal sequence, and part of the gene of Serratia protease SSP-1 in the DNA fragment corresponding to.

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Claims (1)

[Claims] 1. A hybrid plasmid comprising a DNA encoding a signal amino acid sequence of an extracellular protein of a Serratia bacterium, which comprises a DNA encoding the following amino acid sequence. Met-Ile-Leu-Asn-Lys-Arg-Leu-Lys- Leu-Ala-Tyr-Cys-Val-Phe- Leu-Gly-Cys-Tyr-Gly-Leu-Ser- Ile-His-Ser-Leu- Ala [Claim 2] The plasmid according to claim 1, wherein the extracellular protein is Serratia protease. 3. The plasmid according to claim 2, wherein the Serratia protease is Serratia protease SSP-1.