JPH1146765A - Method for determining topology of membrane protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a method for determining topology of membrane protein simply and surely. SOLUTION: The method, which allows determination of topology of membrane protein having (n) membrane-penetrating domains [(n) is an integer >=2], is obtained by preparing (n) kinds of DNA fragments containing initiation codon and first to (n)th membrane-penetrating domains serially from a gene coding membrane protein, fusing a gene coding a reporter protein to each 3'-end of these DNA fragments, introducing each of these fused genes into a separate animal cell to express fused protein, followed by the indexing of the location of the reporter protein in each cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for determining the topology of a membrane protein. For more information,
The present invention relates to a method for easily and reliably determining a topology as structural information of a membrane protein important for developing a drug or the like.

[0002]

2. Description of the Related Art Membrane proteins play important functions as receptors, ion channels, transporters, etc., in information transmission and substance transport through cell membranes. Certain membrane proteins are specifically expressed in cancer cells, and these are useful in developing various diagnostic agents and anticancer agents as tumor antigens. In addition, cells in which a specific membrane protein has been expressed in large amounts by gene transfer can be used for searching for ligands and analogs corresponding to the membrane protein and for measuring their physiological activities.

[0003] Membrane proteins are roughly divided into two types depending on their structure. One is a superficial membrane protein bound to the cell membrane surface and the other is an integral membrane protein embedded in the lipid bilayer of the cell membrane. The structure of an integral membrane protein is composed of an extracellular region, an intracellular region and a transmembrane domain. The transmembrane domain is rich in hydrophobic amino acids and is thought to form an alpha helix within the membrane. In the case of a protein having a plurality of transmembrane domains,
When the membrane protein is biosynthesized, it functions as a signal sequence that starts the permeation of the nascent peptide chain following the C-terminal side of the membrane protein or a stop transfer sequence that stops the permeation. In many cases, domains having respective functions are alternately arranged, and extracellular regions and intracellular regions are alternately arranged via the domains. The topology of such a membrane protein, that is, which part of the membrane protein is located extracellularly or intracellularly, is determined by whether each transmembrane domain functions as a signal sequence or a stop transfer sequence.

[0004] Information on the topology of membrane proteins is important in drug development. For example, when an analog of a receptor-specific ligand or an antibody against a tumor antigen is used as a medicament, it is essential to specify an extracellular region serving as a binding site between them. When developing a therapeutic drug for a disease caused by a mutant membrane protein, the location of the mutation site in a cell is the most basic information.

[0005] The presence of the transmembrane domain can be predicted to some extent from the deduced amino acid sequence of the membrane protein, and from such sequence information, the topology of the membrane protein can be inferred. Although a method of experimentally verifying this has been considered, each of the conventional topology analysis methods has a problem. For example, in the case of a method for chemically modifying a protein or a method for examining resistance to a protease, depending on the structure of the protein, it may be difficult to undergo chemical modification or protease degradation, or an antibody may be required for protein detection. There is a problem.

[0006] A method of expressing a fusion gene of a membrane protein gene and a reporter gene and analyzing the topology from the localization of the reporter gene product is also widely used. In these methods, the fusion gene is expressed in E. coli, in vitro translation systems, Xenopus eggs, animal-derived cultured cells, and the like. Among these, alkaline phosphatase is frequently used as a reporter in E. coli expression systems [Manoil, C. and Beckwith, J., Science, 23
3, 1403-1408, (1986), etc.]. The activity of this enzyme is high when present in the periplasm and low in the cytoplasm. The topology is determined using the difference in enzyme activity due to this localization. However, such a method of determining the topology by directly detecting the activity of the reporter gene product has not yet been reported in an in vitro translation system, an expression system of Xenopus eggs or cultured cells. In these expression systems, the reporter is often detected indirectly by using an antibody against the reporter. Recently, a fusion protein in which an epitope tag was inserted between two transmembrane domains was expressed in Xenopus eggs, and immunostaining was performed on the inside and outside of cells using a fluorescently labeled antibody against this tag. Have been reported to determine which side of the cell is located [Shih, TM, and Goldin, AL, J. Cell.
Biol. 136, 1037-1045, (1997)]. This method is excellent in that it does not require a radioisotope and does not affect the interaction between transmembrane domains, but has problems such as complicated construction of a fusion gene and operation of immunostaining.

[0007] A method for detecting a secretion signal using urokinase as a reporter has been reported [Yokoyama-Kobayashi, M. et al., Gene 163: 193-196].
(1995)], there is no report that this method was used to determine the topology of membrane proteins.

[0008]

Information on the topology of membrane proteins is extremely important in drug development and the like, but it has been difficult to determine the topology of membrane proteins simply and reliably by conventional methods. The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a new method that can easily and reliably determine the topology of a membrane protein.

[0009]

Means for Solving the Invention The inventors of the present invention
DN encoding two or more transmembrane domains of a membrane protein
When a fusion gene of fragment A and a reporter protein gene is expressed in animal cells, it is found that the reporter protein is exposed on the membrane surface or remains in the cell depending on the topology of the membrane protein, and this phenomenon is utilized. Thus, a method for determining the topology of membrane proteins was completed.

That is, the present invention is a method for determining the topology of a membrane protein having n (n is an integer of 2 or more) transmembrane domains, comprising the steps of: N types of DNA fragments containing the transmembrane domains from the nth to the nth sequence in sequence are prepared, and a gene encoding a reporter protein is fused to each C-terminus of these DNA fragments. Are introduced into animal cells to express the fusion protein, and the location of the reporter protein in each cell is used as an index to determine the topology of the membrane protein.

[0011] In a preferred embodiment of the method of the present invention, the reporter protein is a plasminogen activator.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The method of the present invention basically includes the following three steps. 1: A DNA fragment prepared from a gene encoding a membrane protein whose topology is to be determined is fused with a gene encoding a reporter protein to prepare a fusion gene.

2: The prepared fusion gene is introduced into animal cells using a transfection method. 3: The cells into which the fusion gene has been introduced are cultured, the fusion gene is expressed, and the presence or absence of a reporter protein on the cell membrane surface is examined. Each step is described in detail below. Step 1 Kyte & Doolittel method [Kyte, J. & Doolittle, RF, J.
M. Biol. 157: 105-132 (1982)] to estimate the location of the transmembrane domain in the membrane protein whose topology is to be determined (hereinafter, sometimes referred to as “target protein”). I do. Next, from the target protein gene, n types of DNA fragments containing an initiation codon and downstream of the n transmembrane domains one by one are prepared. The preparation of such a DNA fragment is performed, for example, by
It can be performed by digestion with an appropriate restriction enzyme or the like. If there is no appropriate restriction enzyme site between the transmembrane domains, an oligonucleotide complementary to the sequence before and after the target region is synthesized, and this is used as a primer for polymerase chain reaction (PCR). DN
A fragment can also be prepared. The DNA fragment is
It can be prepared from any form of DNA such as cDNA, genomic DNA, and synthetic DNA.

Next, the obtained DNA fragment and the reporter protein gene are ligated to prepare a fusion gene encoding a fusion protein between the N-terminal region of the target protein and the reporter protein. Such a fusion gene can be prepared, for example, by inserting a DNA fragment into a reporter protein expression vector to prepare a fusion protein expression vector.

As a reporter protein, if a transmembrane domain is added to its N-terminus, it is transported to the cell surface,
In addition, it is desirable that the difference between those transported and expressed on the cell surface and those remaining inside the cell can be easily detected.
For example, enzymes such as urokinase-type plasminogen activator, tissue plasminogen activator, and blood coagulation factor can be exemplified. When the reporter protein is originally a secretory protein, it is preferable to use a protein from which a secretory signal sequence peptide derived from these proteins has been removed. Step 2 In order to introduce the fusion gene prepared in Step 1 into animal cells, for example, after transforming Escherichia coli with the fusion protein expression vector, the colony is transformed into a single colony, and the plasmid, single-stranded DNA, or single-stranded DNA prepared from each is transformed. Transfection of animal cells is carried out using the main chain phage or the like.

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

As the animal cell, any cell may be used as long as it does not express an endogenous reporter protein and the replication origin or promoter incorporated in the vector works. Note that SV as shown in the embodiment
When using 40 replication origins and promoters, COS cells expressing T antigen are suitable. Step 3 After culturing animal cells into which the fusion gene has been introduced as an expression vector or the like under appropriate conditions, removing the culture solution, washing the cells sufficiently, and detecting whether the reporter protein remains on the membrane surface. For this detection, a detection method according to the reporter protein used is used. In the case of enzymes,
Use the corresponding enzyme activity detection method. When urokinase is used as the reporter protein, a method using an artificial substrate peptide, a fibrin plate method, or the like can be used. For example, the activity of urokinase present on the cell surface can be measured by bringing an agar sheet containing fibrin into direct contact with animal cells. Also,
After crushing the cells, the activity of the lysate may be measured. When urokinase remains in the cell, no urokinase activity is detected in these assays.

According to the above method, when a fusion gene of each DNA fragment having a start codon and a transmembrane domain sequentially from the N-terminus and a reporter gene is expressed in a cell, urokinase present at the C-terminus of the fusion protein is expressed as It can be determined whether or not it is on the film surface. Therefore, by comprehensively judging these results,
One can determine how the transmembrane domain of the target protein is oriented in the cell membrane and ultimately determine the topology of the membrane protein. .

[0019]

EXAMPLES Next, the present invention will be described in more detail and specifically with reference to examples, but the present invention is not limited to these examples. The basic operation and enzyme reaction for DNA recombination are described in the literature ("Molecular Clonin
g. A Laboratory Manual ", Cold Spring Harbor Laborat
ory, 1989). 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. Reference Example 1: Cloning of Membrane Protein Gene HP00966 Having Four Transmembrane Domains As a result of large-scale nucleotide sequencing of a cDNA clone selected from a human osteosarcoma cell line Saos-2 cell cDNA library, a clone HP00966 of a membrane protein gene was obtained. I got This clone consists of the nucleotide sequence shown in SEQ ID NO: 1,
It had a 5 bp 5 ′ untranslated region, a 762 bp open reading frame, and an 805 bp 3 ′ untranslated region. The open reading frame encodes a protein consisting of 253 amino acid residues. When this sequence was used to search a protein database, a human CD63 antigen [Metzelaar, MJ et al.,
J. Bio. Chem. 266: 3239-3245 (1991)]. Figure 1 shows Kyte & D
oolittle method [Kyte, J. & Doolittle, RF, J. Mol. Bi
157: 105-132 (1982)], showing the hydrophobicity profile of this protein. Highly hydrophobic regions, which are likely to be transmembrane domains, are found at four locations. This pattern is a feature common to other TM4 superfamilies. Example 1 Construction of Expression Vector for Urokinase Fusion Protein Recombinant Vector Using Four Kinds of Fusion Genes Each Structure is schematically shown in FIG. (1) Preparation of pSSD1 vector fragment 1 μg of pSSD1 plasmid was digested with 10 units of restriction enzyme SnaBI and 10 units of EcoRV15 restriction enzyme, and the terminal phosphate group was removed with 0.6 unit of alkaline phosphatase derived from Escherichia coli. This was subjected to 0.7% agarose gel electrophoresis, and a fragment of about 4,500 base pairs was isolated. (2) Construction of pSSD1-966.1 Containing the Translation Region of the First Transmembrane Domain of HP00966 After digestion of 2 μg of the HP00966 clone with 10 units of the restriction enzyme SnaBI and 10 units of the restriction enzyme AfaI, 1.5%
A 0.3 kb fragment was isolated by agarose gel electrophoresis. About 20 ng of this fragment and about 10 ng of the vector fragment obtained in the above (1) were mixed and ligated using a DNA ligation kit manufactured by Takara Shuzo. Using this reaction product, Escherichia coli JM109 was transformed. Six strains were selected from the obtained ampicillin-resistant colonies and cultured overnight at 37 ° C. in 2 ml of LB medium. Approximately 0.5 μg of plasmid extracted from these cells was used for 2 units of restriction enzyme Nc
After digestion with oI, it was subjected to 1.0% agarose gel electrophoresis. From this electrophoresis pattern, a clone in which the HP00966-derived cDNA fragment was inserted in the forward direction was selected and designated as pSSD1-966.1. (3) Construction of pSSD1-966.2 containing the translation regions of the first and second transmembrane domains of HP00966 Using the HP00966 clone as a template, a synthetic DNA primer
5'-GCGAGATCTGTATTTAATACGACTCACTATAGGG-3 'and 5'-C
PCR was performed using GGATATCGACTTTCCCGGATTGTGGC-3 'and 5 units of Amplitaq manufactured by PerkinElmer, Inc., and the reaction product was purified by ethanol precipitation. After digesting this product with the restriction enzyme SnaBI 10 units and the restriction enzyme EcoRV15 unit, the product is subjected to 1.5% agarose gel electrophoresis, about 0.4 k
The fragment of b was isolated. About 20ng of this fragment
And about 10 ng of the vector fragment obtained in (1) above, and ligated using a DNA ligation kit manufactured by Takara Shuzo. Using this reaction product, E. coli JM109 was transformed, and 6 ampicillin-resistant colonies were obtained.
A strain was selected and cultured overnight at 37 ° C. in 2 ml of LB medium. About 0.5 μg of the plasmid extracted from these cells was digested with 2 units of restriction enzyme NcoI and subjected to 1.0% agarose gel electrophoresis. From this electrophoresis pattern, HP00966
A clone in which the derived cDNA fragment was inserted in the forward direction was selected and designated as pSSD1-966.2. (4) Construction of pSSD1-966.3 containing the coding regions of the first to third transmembrane domains of HP00966 After digesting 2 μg of the HP00966 clone with 10 units of the restriction enzyme SnaBI and 10 units of the restriction enzyme EcoT22, DNA blunting kit manufactured by Takara Shuzo Was used to blunt the ends. This is subjected to 1.5% agarose gel electrophoresis,
An approximately 0.8 kb fragment was isolated. About 20 ng of this fragment and about 20 ng of the vector fragment obtained in the above (1)
10 ng were mixed and ligated using Takara Shuzo DNA Ligation Kit. Using this reaction product, Escherichia coli JM109 was transformed. Six strains were selected from the obtained ampicillin-resistant colonies and cultured overnight at 37 ° C. in 2 ml of LB medium. About 0.5 μg of the plasmid extracted from these cells was digested with 2 units of restriction enzyme NcoI and subjected to 1.0% agarose gel electrophoresis. From this migration pattern, HP
A clone in which the cDNA fragment derived from the 96966 was inserted in the forward direction was selected and designated as pSSD1-966.3. (5) Construction of pSSD1-966.4 Containing Coding Regions of HP00966 First to Fourth Transmembrane Domains After digesting 2 μg of the HP00966 clone with 10 units of the restriction enzyme SnaBI and 15 units of the restriction enzyme SacI, a DNA branding kit manufactured by Takara Shuzo Was used to blunt the ends. This was subjected to 1.5% agarose gel electrophoresis,
A 0.9 kb fragment was isolated. About this fragment
20 ng and about 10 vector fragments obtained in (1) above
ng were mixed and ligated using a DNA ligation kit manufactured by Takara Shuzo. Using this reaction product, Escherichia coli JM109 was transformed. Six strains were selected from the obtained ampicillin-resistant colonies and cultured overnight at 37 ° C. in 2 ml of LB medium. About 0.5 μg of the plasmid extracted from these cells was digested with 2 units of restriction enzyme NcoI and subjected to 1.0% agarose gel electrophoresis. From this migration pattern, HP00
A clone in which the 966-derived cDNA fragment was inserted in the forward direction was selected and designated as pSSD1-966.4. Example 2: Expression of various fusion genes and detection of urokinase activity (1) Expression of various derivatives of pSSD1 Reference [Yokoyam-Kobayashi, M. et al., Gene 163: 193-1]
96 (1995)] according to the method described in about 100 ng of pSSD1-966.
1. A phage particle containing single-stranded DNA of pSSD1-966.2, pSSD1-966.3, and pSSD1-966.4, and a phage particle containing the same amount of single-stranded DNA of pSSD1 as a control were each mixed with 7.5 μg of lipopolyamine (TRANSFECTAM). And introduced into a monkey kidney-derived cultured cell line COS7 (10 ⁵ cells).
These cells were cultured under conditions of 37 ° C. and 5% CO ₂ for 5 days. (2) Detection of urokinase activity in culture medium [Yokoyam-Kobayashi, M. et al., Gene 163: 193-1]
96 (1995)] on a fibrin plate prepared according to the method described in
Incubated overnight at ℃. As a result, urokinase activity was not detected in the culture medium of any of the cells. (3) Detection of urokinase activity on cell surface Using a 50 mM phosphate buffer (pH 7.4), a 2% solution of bovine fibrinogen and a 0.5% agarose solution were prepared, and 1 ml of each solution contained 2 μl of 1M calcium chloride and thrombin. Add 2 units, pour into a 3.5cm diameter plate,
Polymerization was performed at 37 ° C. for 1 hour. The polymerized fibrin was peeled off from this plate to obtain a thin fibrin (fibrin sheet). Next, the medium was removed from the cultured cells of the above (1), and a physiological saline solution (PBS) containing a phosphate buffer solution was used.
To wash the cell surface. 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, pSSD1, pSSD1-966.2, pSSD1-96
No change was seen in the fibrin sheet for cells expressing 6.4. On the other hand, pSSD1-966.1, pSSD1-966.
Lysed spots were observed on the fibrin sheet in cells expressing 3 (FIG. 3). From the above results, as shown in FIG. 4, the membrane protein encoded by HP00966 has the N-terminus, the portion between the first and third transmembrane domains, and the C-terminus on the cytoplasmic side. The portion between the first and second transmembrane domains and the third
It was found that the portion between the transmembrane domain and the fourth transmembrane domain had a structure that was extracellular. In addition, this structure was consistent with what was predicted as the topology of the TM4 superfamily membrane proteins. (4) Detection of urokinase activity from cells disrupted by sonication After removing the medium from the cultured cells in the above (1), washing the cell surface with PBS, and culturing the cells using a cell scraper (Falcon) Peeled off. The cells were suspended in 0.2 ml of PBS, and subjected to sonication using an ultrasonic cell disrupter BioRaptor (manufactured by Cosmo Bio) under the conditions of output H, level 4, 15 seconds, and 10 times. After the treatment, 10 µl of the suspension was placed on a fibrin plate and incubated at 37 ° C overnight. As a result, pSSD
1. No urokinase activity was detected in cells expressing pSSD1-966.2 and pSSD1-966.4. On the other hand, pS
Urokinase activity was detected in cells expressing SD1-966.1 and pSSD1-966.3. The above results were consistent with the results of the above (3) in which the activity of the film surface was directly measured.

[0020]

As described in detail above, the method of the present invention makes it possible to easily and reliably determine the topology of a membrane protein. The determined structure of the membrane protein provides useful information when the membrane protein is used as a drug or when used as a drug target.

[0021]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1722 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin: Organism name: Homo-Sapiens Cell type: Osteosarcoma Cell line : Saos-2 Clone name: HP00966 Sequence features: Symbol indicating feature: CDS Location: 156. . 917 Method of Characterization: E Sequence ACTTGCTGGG GTCGGGGCTG CGCGACGGCG CAGGGGCTGC GGGGAGCGCC GCGCAGGCCG 60 TGCAGTTCCT AGCGAGGAGG CGCCGCCGCC ATTGCCGCTC TCTCGGTGAG CGCAGCCCCG 120 CTCTCCGGGC CGGGTCGC CGGGTCGCGGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGGCGGCGCGCGCGCGCGCGCGCGCGCGCGGCGGCGCGCGCGCGGCGGCGGCGGCGGCGGCGCGCGGCGGCGGCGGCC TTT CTC AAC CTC ATC TTC TGG GGG 221 Thr Ser Ser Lys Thr Val Leu Val Phe Leu Asn Leu Ile Phe Trp Gly 10 15 20 GCA GCT GGC ATT TTA TGC TAT GTG GGA GCC TAT GTC TTC ATC ACT TAT 269 Ala Ala Gly Ile Leu Cys Tyr Val Gly Ala Tyr Val Phe Ile Thr Tyr 25 30 35 GAT GAC TAT GAC CAC TTC TTT GAA GAT GTG TAC ACG CTC ATC CCT GCT 317 Asp Asp Tyr Asp His Phe Phe Glu Asp Val Tyr Thr Leu Ile Pro Ala 40 45 50 GTA GTG ATC ATA GCT GTA GGA GCC CTG CTT TTC ATC ATT GGG CTA ATT 365 Val Val Ile Ile Ala Val Gly Ala Leu Leu Phe Ile Ile Gly Leu Ile 55 60 65 70 GGC TGC TGT GCC ACA ATC CGG GAA AGT CGC TGT GGA CTT GCC ACG TTT 413 Gly Cys Cys Ala Thr Ile Arg Glu Ser Arg Cys Gly Leu Ala Thr Phe 75 80 85 GTC ATC ATC CTG CTC TTG GTT TTT GTC ACA GAA GTT GTT GTA GTG GTT 461 Val Ile Ile Leu Leu Leu Val Phe Val Thr Glu Val Val Val Val 90 95 100 TTG GGA TAT GTT TAC AGA GCA AAG GTG GAA AAT GAG GTT GAT CGC AGC 509 Leu Gly Tyr Val Tyr Arg Ala Lys Val Glu Asn Glu Val Asp Arg Ser 105 110 115 ATT CAG AAA GTG TAT AAG ACC TAC AAT GGA ACC AAC CCT GAT GCT GCT GCT 557 Ile Gln Lys Val Tyr Lys Thr Tyr Asn Gly Thr Asn Pro Asp Ala Ala 120 125 130 AGC CGG GCT ATT GAT TAT GTA CAG AGA CAG CTG CAT TGT TGT GGA ATT 605 Ser Arg Ala Ile Asp Tyr Val Gln Arg Gln Leu His Cys Cys Gly Ile 135 140 145 150 CAC AAC TAC TCA GAC TGG GAA AAT ACA GAT TGG TTC AAA GAA ACC AAA 653 His Asn Tyr Ser Asp Trp Glu Asn Thr Asp Trp Phe Lys Glu Thr Lys 155 160 165 AAC CAG AGT GTC CCT CTT AGC TGC TGC AGA GAG ACT GCC AGC AAT TGT 701 Asn Gln Ser Val Pro Leu Ser Cys Cys Arg Glu Thr Ala Ser Asn Cys 170 175 180 AAT GGC AGC CTG GCC CAC CCT TCC GAC CTC TAT GCT GAG GGG TGT GAG 749 Asn Gly Ser Leu Ala His Pro Ser Asp Leu Tyr Ala Glu Gly Cys Glu 185 190 1 95 GCT CTA GTA GTG AAG AAG CTA CAA GAA ATC ATG ATG CAT GTG ATC TGG 797 Ala Leu Val Val Lys Lys Leu Gln Glu Ile Met Met His Val Ile Trp 200 205 210 GCC GCA CTG GCA TTT GCA GCT ATT CAG CTG CTG GGC ATG CTG TGT GCT 845 Ala Ala Leu Ala Phe Ala Ala Ile Gln Leu Leu Gly Met Leu Cys Ala 215 220 225 230 TGC ATC GTG TTG TGC AGA AGG AGT AGA GAT CCT GCT TAC GAG CTC CTC 893 Cys Ile Val Leu Cys Arg Arg Ser Arg Asp Pro Ala Tyr Glu Leu Leu 235 240 245 ATC ACT GGC GGA ACC TAT GCA TAGTTGACAA CTCAAGCCTG AGCTTTTTGG 944 Ile Thr Gly Gly Thr Tyr Ala 250 253 TCTTGTTCTG ATTTGGAAGG TGAATTGAGC AGGTCTGCTG CTGTTGGCCT CTGGAGTTCA 1004 TTTAGTTAAA GCACATGTAC ACTGGTGTTG GACAGAGCAG CTTGGCTTTT CATGTGCCCA 1064 CCTACTTACC TACTACCTGC GACTTTCTTT TTCCTTGTTC TAGCTGACTC TTCATGCCCC 1124 TAAGATTTTA AGTACGATGG TGAACGTTCT AATTTCAGAA CCAATTGCGA GTCATGTAGT 1184 GTGGTAGAAT TAAAGGAGGA CACGAGCCTG CTTCTGTTAC CTCCAAGTGG TAACAGGACT 1244 GATGCCGAAA TGTCACCAGG TCCTTTCAGT CTTCACAGTG GAGGCTAGGGCTAG CTGGTTAAGG TTAACACCAG ATGGTGCCCC 1364 TCATTGGTGT CCTTTTAAAA AATATTTACT GTAGTCCAAT AAGATAGCAG CTGTACAAAA 1424 TGACTAAAAT AGATTGTAGG ATCATATGGC GTATATCTTG GTTCATCTTC AAAATCAGAG 1484 ACTGAGCTTT GAAACTAGTG GTTTTTAATC AAAGTTGGCT TTATAGGAGG AGTATAATGT 1544 ATGCACTACT GTTTTAAAAG AATTAGTGTG AGTGTGTTTT TGTATGAATG AGCCCATTCA 1604 TGGTAAGTCT TAAGCTTGTT GGAAATAATG TACCCATGTA GACTAGCAAA ATAGTATGTA 1664 GATGTGATCT CAGTTGTAAA TAGAAAAATC TAATTCAATA AACTCTGTAT CAGCCCCC 1722

[Brief description of the drawings]

FIG. 1 is a graph showing the hydrophobicity / hydrophilicity profile in the amino acid sequence of the membrane protein gene clone HP00966.

FIG. 2 is a schematic diagram showing the structure of a fusion gene of membrane protein HP00966 and urokinase.

FIG. 3 is a photograph instead of a drawing showing urokinase activity on the cell surface, where each cell is pSSD1, pSSD1-96 from the upper left.
61, pSSD1-9662, pSSD1-10085, pSSD1-966 from bottom left
3. Transformant with pSSD1-9664.

FIG. 4. Membrane protein H determined by the method of the invention.
It is a schematic diagram which shows the topology of P00966.

Continued on the front page (72) Inventor Seishi Kato 3-46-50 Wakamatsu, Sagamihara City, Kanagawa Prefecture

Claims (2)

[Claims] 1. A method for determining the topology of a membrane protein having n (n is an integer of 2 or more) transmembrane domains, comprising: a gene encoding a membrane protein; N types of DNA fragments containing the transmembrane domains up to the first in order are prepared, and a gene encoding a reporter protein is fused to the C-terminus of each of these DNA fragments. And expressing the fusion protein, and determining the topology of the membrane protein using the location of the reporter protein in each cell as an index. 2. The method according to claim 1, wherein the reporter protein is a plasminogen activator.