JP2001231570A - Protein having function to control localization of lipoprotein in cell of microorganism belonging to gram- negative bacterium and dna encoding the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To identify and provide proteins each having such a general function as to isolate from cell membrane and deliver to localization mechanism (LolA- LolB mechanism) to outer membrane a matured lipoprotein after processing in microorganisms belonging to Gram-negative bacteria. SOLUTION: The protein, proteins (LolC, LolD and LolE) each having the function to control the localization of a lipoprotein in Escherichia coli, are identified, and their amino acid sequences are identified respectively. These amino acid sequences are compared, on a database, with the gene arrangements of other Gram-negative bacteria and the corresponding homologous proteins are searched. By controlling the expression of the objective protein, it is hopeful of raising the secretive production efficiency of useful protein using microorganisms; and by searching a substance capable of specifically inhibiting the function, it is thought that antibacterial agents for Gram-negative bacteria can efficiently be obtained; furthermore, using antibodies to the above proteins, it is also hopeful of constructing a specific detective system for Gram-negative bacteria.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a protein having a function of controlling lipoprotein localization in cells of a microorganism belonging to Gram-negative bacteria.

[0002]

2. Description of the Related Art Escherichia coli has a protein (hereinafter, referred to as lipoprotein) to which at least 10 kinds of lipids are added, present in an outer membrane or a cytoplasmic membrane. All lipoproteins are biosynthesized as precursors of signal peptide fused to the N-terminus, similar to secreted proteins. Glyceride is bound by a thioester bond, and the signal peptide is processed by lipoprotein-specific signal peptidase II to become a mature form (Microbio).
l. Rev .. , Vol. 57 pp. 50-108, 1
993). The Cys residue exposed by the processing is further subjected to aminoacylation, and finally becomes a mature lipoprotein with three molecules of fatty acids added to the N-terminal. Of these lipoproteins, the second amino acid residue following the Cys residue is As
It is known that if it is a p residue, it stays in the cytoplasmic membrane, and all other residues are transported to the outer membrane (Cel).
1, Vol. 53 pp. 423-432,198
8).

In recent years, lipoproteins localized in the outer membrane have been
A and LolB were found to be transported by the mediation of two factors (EMBO J., Vol. 16, p.
p. 6947-6955, 1997). LolA is a periplasm-specific chaperone molecule that forms a soluble complex with lipoproteins located in the outer membrane (EMBO
J. , Vol. 14, pp. 3365-3372,19
95). Lipoproteins are water-insoluble because they have a lipid site at the N-terminus, and must form a water-soluble complex to cross the periplasm. Lipoprotein and LolA
Binds to the lipoprotein LolB present in the outer membrane, the lipoprotein is passed from LolA to LolB, and the lipoprotein is incorporated into the outer membrane. The localization mechanism of lipoproteins by the LolA-LolB system in E. coli is universal (J. Biol. Chem., Vo).
l. 274, pp. 30995-30999,199
9).

It is known that the mechanism by which lipoproteins are detached from the cytoplasmic membrane and transported to the outer membrane requires the energy of ATP. However, LolA-Lol
In the localization mechanism by the B system, there is no reaction involving ATP hydrolysis activity. That is, LolA and LolB, which have not yet been found in the mechanism of lipoprotein outer membrane localization.
Other factors were suggested to be involved.

[0005] The secretory production of useful proteins using Escherichia coli has already been put to practical use as seen in the case of human growth hormone (FEBS Lett., V.
ol. 204, pp. 145-150, 1986). In addition, since the secretory mechanism of secreted proteins has been elucidated in detail, several examples of improved productivity such as co-expression of SEC factors have been reported (BIO / TECHN).
OLOGY, Vol. 12, pp. 175-180; JP-A-9-313191). However, there is no known report on the improvement of productivity of lipoproteins because the mechanism of localization is unknown.

At present, colistin and polyxin B are typical antibacterial agents against gram-negative bacteria, other aminoglycoside antibiotics such as spectinomycin and paromomycin, and broad antibacterial antibiotics tetracycline and chloram. Phenicol is used as a clinical and feed-added antibacterial agent. However, toxicity and side effects have been reported on the human body, livestock, and poultry for all of these drugs, and there has been a problem of emergence of resistant bacteria due to continuous use. Therefore, development of new antibacterial agents has been demanded.

[0007] Representative causative bacteria of infectious food poisoning include pathogenic Escherichia coli, Salmonella, and cholera.
There is also a social demand for a method for rapidly detecting these pathogenic Gram-negative bacteria.However, specific antibodies that conventionally react universally with microorganisms belonging to Gram-negative bacteria and do not cross with other microorganisms are available. Not obtained.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to release the mature lipoprotein after processing in a microorganism belonging to Gram-negative bacteria from the membrane and localize it to the outer membrane (Lol).
(A-LolB mechanism) to identify and provide a protein having a universal function. Another object of the present invention is to identify and provide a protein necessary for enhancing secretory productivity when secretory production of lipoprotein is performed using gram-negative bacteria. A further object of the invention is
An object of the present invention is to identify and provide a protein having a specific function to be a target when searching for a new antibacterial agent effective against Gram-negative bacteria. Still another object of the present invention is to identify and provide a protein that serves as an antigen necessary for obtaining an antibody that universally reacts with microorganisms belonging to Gram-negative bacteria and does not cross other microorganisms. .

[0009]

Means for Solving the Problems The present inventors have constructed a system for reconstituting the lipoprotein localization mechanism in Escherichia coli on proteoliposomes and belonged to a new ABC transporter requiring ATP hydrolysis. The protein complex LolCDE containing the protein LolD was successfully obtained. The amino acid sequence was queried from the NCBI gene database, and it was found that these protein complexes are essential proteins that are universally present in Gram-negative bacteria such as Salmonella, Haemophilus influenzae and Cholera.

That is, the present invention is as follows. (1) having the function of controlling the localization of lipoproteins in the cells of microorganisms belonging to Gram-negative bacteria, substituting the amino acid sequence of SEQ ID NO: 1 or one or more amino acids in the sequence; LolC protein or its homologous protein obtained by insertion or deletion. (2) A DNA encoding the protein of (1). (3) the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or the DNA of (2), which hybridizes with the DNA under stringent conditions and encodes a protein having the same function. (4) having the function of controlling the localization of lipoproteins in the cells of microorganisms belonging to Gram-negative bacteria, substituting the amino acid sequence of SEQ ID NO: 2 or one or more amino acids in the sequence; LolD protein or its homologous protein obtained by insertion or deletion. (5) A DNA encoding the protein of (4). (6) the DNA comprising the nucleotide sequence of SEQ ID NO: 2 or the DNA of (5), which hybridizes with the DNA under stringent conditions and encodes a protein having the same function. (7) having the function of controlling the localization of lipoproteins in the cells of microorganisms belonging to Gram-negative bacteria, substituting the amino acid sequence of SEQ ID NO: 3 or one or more amino acids in the sequence; LolE protein or its homologous protein obtained by insertion or deletion. (8) A DNA encoding the protein of (7). (9) The DNA according to (8), which has a nucleotide sequence represented by SEQ ID NO: 3 or encodes a protein which hybridizes with the nucleotide sequence under stringent conditions and has the same function. (10) A recombinant vector containing the DNA of (2), (3), (5), (6), (8) or (9). (11) A transformant containing the recombinant vector according to (10). (12) A monoclonal antibody reactive with the protein of (1), (4) or (7). (13) A monoclonal antibody having reactivity with a protein having the amino acid sequence of SEQ ID NO: 1, 2, or 3. (14) The protein according to (1), (4) or (7), wherein the gram-negative bacterium is Escherichia coli. (15) The gram-negative bacterium is Escherichia coli (2), (3),
(5) The DNA according to (6), (8) or (9).

The LolC protein, LolD protein and LolE protein of the present invention can be applied using genetic engineering techniques. Examples of applications using genetic engineering techniques include, for example, to obtain an antigen, or to regulate the expression level in cells to improve secretory productivity of lipoproteins, or to transduce a heterologous microorganism, or amino acid Functional enhancement by substitution can be considered.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, examples of microorganisms belonging to Gram-negative bacteria include Escherichia coli, Salmonella, Influenza, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Cholera, Pasteurella, Neisseria gonorrhoeae, Meningococcus, Pertussis Fungi, pestis, Actinobacillus, Shewanella, and the like.

In the present invention, the term lipoprotein in a broad sense means a complex of protein and lipid, and the binding method of the protein and lipid includes a thioether group at a thiol group of a cysteine residue and a prenyl group of a lipid. An amide bond is known between the N-terminal glycine or lysine ε-amino group and the carbonyl group of the fatty acid. In addition, the LolC of the present invention
As long as the lipoprotein can act on the DE complex, the lipoprotein may be derived from microorganisms other than Gram-negative bacteria or eukaryotic cells, or may be derived from organelles such as mitochondria. Furthermore, artificial proteins modified by adding lipids to enhance solubility can also be included in the category of lipoproteins of the present invention. The artificial protein modified by adding lipid as used herein includes not only those synthesized using the lipid addition function of cells but also those prepared by using a chemical synthesis technique in a test tube. Gram-negative bacteria include major outer membrane lipoprotein Lpp and outer membrane lipoprotein Pal
And the like.

In the present invention, lipoprotein localization means
This means that the lipoprotein precursor synthesized in the cytoplasm passes through the cytoplasmic membrane, undergoes lipid modification to become a mature form, and is localized in the cytoplasmic membrane and outer membrane. The function of controlling lipoprotein localization means the ability to mediate physicochemical reactions required for the localization process. More specifically, the function is to release mature lipoprotein on the cytoplasmic membrane from the cytoplasmic membrane and pass the lipoprotein to LolA, which is a periplasmic chaperone. Having the function of controlling lipoprotein localization means that the lipoprotein itself controls the localization of lipoprotein as well as the case where the function is exerted only after forming a complex with another protein. included. In addition, this function involves the hydrolysis of ATP. The function of detaching the lipoprotein from the cytoplasmic membrane can be measured in Example 3.

The amino acid sequences of SEQ ID NOs: 1, 2 and 3 and the nucleotide sequences of DNA represent the amino acid sequences of LolC, LolD and LolE proteins of Escherichia coli and the nucleotide sequences of DNAs encoding them, respectively. As long as the protein has the function of controlling lipoprotein localization in the cells of microorganisms belonging to Gram-negative bacteria, one, two or more amino acids and DNA have been substituted, inserted, and deleted in the amino acid sequence and DNA base sequence. Those are also included in the scope of the present invention.

[0016] Amino acid substitution, insertion,
To introduce a mutation for deletion, the Kunkel method or G
A known genetic engineering method such as an applied Duplex method or a method similar thereto may be used. Alternatively, a method of introducing a mutation that is non-selectively generated by treatment with ultraviolet light or a chemical agent may be used.

In the present invention, the hybridizing DN
A is D having the DNA sequence of Sequence Listing 1, 2 or 3.
NA means DNA that hybridizes using genetic engineering techniques such as Southern hybridization,
Stringent conditions include, for example, a sodium concentration of the hybridization solution of 15 to 150 mM, preferably 15 to 30 mM, and a temperature of 37 to 60 ° C.
The condition is preferably 60 ° C.

The recombinant vector of the present invention can be obtained by ligating (inserting) the DNA of the present invention into an appropriate vector. The vector into which the DNA of the present invention is inserted is not particularly limited as long as it can be replicated in a host. For example, plasmid DNA and phage DNA
And the like, and can be directly integrated into chromosomal DNA.

As the plasmid DNA, a plasmid derived from Escherichia coli, for example, pBR322, pBR325, pU
C18, pUC19, pUC118, pUC119, p
KK223-3 and the like. In addition, all plasmids capable of replicating in Gram-negative bacterial hosts are available. Examples of phage DNA include lambda phage and M13
Phage and the like. In order to insert the DNA of the present invention into these vectors, a method in which the purified DNA is first cut with an appropriate restriction enzyme and ligated to a restriction enzyme cleavage site or a multiple cloning site in an appropriate vector can be mentioned.

[0020] The DNA of the present invention can be obtained from the sequence listing 1, 2 or 3
Must be incorporated into the vector so that the protein consisting of the amino acid sequence can be correctly translated. Thus, in addition to the DNA of the present invention, a DNA containing a promoter, an enhancer, a ribosome binding sequence, a transcription terminator, a drug selection marker and the like can be ligated to the vector of the present invention. In addition, as a drug selection marker,
Examples include an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, and a kanamycin resistance gene.

The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the target protein can be expressed. Here, the host is not particularly limited as long as it can express the DNA of the present invention. For example, E. coli (Escheri)
chia coli), Salmonella, Pseudomonas aeruginosa and the like.

When Escherichia coli is used as a host, the recombinant vector of the present invention is capable of autonomous replication in the cells,
It is preferred that the promoter, ribosome binding sequence, and transcription terminator function efficiently. Further, a gene controlling a promoter may be included. As Escherichia coli, for example, Escherichia coli K
There are 12 strains and HB101 strain.

Any promoter can be used as long as it can be expressed in a host such as Escherichia coli. For example, trp promoter, lac promoter, PL
Examples of the promoter include a promoter derived from Escherichia coli or a phage such as a promoter. Furthermore, artificially designed and modified promoters such as the tac promoter may be used.

The method for introducing a recombinant vector into a host is not particularly limited as long as it is a method for introducing DNA into bacteria. For example, a method using calcium, an electroporation method, and the like can be given.

The expression of the protein of the present invention can be obtained by culturing the transformant and collecting from the culture. The term “culture” means any of a culture supernatant, a cultured microbial cell or a crushed microbial cell.
The method for culturing the transformant of the present invention is performed according to a usual method used for culturing a host.

The medium for culturing the transformant includes:
As long as the medium contains a carbon source, nitrogen source, inorganic salts, and the like that can be assimilated by the microorganism, and can efficiently culture the transformant,
Either a natural medium or a synthetic medium may be used. As the carbon source, carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used.

As the nitrogen source, there may be used ammonium salts of inorganic acids such as ammonia, ammonium chloride, ammonium sulfate and ammonium phosphate, or ammonium salts of organic acids, or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor and the like. Can be As inorganic substances,
Potassium potassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used. If necessary, vitamins and nucleic acid-related substances may be added.

In the case of a liquid medium, the culture is generally carried out at 37 ° C. for 6 hours under aerobic conditions such as shaking culture or aeration and stirring culture.
It is performed for up to 24 hours, but anaerobic culture is also used depending on the nature of the microorganism. When Escherichia coli is used as the transformant host, the pH is preferably maintained at 7.0 to 7.5 during the culture period. Adjustment of the pH is performed using an inorganic or organic acid, an alkaline solution or the like. If necessary, antibiotics such as ampicillin and tetracycline may be added to the medium.

When culturing a microorganism transformed with an expression vector using a temperature-sensitive inducible promoter as a promoter, the culturing temperature may be changed as necessary. When culturing a microorganism transformed with an expression vector using a compound-inducible promoter, an inducer may be added to the medium as needed. For example, when culturing a transformed microorganism using an expression vector using a Lac promoter, isopropyl-β-D-thiogalactopyranoside (IPTG) or the like is used to transform the microorganism using an expression vector using a trp promoter. When culturing the isolated microorganism, indoleacrylic acid (IAA) or the like may be added to the medium.

In the present invention, LolC protein and Lo
It is possible to create a method for screening for a substance that inhibits lipoprotein localization using the ID protein or LolE protein. Since strains deficient in these proteins are lethal, substances that inhibit these functions can be expected to be useful as antibacterial agents against Gram-negative bacteria. In this screening method, the amino acid sequence portion of these proteins that is well conserved among microorganisms belonging to Gram-negative bacteria is prepared by chemical synthesis or genetic engineering techniques and strongly binds to these peptide fragments. A method of searching for a substance is conceivable. Specifically, use of an affinity column or biosensor on which the peptide is immobilized can be considered.

In the present invention, an antibody against the LolC protein, LolD protein or LolE protein of the present invention can also be prepared. The term “antibody” refers to an entire antibody molecule or a fragment thereof (eg, Fab or F (ab ′) 2 fragment) capable of binding to each protein of the present invention, which is an antigen. It may be. The antibodies of the present invention can be produced by any of various methods. Methods for producing such antibodies are well known in the art. For example, Sambr
book, J.K. et al, Molecular Clo
ning, Cold Spring Laborato
ry Press (1989) or the like.

LolC protein, LolD protein and Lol that control lipoprotein outer membrane localization in Escherichia coli
A method for isolating the E protein, a method for confirming its function, and a method for searching for a homologous gene (ORF) from a DNA database of microorganisms belonging to Gram-negative bacteria will be described in detail below.

The inventor of the present invention disclosed a reconstituted proteoliposome composed of phospholipids of Escherichia coli and purified LolA and LolB from each fraction obtained by solubilizing a protein localized in a cytoplasmic membrane and various column chromatography. Above, a protein that promotes detachment of the lipoprotein Pal located in the outer membrane from the membrane was screened. The N-terminal amino acid sequence of the protein in the fraction whose activity was confirmed was identified, and a matching ORF frame was searched from the DNA database of E. coli. An ORFyc having a domain exhibiting ATP dependency in the amino acid sequence translated from these ORFs
fV was found and the product was named LolD. next,
Similarly, ORFycfU and ORFycfW for two proteins that form a complex during purification and behave together
From each other, and identify each product as L
Named olC and LolE. These three proteins were purified, and the above-mentioned phospholipid of Escherichia coli and purified LolA
As a result of examining the elimination of lipoprotein Pal from the membrane on the reconstituted proteoliposome composed of ATP and LolB, the elimination reaction was confirmed in the presence of ATP. In addition, when mutant Escherichia coli expressing these proteins in a temperature-dependent manner was prepared and cultured at a non-permissive temperature, it was confirmed that the cells could not grow. From the above results, the LolCDE complex was found to be LolA-
These factors are responsible for all reactions in which the localization mechanism of the outer membrane lipoprotein by the LolB system has not been discovered, and no other factors are required. In addition, since deletion of these genes is lethal in Escherichia coli, it can be inferred that this is the only mechanism without an alternative function.

Furthermore, the inventors have determined the amino acid sequence of each protein constituting the LolCDE complex and
The amino acid sequence was translated from the ORF of Gram-negative bacteria registered in the NA database and queried. As a result, this Lo
It has been revealed that proteins having high homology to the ICDE complex are universally conserved in microorganisms belonging to Gram-negative bacteria.

[0035]

The present invention will now be described more specifically with reference to examples.

[Example 1] Isolation of LolC protein, LolD protein and LolE protein (1) Solubilization of membrane protein and fractionation by column chromatography Escherichia coli K12
Was fractionated by centrifugation, and 2% (w /
v) Sucrose monoca plate (Dojindo Laboratories), 50 mM containing 5 mM magnesium sulfate, 2 mM ATP and 1 mM DTT (dithiothreitol)
4 ° C., 10 ° C. in Tris-HCl buffer (pH 7.5)
Solubilized for minutes. This lysate was centrifuged at 100,000 G for 30 minutes, and the resulting supernatant was subjected to 0.5% (w / v) sucrose monoca plate, 2 mM magnesium sulfate,
50 mM Tris-HCl buffer containing 0.2 mM ATP and 1 mM DTT (dithiothreitol) (p
H7.5) was added to a MonoQ anion exchange resin column (Pharmacia) equilibrated with NaCl concentration of 0 to 1
Apply a linear gradient up to 50 mM, 4 m / min
Eluted at a flow rate of 1 l. The fraction eluted around 100 mM NaCl concentration was adjusted with 0.1 volume of 1 M potassium phosphate buffer (pH 7.5), and 0.5% (w / v) sucrose monoca plate, 2 mM magnesium sulfate, Econopack CHT-II equilibrated with 10 mM potassium phosphate buffer (pH 7.5) containing 0.2 mM ATP and 1 mM DTT (dithiothreitol)
It was added to a hydroxyapatite column (Bio-Rad). Elution was performed at a potassium phosphate concentration of 10 mM to 500 mM.
A linear gradient to mM was performed at a flow rate of 1 ml per minute. The fraction having a potassium phosphate concentration of about 400 mM was concentrated using Macrosep 30K (Nippon Pall). This concentrated solution was added to a 0.5% (w / v) sucrose monoca plate, 2 mM magnesium sulfate, 0.2 mM AT.
It was added to a Superose 12 gel filtration column (Pharmacia) equilibrated with 50 mM Tris-HCl buffer (pH 7.5) containing P and 1 mM DTT (dithiothreitol), and eluted at a flow rate of 0.5 ml per minute. Was done.

(2) Preparation of proteoliposome Proteoliposome is described in Eur. J. Biochem.
Vol. 192, pp. 583-589 (1990). First, 0.8 mg of phospholipid of Escherichia coli washed with acetone and purified lipoprotein (Pal
Or Lpp) 0.5-1 μg on ice for 10 min.
μl of 1.2% (w / v) sucrose monoca plate, 50 mM containing 2 mM magnesium sulfate, 0.5 mM ATP and 1 mM DTT (dithiothreitol)
It was mixed with mM Tris-HCl buffer (pH 7.5). This mixture was mixed with 1 ml of buffer A (100 mM
50 mM with NaCl and 5 mM magnesium sulfate
The mixture was diluted with potassium phosphate (pH 7.5) and dialyzed against buffer A overnight in the presence of 2 mM ATP. The proteoliposome was recovered by centrifuging the dialysate at 200,000 G for 1 hour. After the collected fraction was dissolved in 100 μl of buffer A containing 2 mM ATP, freezing, thawing and sonication were performed while adding ATP.

(3) Acquisition of protein having elimination activity in proteoliposome The elimination activity of lipoprotein from cell membrane is described in J. Am. Biol. C
hem. Vol. 274, pp. 30995-3099
9 (1999). That is, 2 mM AT was added to 50 μl of the above proteoliposome suspension.
Mix 250 μl of buffer A to which P was added, and mix at 30 ° C.
And cultured. The reaction solution was centrifuged at 200,000 G for 1 hour.
The precipitate fraction and the supernatant fraction are 10% trichloroacetic acid (TCA)
, And immunostained using an anti-Pal antibody or an anti-Lpp antibody after separation by SDS-PAGE. As a result, S
A fraction that promoted lipoprotein desorption was found in the eluted fraction separated using the upperose 12 gel filtration column (FIG. 1).

(4) Identification of Obtained Protein The protein eluted from the gel filtration column (Fr. 15-19) was analyzed by SDS-PAGE, and this fraction contained 9 types of proteins. I was N-terminal amino acid sequences could be decoded for seven of these, and ORFs encoding each could be identified. Of these ORFs, only ycfV is ABC
It had Walker A and B motifs that predicted it belonged to the transporter family.

(5) Identification of Carried Protein The molecular size of the above-mentioned ycfV gene product was expected to be 130 to 140 kDa from the gel filtration elution time, but this value was larger than the molecular size calculated from the ORF. According to SDS-PAGE analysis, this protein had 2 other
It has been found that proteins of two molecular sizes associate and are always purified together with their behavior. The N-terminal amino acid sequence of these proteins could also be decoded, and the ORFs, ycfU and ycfW encoding the respective proteins could be identified. These ORFycfU, ycfV and y
The present inventors have determined that cfW-derived products are LolC and Lo, respectively.
Named ID and LolE.

[Example 2] Confirmation of function of LolCDE complex (1) Preparation of strain overexpressing LolCDE complex 8.5 kb Sma containing peripheral gene of ycfV gene
The I-digested DNA fragment (FIG. 2) was transferred to a Kohara library (Cell, Vol. 50, pp. 495-508, 1).
987) and lambda phage clone 238 (E4C
Obtained from 2). This SmaI fragment was replaced with plasmid pU.
It was incorporated into C19 to construct pJY310.

(2) Measurement of elimination activity A protein fraction extracted from the cytoplasmic membrane of Escherichia coli K003 transfected with plasmid pJY310 (K003 / pJY310 strain), and a protein fraction extracted from the cytoplasmic membrane of Escherichia coli K003 transfected with pKM001 When the protein fraction extracted from the cytoplasmic membrane of Escherichia coli K003 strain into which pUC19 had been introduced was reacted with proteoliposomes at 30 ° C. at 20 μg each at 30 ° C., the elimination activity of Pal increased about 10-fold in 5 minutes. (FIG. 3). This SmaI fragment is ycfU, yc
It was suggested that fV and ycfW formed an operon. Further, when plasmid pKM001 containing only ycfU, ycfV and ycfW was prepared,
003 / pKM001 strain-derived cytoplasmic extract fraction showed the same effect as pJY310.
It has been found that these three gene products alone are sufficient for enhancing the elimination activity of l.

(3) Purification of LolCDE complex LolCDE complex overexpressing strain (K003 / pJY31)
LolCDE complex was purified from the cytoplasmically extracted protein fraction of Strain No. 0). The final purified sample contained three proteins, which eluted as a 130-140 kDa complex in gel filtration chromatography. The purified sample was separated by SDS-PAGE, and the N-terminal amino acid sequence of each protein was determined.
It was confirmed that they were cfV and ycfW.

Example 3 Gene Analysis (1) Search for Homologous Genes In order to confirm that the LolCDE complex is an essential gene product universally present in Gram-negative bacteria,
CBI genome database (http://www.ncbi.nlm.ni
h.gov/BLAST/unfinishedgenome.html)
Homologous genes were searched using a homology search program. As a result, homologous genes were searched from representative microorganisms belonging to Gram-negative bacteria.

(2) Search for homologous region L found from a representative microorganism belonging to Gram-negative bacteria
When the homologous proteins of the olC protein, LolD protein and LolE protein were compared, it was found that two or more regions having high homology existed in any of the proteins. These regions are LolCDE
It is presumed that this is an important region for expressing the function of the complex. The region of high homology in the LolC protein includes the amino acid sequence from the 20th to the 35th from the N-terminal, the 245th to the 295th from the N-terminal, and the 37th from the N-terminal.
As the region of high homology in the LolD protein, which includes the amino acid sequence from the 5th to the 399th amino acid sequence (FIG. 5), the Walker motif A and the ABC transporter motif 1 which have been known so far were found (FIG. 4) ). A, B, ABC1, ABC
2 and LolD motifs are shown by solid lines. LolE
The region of high homology in the protein is 3
Amino acid sequence from 0th to 65th and 26 from N-terminal
0th to 310th and 365th to 40th from the end of N
The fifth amino acid sequence is included (FIG. 6).

[0046]

[Sequence List] Sequence Listing <110> MITSUI CHEMICALS, INC. <120> Novel proteins and DNA encoding them which are mediating the out er membrane-localization of lipoproteins in Gram-negative bacteria <210> 1 <211> 1200 <212> DNA <213> Escherichia coli <220> <221> CDS <222> 1..1200 <400> 1 atg tac caa cct gtc gct cta ttt att ggc ctg cgt tac atg cgt ggg 48 Met Tyr Gln Pro Val Ala Leu Phe Ile Gly Leu Arg Tyr Met Arg Gly 1 5 10 15 cgt gca gcg gat cgc ttc ggt cgt ttc gtc tcc tgg ctt tct acc atc 96 Arg Ala Ala Asp Arg Phe Gly Arg Phe Val Ser Trp Leu Ser Thr Ieu 20 25 30 ggc att acc ctc ggg gtg atg gcg ctg gtc aca gta ttg tca gtg atg 144 Gly Ile Thr Leu Gly Val Met Ala Leu Val Thr Val Leu Ser Val Met 35 40 45 aac ggc ttt gag cgc gag ctg caa aac aac atc ctt ggc ctg atg Asn Gly Phe Glu Arg Glu Leu Gln Asn Asn Ile Leu Gly Leu Met Pro 50 55 60 cag gca att ctc tct tct gag cat ggc tct ctt aac ccg cag caa ctc 240 Gln Ala Ile Lele Ser Ser Glu His Gly Ser Leu Asn Pro Gln Gln Leu 65 70 75 80 cca gaa acg gca gtc aaa ctg gac ggc gtt aat cgc gtc gca cct att 288 Pro Glu Thr Ala Val Lys Leu Asp Gly Val Asn Arg Val Ala Pro Ile 85 90 95 act acc ggt gat gtg gta ctg caa agc gcg cgc ggc gg ggt ggg 336 Thr Thr Gly Asp Val Val Leu Gln Ser Ala Arg Ser Val Ala Val Gly 100 105 110 gtg atg ctc ggt atc gac ccg gcg caa aaa gat cca ctt aca ccg tat 384 Val Met Leu Gly Ile Ala Pro Ala Gln Lys Asp Pro Leu Thr Pro Tyr 115 120 125 ctg gtc aat gtg aaa caa act gac ctc gag ccg ggg aaa tat aat gtc 432 Leu Val Asn Val Lys Gln Thr Asp Leu Glu Pro Gly Lys Tyr Asn Val 130 135 140 atc ctc ggc gaa caa ctt gcc tca cag cta ggc gtt aat cgc ggt gat 480 Ile Leu Gly Glu Gln Leu Ala Ser Gln Leu Gly Val Asn Arg Gly Asp 145 150 155 160 caa atc cgc gtg atg gta cca tct gcc agc cag ttc acg ccg atg ggg Ggg 528 Val Met Val Pro Ser Ala Ser Gln Phe Thr Pro Met Gly 165 170 175 cgt att cca agc cag cgc ctg ttc aat gtg att ggc act ttc gcc gcc 576 Arg Ile Pro Ser Gln Arg Leu Phe Asn Val Ile Gly Thr Phe Ala Ala 180 18 5 190 aac agt gaa gtc gat ggc tat gaa atg ctg gtg aat att gag gat gcc 624 Asn Ser Glu Val Asp Gly Tyr Glu Met Leu Val Asn Ile Glu Asp Ala 195 200 205 tcg cgt ctg atg cgt tat ccg atcacc atc ggc tgg cgt ttg 672 Ser Arg Leu Met Arg Tyr Pro Ala Gly Asn Ile Thr Gly Trp Arg Leu 210 215 220 tgg ctg gat gag ccg ctg aaa gtc gac tca tta agt cag caa aaa ctg 720 Trp Leu Asp Glu Pro Leu Lys Val Ser Leu Ser Gln Gln Lys Leu 225 230 235 240 cct gaa ggc agc aaa tgg cag gac tgg cgt gat cgt aaa ggc gag ttg 768 Pro Glu Gly Ser Lys Trp Gln Asp Trp Arg Asp Arg Lys Gly Glu Leu 245 250 255 ttc cag gta cgc atg gaa aaa aat atg atg ggt tta ctg ctg agc 816 Phe Gln Ala Val Arg Met Glu Lys Asn Met Met Gly Leu Leu Leu Ser 260 265 270 ctg att gtc gcc gtt gcg gcg ttt aac att atg tc acct Leu Ile Val Ala Val Ala Ala Phe Asn Ile Ile Thr Ser Leu Gly Leu 275 280 285 atg gta atg gag aag cag ggc gaa gta gcg atc ctg caa acg caa ggc 912 Met Val Met Glu Lys Gln Gly Glu Val Ala Ile Leu Gln Gl n Gly 290 295 300 tta act ccg cga caa atc atg atg gtc ttt atg gtg caa ggg gcc agc 960 Leu Thr Pro Arg Gln Ile Met Met Val Phe Met Val Gln Gly Ala Ser 305 310 315 320 gcc ggg att atc ggt gcg atc ctc gga gcg gcg ctt ggc gcc ctg ctt 1008 Ala Gly Ile Ile Gly Ala Ile Leu Gly Ala Ala Leu Gly Ala Leu Leu 325 330 335 gcc agc cag tta aat aat ctg atg ccg ata atc ggc gtc Lec g Ast Gat Gn gct gc gat gln Asn Leu Met Pro Ile Ile Gly Val Leu Leu Asp 340 345 350 ggc gcg gcg ctg ccg gtg gct atc gaa cct tta cag gtc att gtt att 1104 Gly Ala Ala Leu Pro Val Ala Ile Glu Pro Leu Gln Val Ile Val Ile 355 360 gcg ctg gtg gcg atg gct atc gcg ctg ctg tct acg ctt tac cct tca 1152 Ala Leu Val Ala Met Ala Ile Ala Leu Leu Ser Thr Leu Tyr Pro Ser 370 375 380 tgg cgc gct gcc gcc act caa ccc gct ggt gct tta gaa taa 1200 Trp Arg Ala Ala Ala Thr Gln Pro Ala Glu Ala Leu Arg Tyr Glu 385 390 395 <210> 2 <211> 702 <212> DNA <213> Escherichia coli <220> <221> CDS <222> 1. .702 <400> 2 atg aat aag atc ctg ttg c aa tgc gac aac ctg tgc aaa cgc tat cag 48 Met Asn Lys Ile Leu Leu Gln Cys Asp Asn Leu Cys Lys Arg Tyr Gln 1 5 10 15 gaa ggc agt gtg caa acc gat gtg ctg cac aat gtc agt gtc ag gtc ag gtc ag gtc glut Ser Val Gln Thr Asp Val Leu His Asn Val Ser Phe Ser Val 20 25 30 ggc gaa ggt gaa atg atg gcg atc gtc ggt agc tct ggt tcc ggt aaa 144 Gly Glu Gly Glu Met Met Ala Ile Val Gly Ser Ser Gly Ser Gly Lys 35 40 45 agt acc ttg ctg cac ctg ctg ggc ggg ctg gat acg cca acc tcc ggc 192 Ser Thr Leu Leu His Leu Leu Gly Gly Leu Asp Thr Pro Thr Ser Gly 50 55 60 gat gtg att ttt aat ggt cag cca atg agc aaa ctg tcg tcg gca gcg 240 Asp Val Ile Phe Asn Gly Gln Phe Met Ser Lys Leu Ser Ser Ala Ala 65 70 75 80 aaa gct gaa ctg cgc aac cag aag ctg ggc ttt att tat cag ttt cac 288 Lys Ala Glu Leu Arg Asn Lys Leu Gly Phe Ile Tyr Gln Phe His 85 90 95 cac ctg ctg ccg gat ttt acc gcc ctg gaa aac gtg gct atg ccg ctg 336 His Leu Leu Pro Asp Phe Thr Ala Leu Glu Asn Val Ala Met Pro Leu 100 105 110 ctg att ggc aag aaa aag ccc gct g aa atc aac agc cgt gca ctt gag 384 Leu Ile Gly Lys Lys Lys Pro Ala Glu Ile Asn Ser Arg Ala Leu Glu 115 120 125 atg tta aaa gcg gtg ggg ctg gat cat cgt gcg aat cac cgc cca tct 432 Met Valu Lys A Gly Leu Asp His Arg Ala Asn His Arg Pro Ser 130 135 140 gaa ctt tct ggc ggc gaa cgc cag cgt gtg gcg att gcc cgt gcg ctg 480 Glu Leu Ser Gly Gly Glu Arg Gln Arg Val Ala Ile Ala Arg Ala Leu 145 150 160 gtg aat aac ccg cgc ctg gta ctg gcg gat gaa cct acc ggt aac ctc 528 Val Asn Asn Pro Arg Leu Val Leu Ala Asp Glu Pro Thr Gly Asn Leu 165 170 175 gat gcg cgt aac gcc gac agc atc ttc cag gaa ttg aat 576 Asp Ala Arg Asn Ala Asp Ser Ile Phe Gln Leu Leu Gly Glu Leu Asn 180 185 190 cgc ttg cag ggc acc gcc ttc ctg gtg gtt act cac gac ctg caa ctg 624 Arg Leu Gln Gly Thr Ala Phe Thr His Asp Leu Gln Leu 195 200 205 gcg aaa cgt atg agc cgc cag tta gaa atg cgt gat ggt cgt ctg acg 672 Ala Lys Arg Met Ser Arg Gln Leu Glu Met Arg Asp Gly Arg Leu Thr 210 215 220 gcg gaa ctg ag ctg ag a tg ggg gcg gag taa 702 Ala Glu Leu Ser Leu Met Gly Ala Glu 225 230 <210> 3 <211> 1245 <212> DNA <213> Escherichia coli <220> <221> CDS <222> 1..1245 <400 > 3 atg gcg atg cct tta tcg tta tta att ggc ctg cgt ttt agt cgc gga 48 Met Ala Met Pro Leu Ser Leu Leu Ile Gly Leu Arg Phe Ser Arg Gly 1 5 10 15 cgg cgg cgc ggc ggc atg gtg tcg ccg gtt att tct acc att 96 Arg Arg Arg Gly Gly Met Val Ser Leu Ile Ser Val Ile Ser Thr Ile 20 25 30 ggc att gcc ctc ggc gtg gcg gta ttg atc gtc ggc tta agc gcg atg 144 Gly Ile Ala Leu Gly Val Ala Val Leu Ile Val Gly Leu Ser Ala Met 35 40 45 aac ggc ttt gaa cgc gaa ctg aac aac cgt att ctg gcg gtg gtg ccg 192 Asn Gly Phe Glu Arg Glu Leu Asn Asn Arg Ile Leu Ala Val Val Phe 50 55 60 cat ggt atc gag gcg gtg gat cag ccg tgg act aac tgg cag gaa 240 His Gly Glu Ile Glu Ala Val Asp Gln Phe Trp Thr Asn Trp Gln Glu 65 70 75 80 gca ctg gat cac gtg caa aaa gtg ccg ggt att gcc gct gcc gc 288 Ala Leu Asp His Val Gln Lys Val Pro Gly Ile Ala Ala Ala Ala Pro 85 9 0 95 tat atc aat ttc acc ggg ctg gtg gaa agt ggc gcg aat ctt cgc gca 336 Tyr Ile Asn Phe Thr Gly Leu Val Glu Ser Gly Ala Asn Leu Arg Ala 100 105 110 atc cag gtg aag ggc gtt aac ccg caa cag cgt ctg agc gca 384 Ile Gln Val Lys Gly Val Asn Pro Gln Gln Glu Gln Arg Leu Ser Ala 115 120 125 tta ccc tcg ttt gtc cag ggc gat gcg tgg cgc aat ttt aaa gcg ggc 432 Leu Pro Ser Phe Val Gln Gly Asp Trp Arg Asn Phe Lys Ala Gly 130 135 140 gaa cag caa att atc atc ggc aaa ggc gtg gcg gat gcg ctg aaa gtg 480 Glu Gln Gln Iln Ile Ile Ile Gly Lys Gly Val Ala Asp Ala Leu Lys Val 145 150 gg 160 aag gat tgg gtg tcg att atg atc ccc aac tcg aat ccc gag 528 Lys Gln Gly Asp Trp Val Ser Ile Met Ile Pro Asn Ser Asn Pro Glu 165 170 175 cat aaa ctg atg cag cca aaa cgt gtg cgt ttg cac gtt gcc ggtt His Lys Leu Met Gln Pro Lys Arg Val Arg Leu His Val Ala Gly Ile 180 185 190 ttg cag ttg agt ggt caa ctc gat cac agt ttt gcc atg atc ccg ctg 624 Leu Gln Leu Ser Gly Gln Leu Asp His Ser Phe Ala Met Ile Pro Leu 195 200 205 gcg gat gcc caa caa tat ctt gat atg ggt tcc agc gtg tca ggt att 672 Ala Asp Ala Gln Gln Tyr Leu Asp Met Gly Ser Ser Val Ser Gly Ile 210 215 220 gcc ctt aaa atg acg gat gtt ttc aac gcc aat aag ctg gta cgc gat 720 Ala Leu Lys Met Thr Asp Val Phe Asn Ala Asn Lys Leu Val Arg Asp 225 230 235 240 gcg ggt gaa gtg acc aac agc tat gtt tat att aaa agc tgg att ggt 768 Ala Gly Glu Val Thrn Ser Tyr Val Tyr Ile Lys Ser Trp Ile Gly 245 250 255 act tac ggc tat atg tat cgc gat atc caa atg atc cgc gcc att atg 816 Thr Tyr Gly Tyr Met Tyr Arg Asp Ile Gln Met Ile Arg Ala Ile Met 260 265 270 tat ctg gcg atg gta ctg gtg att ggc gtg gcc tgt ttc aac atc gtc 964 Tyr Leu Ala Met Val Leu Val Ile Gly Val Ala Cys Phe Asn Ile Val 275 280 285 tcc acc tta gtg atg gcg gtg aaa gac gag atg gta 912 Ser Thr Leu Val Met Ala Val Lys Asp Lys Ser Gly Asp Ile Ala Val 290 295 300 tta aga acg ctg ggg gcg aaa gat ggt tta att cgc gcc atc ttt gtc 960 Leu Arg Thr Leu Gly Ala Lys Asp Gly Leu Ile Arg Ala Ile Phe Val 305 310 315 320 tgg tat gga ttg ctg gca ggg ctg ttc ggc agc ctg tgt ggt gtg att 1008 Trp Tyr Gly Leu Leu Ala Gly Leu Phe Gly Ser Leu Cys Gly Val Ile 325 330 335 atc ggc gta ctg caa ctt acc ccg att att gag tgg att 1056 Ile Gly Val Val Val Ser Leu Gln Leu Thr Pro Ile Ile Glu Trp Ile 340 345 350 gaa aag ttg atc ggt cat cag ttc ctc tcc agc gat atc tat ttt att 1104 Glu Lys Le Ile Gly His Gln Phe Leu Ser Ser Asp Ile Tyr Phe Ile 355 360 365 gat ttc ctg cca tcg gaa ttg cac tgg ctg gac gtc ttc tac gta ctg 1152 Asp Phe Leu Pro Ser Glu Leu His Trp Leu Asp Val Phe Tyr Leu 375 380 gtc aca gca ttg ttg ctg agt ctt ttg gca agt tgg tat ccg gcg cgg 1200 Val Thr Ala Leu Leu Leu Ser Leu Leu Ala Ser Trp Tyr Pro Ala Arg 385 390 395 400 cgc gcc agt aat att gac cgt gcg cg agc ggc cag taa 1245 Arg Ala Ser Asn Ile Asp Pro Ala Arg Val Leu Ser Gly Gln 405 410

[Brief description of the drawings]

FIG. 1 is a schematic diagram of electrophoresis of a gel filtration fraction of a cytoplasmic membrane extract. “+” Indicates a proteoliposome containing a fractionated cytoplasm-derived protein fraction, a proteoliposome not containing a cytoplasm-derived protein fraction, and “Pal” indicates electrophoresis. Outer membrane lipoprotein P after
The al band is schematically shown.

FIG. 2 is a diagram showing genes incorporated into recombinant vectors pJY310 and pKM001, which are amplified by a lipoprotein detachment device.

FIG. 3 is a graph showing changes in lipoprotein withdrawal activity with time. Open circles indicate cytoplasmic membrane extracts prepared from Escherichia coli K003 strain that retain pKM001, and black circles indicate p.
The cytoplasmic membrane extract prepared from Escherichia coli K003 strain holding JY310, and the squares represent E. coli K003 holding pUC19.
3 shows that each of the cytoplasmic membrane extracts prepared from the 003 strain was used.

FIG. 4. LolD in microorganisms belonging to Gram-negative bacteria
FIG. 4 is a diagram showing the homology of the amino acid sequences of the above. The underlined part is Lo
Regions that are very well conserved between ID homologous proteins are shown. Amino acid residues conserved in six or more types of microorganisms are shown in bold letters. The following abbreviations indicate homologous proteins from each microorganism. Eco, Esc
herichia coli; Sty, Salomo
nella typhi; Ype, Yersinia
pestis; Hin, Haemophilus
influenzae; Aac, Actinobac
illus actinomycetemcomita
ns; Vch, Vibrio cholerae;
Spu, Shewanella putrefacie
ns; Pae, Pseudomonas aerug
inosa; Bpe, Bordetella per
tussis; Ngo, Neisseria gon
ormee; Nme, Neisseriam
eningitidis; Pmu, Pasture
lla multitocida

FIG. 5: LolC in microorganisms belonging to Gram-negative bacteria
FIG. 4 is a diagram showing the homology of the amino acid sequences of the above. E. coli Lo
The transmembrane domain (TM) of 1C is shown in the figure. Amino acid residues conserved in more than half of the microorganisms are shown in bold. Abbreviations are the same as in FIG.

FIG. 6 shows LolE in microorganisms belonging to Gram-negative bacteria.
FIG. 4 is a diagram showing the homology of the amino acid sequences of the above. E. coli Lo
The transmembrane domain (TM) of IE is shown in the figure. Amino acid residues conserved in more than half of the microorganisms are shown in bold. Kpn is Klebsiella pneumo
3 shows a homologous protein of niae, and other abbreviations are the same as in FIG.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/21 C12P 21/02 C 5/10 21/08 // C12P 21/02 (C12P 21/08 21 / 08 C12R 1:91) (C12P 21/08 C12N 15/00 ZNAA C12R 1:91) 5/00 A

Claims (15)

[Claims] The present invention has the function of controlling lipoprotein localization in the cells of a microorganism belonging to Gram-negative bacteria, and comprises the amino acid sequence of SEQ ID NO: 1 or one or more amino acids in the sequence. A LolC protein or a homologous protein thereof obtained by substitution, insertion or deletion. 2. A DNA encoding the protein according to claim 1.
A. 3. A DN comprising the nucleotide sequence of SEQ ID NO: 1.
3. The DNA according to claim 2, which encodes A or a protein that hybridizes under stringent conditions and has the same function. 4. It has a function of controlling lipoprotein localization in cells of a microorganism belonging to Gram-negative bacteria, and has the amino acid sequence of SEQ ID NO: 2 or one or more amino acids in the sequence. A LolD protein or a homologous protein thereof obtained by substitution, insertion or deletion. 5. A DNA encoding the protein according to claim 4.
A. 6. A DN consisting of the nucleotide sequence of SEQ ID NO: 2
The DNA according to claim 5, which encodes A or a protein that hybridizes with the same under stringent conditions and has the same function. 7. It has a function of controlling lipoprotein localization in cells of a microorganism belonging to a gram-negative bacterium, and has the amino acid sequence of SEQ ID NO: 3 or one or more amino acids in the sequence. LolE protein or a homologous protein thereof obtained by substitution, insertion or deletion. 8. A DN encoding the protein according to claim 7.
A. 9. A DN comprising the nucleotide sequence of SEQ ID NO: 3.
The DNA according to claim 8, which encodes A or a protein which hybridizes with the same under stringent conditions and has the same function. 10. A recombinant vector containing the DNA according to claim 2, 3, 5, 6, 8 or 9. [11] A transformant comprising the recombinant vector according to [10]. 12. A monoclonal antibody having reactivity with the protein of claim 1, 4 or 7. 13. A monoclonal antibody reactive with a protein having the amino acid sequence of SEQ ID NO: 1, 2, or 3. 14. The gram-negative bacterium is Escherichia coli.
8. The protein according to 4 or 7. 15. The gram-negative bacterium is Escherichia coli.
DNA according to 3, 5, 6, 8 or 9.