JPH10243784A - Identification of gene coding type ii membrane protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable the identification of the gene coding type II membrane protein that is useful in development of medicines by expressing a fused gene between a specific DNA fragment and a gene coding a reporter protein in mammalian cells. SOLUTION: A DNA fragment to be identified (for example, HP10085 originating from the cDNA library of human lymphoma cell strain U937 resembling human initial activation antigen CD69) and the gene that codes reporter protein (for example, urokinase type plasminogen activator) are fused and the fused gene is recombined with pSSD1 vector and the recombinant vector is used to transform Escherichia coli JM109, from which plasmid DNA is extracted and transfected into mammalian cells (COS-7 cells). The judgment of whether or not the reporter protein is expressed on the membrane surface of the transfected culture cells is used as an indicator to enable the subject identification (by detecting the activity of the corresponding enzyme).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for identifying a gene encoding a type II membrane protein. By this method, a gene encoding a type II membrane protein useful as a medicine can be easily identified.

[0002]

2. Description of the Related Art Type II membrane proteins are used as signal receptors, ion channels, transporters and the like.
It plays an important role in transporting substances and transmitting information through cell membranes. In many cases, these membrane proteins are specifically expressed in a specific cell or cancer cell. If an antibody against the protein is prepared, it can be used as a carrier for various diagnoses and drug delivery. Cells into which these membrane protein genes have been introduced to express the membrane protein can be applied to detection of the corresponding ligand.

At present, many new cDNAs have been cloned with the progress of the genome project. These cDNAs
CDNA encoding type II membrane protein from clones
If a clone can be selected, a commercially available type II membrane protein can be efficiently obtained.

A characteristic of the type II membrane protein is that a highly hydrophobic region existing at the N-terminus penetrates the membrane, and the N-terminus is located on the cytoplasm side and the C-terminus is located on the extracellular side. Therefore, if there is a hydrophobic region at the N-terminus of the protein encoded by the cDNA and this region remains in the membrane, this cDNA will
It can be said that it encodes a type I membrane protein. When the N-terminal hydrophobic region is a secretory signal sequence, it is cleaved on the membrane surface by a signal peptidase, and the C-terminal side is released outside the cell without remaining on the membrane. Several methods for identifying such secretion signals are already known [Tashiro et al., Science 261: 600-603 (1993);
okoyama-Kobayashi et al., Gene 163: 193-196 (199
4)], A method for identifying a transmembrane domain of a type II membrane protein is not yet known.

[0005]

SUMMARY OF THE INVENTION The object of the present invention is to provide
An object of the present invention is to provide a method for identifying a gene encoding a type membrane protein.

[0006]

Means for Solving the Problems As a result of intensive studies, the present inventors have encoded a transmembrane domain of a type II membrane protein.
When the DNA fragment is fused with a reporter protein gene and then introduced into animal cells, the reporter protein is found to remain on the membrane surface, and by using this, the gene encoding the transmembrane domain of the type II membrane protein is used. The present invention was completed by constructing a method for identifying That is, the present invention provides that a fusion gene of a DNA fragment encoding a protein to be identified and a gene encoding a reporter protein is introduced into an animal cell to express the fusion protein, and the reporter protein is expressed on the membrane surface of the cell. Is the index,
A method for identifying a gene encoding a type II membrane protein is provided.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION In the method of the present invention, if a transmembrane domain is added to the N-terminus, the reporter protein is transported to the cell surface, and the transported reporter protein remaining on the cell surface can be easily detected. Things are desirable. Examples thereof include plasminogen activators, such as urokinase-type plasminogen activator, tissue plasminogen activator, and enzymes such as blood coagulation factors. When the reporter protein is originally a secretory protein, a protein from which signal sequence peptides derived from these proteins have been removed is used.

The present invention comprises the following three steps. Step 1 A fusion gene of a DNA fragment to be identified and a gene encoding a reporter protein is recombined into an expression vector for animal cells. Step 2 The obtained vector is introduced into animal cells using a transfection method. Step 3 After culturing the cells into which the vector has been introduced, the presence or absence of a reporter protein in the culture supernatant and on the cell membrane surface is examined.

Each step will be described in detail below.

Step 1 The following two methods are available for recombination of a fusion gene of an arbitrary DNA fragment to be identified with a gene encoding a reporter protein into an expression vector for animal cells.

<Method 1> Using a vector in which a promoter for animal cells, a cloning site (restriction enzyme cleavage site), and a gene encoding a reporter protein are connected in series, a cDNA fragment is inserted into the cloning site.

<Method 2> A vector containing a full-length cDNA downstream of an animal cell promoter is cleaved at a restriction enzyme site present in the cDNA, and a gene encoding a reporter protein is inserted into this site.

When Method 1 is used, pSSD1 [Yokoyama-Kobayashi et al., Gene 16
3: 193-196 (1994)]. The DNA fragments to be screened are genomic, cDNA, synthetic D
NA is acceptable. In the case of genomic or cDNA, a cut fragment with an appropriate restriction enzyme may be used, or a DNA fragment prepared by polymerase chain reaction (PCR) using appropriate primers may be used. If an open reading frame starting from the start codon is present in the DNA fragment and the frame of the gene encoding the reporter protein matches, a vector capable of expressing these fusion proteins is completed (FIG. 1).

When Method 2 is used, a cDNA expression library cloned into a vector containing an animal cell promoter can be used as a source of cDNA.
For example, pKA1 (described in JP-A-4-117292), pcDV [Okay
ama and Berg, Mol. Cell. Biol. 3: 280-289 (1983)], pC
A cDNA library prepared using DM8 [Seed, Nature 329: 840-842 (1987)] as a vector can be used.
When using a cDNA library prepared with pKA1, after cutting with one or more restriction enzyme sites not present in the vector, blunt-ending, then cutting with Not I, and inserting a DNA fragment encoding a reporter protein, good.

Step 2 In step 1, a mixture of vectors into which various DNA fragments have been inserted is obtained. After transforming Escherichia coli with this, a single colony was formed, and a plasmid and a single-stranded DN prepared from each were prepared.
Transfection of animal cells is performed using A or single-stranded phage.

To introduce an expression vector into animal cells,
The calcium phosphate method, the DEAE-dextran method, the liposome method, the electroporation method, and the like can be used (for example, Experimental Medicine Supplement “Genetic Engineering Handbook”,
Yodosha, 1991). Alternatively, the gene can be more easily introduced into animal cells by preparing a single-stranded phage and then transfecting the same [Yokoyama-Kobayashi and Kato, Biochem. Bioph.
ys. Res. Commun. 192: 935-939 (1993)].

Any animal cell can be used as long as the origin of replication and the promoter incorporated in the vector work. When using the replication origin or promoter of SV40 as in the examples, C expressing the T antigen
OS cells are suitable.

Step 3 After culturing animal cells into which the expression vector has been introduced under appropriate conditions, a culture supernatant is prepared. Whether or not the reporter protein remains on the membrane surface is examined using the reporter protein detection method used. In the case of enzymes, the corresponding enzyme activity detection method is used.

When urokinase is used as a reporter protein, it can be detected by a method using an artificial substrate peptide or a fibrin plate method. In the examples, a method for measuring the activity of urokinase present on the cell surface by directly contacting an agar sheet containing fibrin on animal cells has been exemplified.

[0020]

EXAMPLES Next, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples. D
The basic operations and enzymatic reactions involved in the recombination of NA
Literature ("Molecular Cloning. A
Laboratory Manual ", Cold S
spring Harbor Laboratory, 1
989). Restriction enzymes and various modifying enzymes used were those manufactured by Takara Shuzo Co., Ltd. unless otherwise specified. The buffer composition and reaction conditions for each enzyme reaction were in accordance with the attached instructions.

Cloning of cDNA encoding type II membrane protein Human lymphoma cell line U937 cDNA library [Kato et a
l., Gene 150: 243-250 (1994)].
When the entire base sequence of the cDNA insert of 0085 was determined,
150 bp 5 'untranslated region, 450 bp ORF, 97
bp 3 ′ untranslated region (Sequence Table 1). The ORF encodes a protein consisting of 149 amino acid residues, and has a single hydrophobic region at the N-terminus which is considered to be a transmembrane domain. FIG. 2 shows the hydrophobicity / hydrophilicity profile of the present protein determined by the Kyte-Doolittle method. When a protein database was searched using the amino acid sequence of this protein, it was similar to human early activation antigen CD69 (SWISS-PROT accession number Q07108), which is one of the type II membrane proteins. Table 1 shows a comparison of the amino acid sequences of the human protein (HP) of the present invention and the human early activation antigen CD69 (CD). -Indicates a gap, * indicates the same amino acid residue as the protein of the present invention,. Represents an amino acid residue similar to that of the protein of the present invention. Both are C-terminal 1
It had 36.6% homology in the region of 12 amino acid residues.

Table 1 ──────────────────────────────── HP MMTKHKKCFI CD MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIA HP IVGVLITTNIITLIVKLTRDSQSLCPYDWIGFQNKCYYFSKEEGDWNSSKYNCSTQHADL * *. **. * . *. *** .. *. .. *. * ... ** .. *. * CD LSVGQYNCPGQYTFSMPSDSHVSSCSEDWVGYQRKCYFISTVKRSWTSAQNACSEHGATL HP TIIDNIEEMNFLRRYKCSSDHWIGLKMAKNRTGQWVDGATFTKSFGMRGSEGCA ** SD. ** .. .... *. *. * .. * .. **. * .. * .... CD AVIDSEKDMNFLKRYAGREEHWVGLKKEPGHPWKWSNGKEFNNWFNVTGSDKCVFLKNTE HP AATARCYTERKWICRKRIH ... * .. ***. * CD VSSMECEKNLYWICNKPYK ──── ─────────────────────────────

Construction of Expression Vector for Urokinase Fusion Protein (1) Preparation of cDNA Fragment Derived from HP10085 2 μg of HP10085 plasmid was digested with 15 units of EcoRI restriction enzyme.
After digestion, the ends were blunt-ended with a DNA branding kit manufactured by Takara Shuzo. This was subjected to 1.5% agarose gel electrophoresis, and a 320 bp fragment was isolated.

(2) Preparation of pSSD1 Vector Fragment 1 μg of pSSD1 plasmid was digested with 10 units of restriction enzyme EcoRV, and the terminal phosphate group was removed with 0.6 units of alkaline phosphatase derived from Escherichia coli. This was subjected to 0.7% agarose gel electrophoresis, and a fragment of about 4500 base pairs was isolated.

(3) Construction of pSSD1-HP10085 About 20 ng of the cDNA fragment obtained in the above (1) and (2)
About 10 ng of the vector fragment obtained in the above was mixed and ligated using a DNA ligation kit manufactured by Takara Shuzo. Using this reaction product, E. coli JM109 was transformed, 6 strains were selected from the obtained ampicillin-resistant colonies, and 2 ml of
The cells were cultured overnight in an LB medium at 37 ° C. About 0.5 μg of the plasmid extracted from these cells was digested with 2 units of restriction enzyme XhoI, and then subjected to 1.5% agarose gel electrophoresis. From this electrophoresis pattern, a clone in which the cDNA fragment derived from HP10085 was inserted in the forward direction was selected, and pSSD1-HP10
085 (FIG. 3).

Transfection of COS-7 cells A sample for transformation was prepared as follows. pSSD1-HP
A small amount was collected with a sterile toothpick from the glycerol stock of the E. coli JM109 transformant having the 10085 plasmid,
The cells were inoculated into 2 ml of 2 × YT containing 100 μg / ml ampicillin. 37 ℃
After incubation for 1 hour and 30 minutes, helper phage M13K07 suspension 50
μl was added, and the cells were further cultured for 16 hours. The culture was centrifuged at 15,000 rpm for 5 minutes twice to obtain a supernatant from which Escherichia coli was completely removed. For 1.2 ml of this supernatant
0.3 ml of a 20% polyethylene glycol and 2.5 M NaCl solution was added, mixed well, allowed to stand at room temperature for 10 minutes, and then centrifuged at 15,000 rpm for 10 minutes to collect a precipitate. The obtained precipitate was suspended in 120 μl of Tris-EDTA (pH 8.0), and this suspension was used for transformation. Alternatively, plasmid DNA was extracted from the Escherichia coli body after the culture by an alkaline lysis method, and this was used as a sample.

Monkey kidney-derived cultured cells COS7 (available from ATCC) were cultured at 37 ° C. in Dulbecco's modified Eagle (DMEM) medium containing 10% fetal bovine serum in the presence of 5% CO ₂ . 0.7
0.80.8 × 10 ⁵ COS7 cells were seeded on a 6-well plate (Nunc, 3 cm in diameter) and cultured at 37 ° C. for 20 to 22 hours in the presence of 5% CO ₂ . After removing the medium, the cell surface is washed with a phosphate buffer, and DMEM containing 50 mM Tris-HCl (pH 7.5) (TDME
Washed again with M). Each cell (1-2 μl of phage suspension or 1-2 μg of double-stranded DNA) containing 0.4 μm
Add 0.8 ml of TDMEM containing g / ml DEAE dextran and add 5% CO
_The cells were cultured in the presence of ₂ at 37 ° C. for 4 hours. After removing the sample solution, the cell surface was washed with TDMEM, and T
1 ml of DMEM was added, and the cells were cultured at 37 ° C. for 3 hours in the presence of 5% CO ₂ . Remove this solution and wash the cell surface with TDMEM.
2 ml of DMEM containing% fetal bovine serum was added per well, and the cells were cultured at 37 ° C. for 5 days to 1 week in the presence of 5% CO ₂ .

Assay of culture supernatant A 50 mM phosphate buffer (pH 7.0) containing 2% bovine fibrinogen (Miles), 0.5% agarose and 1 mM calcium chloride.
4) 10 units of human thrombin (Mochida Pharmaceutical) was added to 10 ml and solidified in a plate having a diameter of 9 cm to prepare a fibrin plate. 10 μl of the culture supernatant of the transfected COS cells was placed on a fibrin plate and incubated at 37 ° C for 15 minutes.
Incubated for hours. The diameter of the obtained dissolved circle was used as an index of urokinase activity. As a result, the expression vector pKA1-UPA containing the entire urokinase translation region [Yokoyama-Koba
yashi and Kato, Biochem. Biophys. Res. Commun. 19
2: 935-939 (1993)], a lysis circle was observed in the medium of cells expressing urokinase, but urokinase activity was not detected in the medium of cells expressing pSSD1 and pSSD1-HP10085 (FIG. 4A). ).

Detection of urokinase activity on cell surface Using 50 mM phosphate buffer (pH 7.4), a 2% solution of bovine fibrinogen and a 0.5% agarose solution are prepared, and 1 ml of each solution is mixed with 2 μl of 1M calcium chloride and thrombin. Two units were added, poured into a plate having a diameter of 3.5 cm, and polymerized at 37 ° C. for 1 hour. The polymerized fibrin was peeled off from this plate to obtain a thin fibrin (fibrin sheet).

The medium was removed from the cultured cells into which the pSSD1-HP10085 plasmid had been introduced, and the cell surface was washed with a physiological saline solution containing a phosphate buffer. A fibrin sheet was placed so as to be in close contact with the surface of the cells, and the cells were incubated at 37 ° C overnight. As a result, lysis plaques were observed on the cell surface expressing pSSD1-HP10085 and pKA1-UPA (FIG. 4B).

From the above results, it can be seen that the N-terminal hydrophobic region of HP10085 functions not as a secretory signal sequence but as a transmembrane domain. In addition, the N-terminal side is in the cytoplasm, and the C-terminal side is extracellular. It has been experimentally proved to be a transmembrane domain of a type II membrane protein.

[0032]

According to the present invention, a gene encoding a type II membrane protein can be efficiently identified. The identified gene encoding a type II membrane protein can be used for mass production of a type II membrane protein useful for the development of a medicine.

[0033]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 697 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin: Organism name: Homo sapiens Cell type: Lymphoma Cell line: U937 clone name: HP10085 Sequence features: Symbol indicating feature: CDS Location: 151. . 600 Method of determining the characteristics: E sequence TATACCTCTA GTTTGGAGCT GTGCTGTAAA AACAAGAGTA ACATTTTTAT ATTAAAGTTA 60 AATAAAGTTA CAACTTTGAA GAGAGTTTCT GCAAGACATG ACACAAAGCT GCTAGCAGAA 120 AATCAAAACG CTGATTAAAA GAAGCAGT TTG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG G GTT TTA ATA ACA ACT AAT ATT ATT ACT CTG ATA 222 Phe Ile Ile Val Gly Val Leu Ile Thr Thr Asn Ile Ile Thr Leu Ile 10 15 20 GTT AAA CTA ACT CGA GAT TCT CAG AGT TTA TGC CCC TAT GAT TGG ATT 270 Val Lys Leu Thr Arg Asp Ser Gln Ser Leu Cys Pro Tyr Asp Trp Ile 25 30 35 40 GGT TTC CAA AAC AAA TGC TAT TAT TTC TCT AAA GAA GAA GGA GAT TGG 318 Gly Phe Gln Asn Lys Cys Tyr Tyr Phe Ser Lys Glu Glu Gly Asp Trp 45 50 55 AAT TCA AGT AAA TAC AAC TGT TCC ACT CAA CAT GCC GAC CTA ACT ATA 366 Asn Ser Ser Lys Tyr Asn Cys Ser Thr Gln His Ala Asp Leu Thr Ile 60 65 70 ATT GAC AAC ATA GAA GAA ATG AAT TTT CTT AGG CGG TAT AAA TGC AGT 414 Ile Asp Asn Ile Glu Glu Met Asn Phe Leu Arg Arg Tyr Lys Cys Ser 75 80 85 TCT GAT CAC TGG ATT GGA CTG AAG ATG GCA AAA AAT CGA ACA GGA CAA 462 Ser Asp His Trp Ile Gly Leu Lys Met Ala Lys Asn Arg Thr Gly Gln 90 95 100 TGG GTA GAT GGA GCT ACA TTT ACC AAA TCG TTT GGC ATG AGA GGG AGT 510 Trp Val Asp Gly Ala Thr Phe Thr Lys Ser Phe Gly Met Arg Gly Ser 105 110 115 120 GAA GGA TGT GCC TAC CTC AGC GAT GAT GGT GCA GCA ACA GCT AGA TGT 558 Glu Gly Cys Ala Tyr Leu Ser Asp Asp Gly Ala Ala Thr Ala Arg Cys 125 130 135 TAC ACC GAA AGA AAA TGG ATT TGC AGG AAA AGA ATA CAC TAA 600 Tyr Thr Glu Arg Lys Trp Ile Cys Arg Lys Arg Ile His 140 145 GTTAATGTCT AAGATAATGG GGAAAATAGA AAATAACATTATCAACTCA TTTGTAC 697

[Brief description of the drawings]

FIG. 1 is a diagram showing the structure of a fusion gene.

Figure 2: Kyt for protein encoded by HP10085
The figure which shows the hydrophobicity / hydrophilicity profile calculated | required by the method of e-Doolittle.

FIG. 3 is a view showing the structure of a plasmid vector pSSD1-HP10085.

FIG. 4 is a photograph showing the results of a fibrin plate assay.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12Q 1/68 C12Q 1/68 Z // (C12N 15/09 ZNA C12R 1:91) (C12P 21/00 C12R 1:91)

Claims (2)

[Claims] 1. A method comprising introducing a fusion gene of a DNA fragment encoding a protein to be identified and a gene encoding a reporter protein into an animal cell to express the fusion protein, and expressing the reporter protein on the membrane surface of the cell. As an indicator,
A method for identifying a gene encoding a type II membrane protein. 2. The method according to claim 1, wherein the reporter protein is a plasminogen activator.