Patent Grant
6866998
The present invention relates to the field of genetic engineering and includes methods for detecting and isolating nucleic acid sequences encoding peptides that contain signal sequences.
So far, genes encoding a hormone or growth factor have been isolated and utilized to produce many recombinant proteins that are commercialized as medicines. Most of them are secretory proteins. Therefore, isolation of a gene encoding a novel secretory protein is an extremely important step in developing a novel medicine. Accordingly, methods for isolating a gene encoding a secretory protein have been developed. For example, Honjo et al. developed a method (unexamined published Japanese patent application No. Hei 6-315380) by utilizing the feature that secretory proteins have a signal sequence that allows intracellularly expressed proteins to translocate to the cell surface. In this method, the signal sequence of the α chain of human IL-2 receptor, a secretory protein, is replaced with a short cDNA fragment corresponding to the 5′-end sequence of mRNA from a target cell or tissue to construct a library, which is then introduced into cells. Among the clones, IL-2 receptor is expressed on the cell surface of clones with a signal sequence, but not those without a signal sequence. The presence of the signal sequence can thus be detected by the anti-IL-2 receptor antibody.
Genetics Institute, Inc., (Cambridge, Mass.) developed a more sophisticated system utilizing a yeast metabolic enzyme (U.S. Pat. No. 5,536,637). Invertase, a yeast metabolic enzyme, is a secretory enzyme that cleaves sucrose in the culture medium to glucose and fructose to transfer energy. A mutant strain that does not secrete this enzyme can not grow in a medium containing sucrose as the sole carbon source without glucose. In this method, which utilizes the phenomenon, invertase gene is ligated with cDNA to construct a library, which is then introduced into a mutant yeast strain lacking the invertase gene. Clones containing the signal peptide are isolated by selecting clones capable of growing in a medium containing only sucrose as a carbon source.
However, the method of Honjo et al. is disadvantageous in that laborious steps are required in selecting positive clones due to the use of an antibody. Furthermore, the detection sensitivity is very low. The method of Genetics Institute, Inc., also has a problem in that a clone with poor secretion efficiency in yeast cannot be isolated. In addition, these methods detect only short DNA because of the potential loss of antigenicity or enzymatic activity when the reporter protein is fused with a large protein. Moreover, the methods fail to detect the type II membrane proteins having their N-terminus within the cell and C-terminus outside the cell.
The present invention provides a method for determining whether or not a tested cDNA encodes a peptide that has secretory activity. The methods also provide for isolating a cDNA encoding a peptide with secretory activity, which can be done using a cDNA encoding a peptide longer than the signal sequence.
Proteins, such as cytokine receptors, translocate to the cell surface, dimerize upon binding their ligands, and induce cell proliferation. The proteins that are translocated to the cell surface are guided by a signal sequence. The present inventors discovered that it was possible to determine whether a desired peptide has secretory activity by removing the signal sequence (and, optionally, additional sequence that encodes an extracellular region of the peptide) from the proteins that translocate to the cell surface and replacing that signal sequence with a desired peptide, thereby creating a sequence encoding a fusion protein, expressing the fusion protein in cells, and examining the proliferation of the cells. If the desired peptide has secretory ability, the fusion protein translocates to the cell surface, dimerizes, and induces cell proliferation. In contrast, if the desired peptide does not have secretory activity, the fusion protein cannot translocate to the cell surface and induce cell proliferation. Thus, one can determine whether a desired peptide has the ability to mediate translocation by simply examining cell proliferation as an index. Moreover, the inventors discovered a way to perform positive screening for a peptide that has secretory ability by selecting cells that proliferate. Thus, the present inventors used mpl (thrombopoietin receptor) as a protein that triggers cell proliferation through translocation to the cell surface and dimerization, and developed a method for detecting and isolating peptides possessing secretory ability.
Specifically, we prepared a DNA encoding human mpl without the secretory ability by removing the secretion signal and most of the extracellular domain from a constitutively active form of mpl, which was found by the present inventors (the mpl is altered to be able to confer autonomous proliferation ability to an IL-3 dependent cell line by the transducing signal in the absence of ligand; Blood 88:1399-1406 (1996)). The DNA was then ligated with cDNA to be tested, a DNA encoding a known secretory protein, or a DNA encoding the secretory peptide from which the secretory signal region was removed. The resulting chimeric genes were expressed in cells, and the proliferation ability of the cells was examined. The results show that the DNA encoding a known secretory protein used as a test cDNA induced cell proliferation whereas no cell proliferation was detected for the DNA encoding the secretory protein from which the secretory signal region was removed. In this way, the inventors found that the system thus developed can be used to easily detect and isolate a DNA encoding a peptide with secretory activity and containing a long peptide coding region using cell proliferation as an index. Indeed, they performed a screening and succeeded in detecting and isolating DNAs encoding secretory proteins including type I membrane proteins and type II membrane proteins.
Thus, the present invention relates to:
The present invention relates to a method for detecting a DNA encoding a peptide having secretory ability. The detection method features the use of a DNA encoding a peptide that induces cell proliferation when dimerized on the cell surface but that lacks its secretory signal. This peptide can be used to detect a peptide with secretory ability. Here, “the peptide that induces, or that is capable of inducing, cell proliferation through dimerization on the cell surface” includes mpl (Proc. Natl. Acad. Sci. USA, 89:5640-5644 (1992)), the alpha chain or beta chain of GM-CSF receptor (Blood 83:2802 (1994)), erythropoietin receptor (Nature 348:647 (1990)), c-kit receptor (Blood 85:790 (1995)), and neu (Nature 339:230 (1989)), but are not limited thereto. In the method of the present invention, a cDNA is constructed to encode the above peptides whose secretory ability is eliminated. The secretory ability is usually removed by deleting a region containing the signal sequence. For example, the signal sequence of the human mpl is the region corresponding to positions 1 to 25 in the amino acid sequence of the protein (Proc. Natl. Acad. Sci. USA, 89:5640-5644 (1992)), and that of the beta chain of the human GM-CSF receptor is the region corresponding to positions 1 to 48 (Proc. Natl. Acad. Sci. USA, 87:9655-9659 (1990)). Preferably, the extracellular domain is also deleted from the peptide.
The peptide encoded by a constructed cDNA is preferably ligand-independent (if the peptide is ligand-dependent, it may lose the ligand-binding ability and become inactive after creating a fusion protein). A method for creating a ligand-independent peptide is to introduce a mutation into the peptide. For example, in the case of mpl, the substitution of Ser at position 498 with Asn can abolish the dependency of mpl on its ligand, thrombopoietin (Blood 88:1399-1406 (1996)). The mpl used in the present method preferably comprises the amino acid sequence as described in SEQ ID NO: 4.
The DNA prepared as described above is inserted into an appropriate expression vector. The expression vector is not limited, and can be a retroviral vector, which can be introduced into a variety of cells with high efficiency through virus infection. Upon expression, the DNA inserted into the vector is stably expressed in the cells. Examples of retroviral vectors include those engineered for cDNA library construction, such as pBabeX (Proc. Natl. Acad. Sci. USA 92:9146-9150 (1995)) or pMX (Exp. Hematol. 24:324-329 (1996)). One can also use viral vectors such as adenovirus, EB virus, and papilloma virus, or plasmid vectors such as pEF-BOS (Nucleic Acid Res. 18 (17)) and pcD SRα296 (Mol. Cell. Biol. January 466-472 (1988)). The expression vectors can have a cloning site for a cDNA to be tested for its secretory ability at the 5′-upstream region of the above DNA insert (i.e., upstream of the sequence encoding the protein that, when under the control of its own signal sequence, is translocated to the cell membrane); upon expression, the sequence encodes a fusion protein of the test signal sequence and the translocatable protein that lacks its own signal sequence. The method for creating a cloning site for a cDNA is known to one of ordinary skill in the art.
As noted above, the prepared vector can include a cDNA to be tested. The test cDNA is ligated into the 5′-upstream region of the DNA that encodes a peptide that induces cell proliferation upon dimerization on the cell surface, which is inserted into the vector. The test cDNA can be any cDNA encoding a peptide whose secretory ability is to be tested. The test cDNA can be ligated with a vector according to standard methods. For example, the ligation method using T4 DNA ligase via an adapter linker (Maniatis T., Molecular Cloning).
The prepared vector is then introduced into a cell. Cells into which the vector is introduced are not limited and include cytokine-dependent proliferating cell such as Ba/F3, OTT-1, FDCP-1, and TF-1 cells. Vectors can be introduced into cells by using standard methods including lipofection, calcium phosphate method, DEAE-dextran method, and electroporation. In retrovirus infection-mediated introduction, the vector is introduced into the packaging cells and integrated into the virus particles. The vector can be introduced by using standard methods such as the calcium phosphate method and lipofection. For example, cells such as BOSC23, Bing (Proc. Natl. Acad. Sci. USA 90:8392-8396 (1993)), NX-E, and NX-A cells (Nolan G. P. Immunity 8:461-471 (1998)) can be used as the packaging cell.
Next, the thus-prepared transformants are cultured and examined for their proliferation ability. When a protein encoded by cDNA inserted into the vector is expressed as a fusion protein with a ligand-independent active cytokine receptor, the transformant is cultured in the medium lacking the cytokine (ligand) on which the cell depends. If a significant cell proliferation is detected, the test cDNA is judged to be a “positive clone” encoding a peptide that contains the secretory ability. Alternatively, if no significant cell proliferation is detected, the cDNA is judged to be a “negative clone” that encodes a peptide lacking the secretory ability. When a protein encoded by the inserted cDNA is expressed as a fusion protein with a ligand-dependent cytokine receptor, the transformant is cultured in the presence of the ligand. If a significant cell proliferation is detected, after comparison with a negative control in the absence of the ligand, if necessary, the test cDNA is judged to be a “positive clone.” Other conditions for culturing transformants can be appropriately selected by one skilled in the art depending on the types of cells into which the vector is inserted and the nature of the fusion protein to be expressed.
The present invention also relates to a method for isolating a cDNA encoding a peptide that contains the secretory ability. In the method, a cDNA library is ligated into the vector instead of the above test cDNA that is used for detecting cDNA encoding a peptide containing the secretory ability. In one specific embodiment of the invention, cDNAs prepared by using a random primer are ligated with the BstXI adapter and inserted between the two BstXI sites; one is of the vector and the other is inserted into the extracellular cleavage site of the active mpl. The source of the cDNA library is not limited to any specific one, but can be a cell or tissue from which a desired peptide containing the secretory ability is to be isolated. Many standard methods can be used to construct a cDNA library. In the present method, cells judged to be capable of proliferation are selected from the cDNA library-introduced cells. The cDNAs contained in the selected cells are supposed to encode a peptide having the secretory ability. cDNA can be isolated from the cells whose proliferation has been detected by, for example, extracting the genomic DNA or RNA, amplifying the cDNA of interest by PCR using primers designed to encompass the cloning sites (in case of RNA, after converting it into DNA-using reverse transcriptase), and recovering the products.
Whether the recovered cDNA is full length or a fragment, or whether it is a cDNA encoding a novel secretory peptide, can be analyzed by comparing the cDNA sequence with those of the known proteins in the database. If the cDNA is not full length, it is used to screen a secondary cDNA library to isolate a full-length cDNA. The secondary cDNA library can be constructed by a method known to one skilled in the art, such as those described in the literature (Molecular Cloning, A Laboratory Manual, 2nd edition. Sambrook J. et al., (1989) Cold Spring Harbor Laboratory Press, New York).
A cDNA encoding a peptide with the secretory ability isolated by the method of the present invention can be utilized to produce a recombinant protein that is useful as a medicine or in gene therapy of related diseases. A recombinant protein from the isolated cDNA can be produced by known methods in the art. For example, the cDNA is inserted into an appropriate vector such as pED (Kaufman et al. Nucleic Acids Res. 19:4484-4490 (1991)), pEF-BOS (Mizushima et al. Nucleic Acids Res. 18:5322 (1990)), pXM, pJl3, and pJL4 (Gough et al. EMBO J. 4:645-653 (1985)), the vector is introduced into a host cell, the resulting transformant is cultured to allow it to express a recombinant protein, and the recombinant protein is purified.
The present invention is illustrated in detail below with reference to examples, but is not to be construed as being limited thereto.
In mouse myeloproliferative leukemia virus, env is ligated to the mouse mpl comprising the extracellular domain consisting of 56 amino acids from the transmembrane domain toward the N-terminus, the transmembrane domain, and the intracellular domain. PCR was performed to obtain a cDNA encoding the corresponding region of the human mpl, which is from Leu (449) to the stop codon (636), having the NotI site immediately before the Leu (449) (a single nucleotide insertion) and the SalI site immediately after the stop codon so as to be in the frame of the GM-CSF cDNA shown below. The “pBabeX MPLM” (Blood 88:1399-1406. (1996)), in which active human mpl cDNA is cloned, was used as a template. Primers used are listed in Table 1.
PCR was performed in a reaction mixture containing 10 μg/ml template DNA, 1 μM each primer, 50 U/ml KOD DNA polymerase (TOYOBO), 1 mM MgCl2, 0.2 mM dNTPs, 120 mM Tris-HCl (pH 8), 10 mM KCl, 6 mM (NH4)2SO4, 0.1% Triton X-100, and 10 μg/ml BSA by using the GeneAmpPCR System (Perkin Elmer) under the following conditions: denaturation at 98° C. for 60 sec, followed by 25 cycles of 98° C. for 15 sec, 60° C. for 10 sec, and 74° C. for 30 sec. The PCR products were analyzed by electrophoresis on an agarose gel, and a gel piece containing a 0.6 kb fragment of interest was excised to extract DNA. The DNA was then phosphorylated at its 5′-termini with T4 polynucleotide kinase (TOYOBO), and ligated by using T4 DNA ligase (TOYOBO) with the pBluescript SK(−) vector (Stratagene) that was pretreated with SmaI (TaKaRa Shuzo) and Bacterial Alkaline Phosphatase (BAP; TaKaRa Shuzo). The nucleotide sequence of the active mpl cDNA inserted in the resulting plasmid was verified with the ABI PRISM 310 Genetic Analyzer (Perkin Elmer). The plasmid was digested with NotI (TaKaRa Shuzo) and SalI (TaKaRa Shuzo), and separated by electrophoresis on an agarose gel to isolate a 0.6 kb fragment. The fragment was ligated with the pMX (Proc. Natl. Acad. Sci. USA 92:9146-9150. (1995)), which was also digested with NotI and SalI, treated with BAP, and purified by agarose gel electrophoresis, using T4 DNA ligase to obtain pMX v-mplM. The plasmid pMX v-mplM contains a cDNA encoding an active mpl lacking the secretory ability. The nucleotide sequence of the cDNA insert and the amino acid sequence of the peptide encoded by the cDNA are shown in SEQ ID NO: 3 and NO: 4, respectively.
Next, to obtain a human GM-CSF cDNA in which the stop codon is replaced with a NotI site, PCR was performed by using the pcDSRα 298 hGM-CSF (Proc. Natl. Acad. Sci. USA 82:4360-4364 (1985)) containing the human GM-CSF cDNA as a template. Primers used are shown in Table 2.
EcoGM was designed to have the translation initiation codon ATG in place of the Ser (17), and, as in EcoGMss and EcoGM, the EcoRI site and the Kozak consensus sequence (J. Cell Biol. 108:29. (1989)) immediately before the ATG codon. Primer pairs of EcoGMss and GM Not were used in PCR to amplify GM-CSF containing the signal sequence, and EcoGM and GM Not were used to amplify GM-CSF lacking the signal sequence. PCR was performed as described above except for using 55° C. for the annealing temperature, and the products were cloned into the pBluescript SK(−). The nucleotide sequence of the DNA inserts was verified by using the ABI PRISM 310 Genetic Analyzer (Perkin Elmer). The plasmids were then digested with EcoRI (TaKaRa) and NotI and inserted into the EcoRI-NotI site of the pMX v-mplM as described above, and “pMX GM(+)v-mplM” and “pMX GM(−)v-mplM” were obtained. The “pMX GM(+)v-mplM” and “pMX GM(−)v-mplM” encode a fusion protein between the C-terminal part of the active mpl starting from Leu (449) and the entire GM-CSF with or without the signal sequence, respectively. The nucleotide sequences of their cDNA inserts are shown as SEQ ID NO: 8 and NO: 10, and the amino acid sequences of the proteins encoded by the cDNAs are shown as SEQ ID NO: 9 and NO: 11.
Each of the above plasmids was introduced into packaging cell-line BOSC23 (Proc. Natl. Acad. Sci. USA 90:8392-8396. (1993)) using LipofectAMINE (Life Technologies). BOSC23 cells were plated into 6-cm dishes (CORNING) with Dulbecco's modified Eagle medium (DMEM; Nissui Pharmaceutical) containing 10% fetal calf serum (FCS; JRH Biosciences). After 6-hr incubation, the cells were washed with OPTI-MEM I reduced serum medium (Life Technologies). Separately, LipofectAMINE (18 μl) diluted in 200 μl OPTI-MEM I was mixed with 3 μg samples of each plasmid diluted in 200 μl OPTI-MEM I. The resulting mixtures were allowed to stand at room temperature for 15 minutes, mixed with 1.6 ml OPTI-MEM I, then added to the cells. After 5 hr, 2 ml of DMEM containing 20% FCS was added to the cells, which were incubated for an additional 19 hr. The medium was then replaced with 3 ml of DMEM containing 10% FCS, and the culture supernatant was recovered after 24 hr. Mouse interleukin-3 (IL-3) and 10 μg/ml polybrene (hexadimethrine bromide, Sigma) were added to the culture supernatant containing the recombinant virus, and Ba/F3 cells were suspended therein for infection. After 24 hr of infection, the cells were washed twice in RPMI1640 (Nissui Pharmaceutical) containing 10% FCS lacking mouse IL-3, and the culture was continued in the same medium.
The cells containing the fusion protein between the entire GM-CSF containing the signal sequence and the active mpl (derived from the pMX GM(+)v-mplM) grew in the absence of IL-3 as well as those containing the active mpl with the secretory ability. In contrast, the cells containing the fusion protein between the GM-CSF lacking the signal sequence and the active mpl (pMX GM(−)v-mplM) did not grow as well as control Ba/F3 cells into which no fusion protein expression vector was introduced (FIG. 1).
The following oligonucleotides (Table 3) were synthesized, and their 5′-termini were phosphorylated using T4 polynucleotide kinase. The oligonucleotides were mixed and denatured at 95° C., and then annealed by gradually cooling them to 40° C. to prepare the cassette DNA.
The pMX GM(−)v-mplM, which was digested with NotI (TaKaRa) and treated with BAP, was mixed with the cassette and ligated using T4 DNA ligase. The direction of the cassette in the resulting plasmid was verified by DNA sequencing to be in the order of BstXI and NotI (pMX GM(−)v-mplM2). Total RNA was prepared from the rat neuroblastic cell line MNS70 using the TRIZOL reagent (GIBCO BRL) and passed through the oligo dT column (Pharmacia) to prepare polyA(+) RNA. Double-stranded cDNA was synthesized with the random hexamer contained in the SuperScript Choice System (GIBCO BRL). The cDNA was blunt-ended, ligated with the BstXI adapter (Invitrogen), and fractionated by using the SizeSep 400 Spun Column (Pharmacia). The cDNA was then mixed and ligated with the pMX GM(−)v-mplM2 which was digested with BstXI (TaKaRa) and treated with BAP, using T4 DNA ligase. The DNA was introduced into DH10B E. coli (GIBCO BRL) by electroporation using Gene Pulser (BioRad) to construct a cDNA library.
Plasmids were extracted from the recombinant E. coli containing a cDNA library and purified by using the JETstar column (GENOMED). The library plasmids were introduced into BOSC23 packaging cells by using LipofectAMINE as described above. Mouse IL-3 (10 ng/ml) and 10 μg/ml polybrene (Hexadimethrine Bromide, Sigma) were added to the culture supernatant containing the recombinant virus, and Ba/F3 cells were suspended therein for infection. After 24-hr infection, the cells were washed twice with phosphate buffer and cultured further in RPMI1640 containing 10% FCS. The genomic DNA was prepared from the clones that grew in the absence of IL-3, and PCR was performed using primers designed to encompass the cDNA insertion site to recover the cDNA fragments.
PCR was performed in 50 μl of the reaction mixture containing 500 ng genomic DNA, 500 pM each primer, 2.5 U TaKaRa LA Taq (TaKaRa), 2.5 mM MgCl2, 0.3 mM dNTPs, and the accompanying buffer using the GeneAmpPCR System2400 in the following process: denaturation at 98° for 60 sec, followed by 30 cycles of 98° for 20 sec and 68° for 120 sec. PCR products were separated by electrophoresis on an agarose gel, the gel pieces containing the amplified fragments were excised, and DNA was purified. The nucleotide sequence of the DNA fragments purified from the resulting 190 clones was determined, and 150 clones were found to be cDNAs encoding a known secretory protein or a membrane protein, or parts thereof. The other 40 clones were found to encode unknown secretory proteins. Some of the thus-obtained known secretory proteins are shown in Table 5, where “length” indicates the length of the ORF of the obtained cDNA fragment by the number of amino acid residues. The average length of the clones encoding a known secretory protein was 273 amino acid residues. “Accession number” indicates the accession number in the GenBank protein database. It should be noted that the background in the present method such as detecting a cDNA encoding a protein other than a secretory protein or cDNA that was inserted in the opposite direction was 1% or less.
The present invention provides a method for detecting and isolating a cDNA encoding a secretory peptide using a peptide capable of triggering cell proliferation through its dimerization on the cell surface but lacking the secretory ability. Since the method utilizes cell proliferation as an index for detection, it is extremely easy and sensitive. Moreover, compared to the conventional methods that enable detecting a short DNA fragment, this method enables detecting and isolating a cDNA containing a longer peptide coding region, and thus provides more information from the first isolated clones. In addition, the method enables detecting and isolating secretory proteins including type I and type II membrane proteins.
| Number | Date | Country | Kind |
|---|---|---|---|
| 9324912 | Nov 1997 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCTJP98/05326 | 11/26/1998 | WO | 00 | 8/22/2000 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO9926978 | 6/3/1999 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5536637 | Jacobs | Jul 1996 | A |
| 5675062 | Haber et al. | Oct 1997 | A |
| Number | Date | Country |
|---|---|---|
| 0 939 119 | Jul 1997 | EP |
| 0 607 054 | Feb 1999 | JP |
| 11 32779 | Feb 1999 | JP |
| WO 9640904 | Dec 1996 | WO |
