CA2113363C

CA2113363C - Process for constructing a cdna library and a novel polypeptide and dna coding the same

Info

Publication number: CA2113363C
Application number: CA002113363A
Authority: CA
Inventors: Tasuku Honjo; Kei Tashiro; Hideaki Tada
Original assignee: Ono Pharmaceutical Co Ltd
Current assignee: Ono Pharmaceutical Co Ltd
Priority date: 1993-01-14
Filing date: 1994-01-13
Publication date: 2003-01-07
Anticipated expiration: 2014-01-13
Also published as: PT607054E; DE69434953D1; ATE359366T1; ES2187516T3; US5525486A; EP1243650A2; KR100271725B1; ATE228163T1; JP2879303B2; EP0607054A2; DE69431736D1; DK0607054T3; CA2381458A1; EP1243650A3; KR940018464A; EP1243650B1; CA2113363A1; JPH06315380A; EP0607054A3; DE69431736T2

Abstract

A process for constructing a cDNA library which has a selectivity for signal peptides, that makes it possible to efficiently identify unknown and useful polypeptide comprising a signal peptide. A novel polypeptide consisting of 89 amino acids (including a signal peptide) produced by a stroma cell line, which is useful as an agent for preventing or treating, for example, anemia, leukopenia or infections and the like, and DNAs coding for said polypeptide, have been identified using the process of the invention.

Description

- i -PROCESS FOR CONSTRUCTING A cDNA LIBRARY
AND A NOVEL POLYPEPTIDE AND DNA CODING THE SAME
This invention relates to a process for constructing a cDNA library, and a novel polypeptide and a DNA coding for the same. More particularly, it relates to an efficient process for constructing a cDNA library having a high selectivity for signal peptides, and a novel polypeptide produced by a specific stroma cell line and a DNA coding for said polypeptide.
In order to obtain specific polypeptides (for example, proliferation and/or differentiation factors) or a DNA coding for the same, there have been generally employed methods comprising confirming the target biological activity in a tissue or a cell culture medium and then cloning of a gene through the isolation and purification of a polypeptide and further methods comprising expression-cloning of a gene with the guidance of the biological activity.
However, it is frequently observed that a gene, which has been cloned with the guidance of a certain activity, codes for a known polypeptide since many physiologically active polypeptides occurring in vivo have various biological activities. Further, most intravital physiologically active polypeptides are secreted only in a trace amount, which makes the isolation and purification thereof and the confirmation of biological activity difficult.
Recent rapid developments in techniques for constructing cDNAs and sequencing techniques have made it possible to quickly sequence a large amount of cDNAs. By utilising these techniques, a process, which comprises constructing cDNA libraries from various cells and tissues, cloning cDNAs at random, identifying the nucleotide sequences thereof, expressing the corresponding ~~iypept?de and then analyzing its physiological functions, is now in ~," ~~133~3 use. Although this process is advantageous in that a gene can be cloned and information on its nucleotide sequence can be obtained without any biochemical or genetic analysis, the target gene can often only be identified by chance.
The present inventors have studied the cloning of genes for proliferation and differentiation factors in hematopoietic and immune systems. They have paid attention to the fact that most secretory proteins such as proliferation and/or differentiation factors (for example various cytokines) and membrane proteins such as receptors thereof (hereinafter these proteins will be referred to generally as secretory proteins and the like) have sequences called signal peptides in the N-termini.
Extensive studies have been conducted to provide a process for efficiently and selectively cloning genes coding for signal peptides. As a result, a process has now been devised whereby an N-terminal fragment can be efficiently amplified and the existence of a signal peptide can be easily examined.
In accordance with the present invention, cDNAs with a high probability of containing a signal peptide are ligated at both their ends to linkers containing restriction enzyme sites which are different from each other. These fragments alone are rapidly produced in a large amount by the polymerase chain reaction (PCR) method so as to elevate the content of the fragments with a high probability of containing a signal peptide. Next, the above-mentioned fragment is introduced into an expression vector containing DNA coding for a known secretory protein or the like but lacking DNA encoding the corresponding signal peptide. Many secretory proteins and the like are secreted or expressed on cell membrane even if the signal peptide has been substituted by a signal peptide of another protein. Therefore, if the known secretory protein or the like is expressed on cell membrane or outside the cells, ~.. 2~~.~~6 - 3 - -.
this confirms that a cDNA fragment corresponding to a signal peptide has been introduced into the expression vector. Thus the inventors have devised a convenient method for detection of the insertion of cDNA encoding a signal peptide.
The polymerase chain reaction used in the present invention is known as a method for amplifying specific DNA
fragments in a large amount. It is also known that many secretory proteins and the like can be expressed even if the signal peptide thereof is substituted by that of another secretory protein or the like. However, to the applicants' knowledge, there has been no suggestion that these techniques be used as the inventors have done to provide a process for selectively cloning a signal peptide.
It is known that hematopoietic cells secrete various proliferation and/or differentiation factors exemplified by interleukin.
The present invention further relates to a novel polypeptide obtained from hematopoietic cells and DNA
coding for the same.
The inventors have sought a novel factor (polypeptide) produced by hematopoietic cells using the process which is the first subject of the present invention. As a result, a novel polypeptide and DNA coding for the same have been identified.
Computer searches to identify polypeptides having sequences identical or highly homologous with that of the polypeptide of the present invention and the DNAs coding for the same have not identified any such sequences. Thus, to the best of the applicants' knowledge, the polypeptide of the present invention and the DNA coding for the same are novel. Further, homology analysis has revealed that the polypeptide of the present invention is a member of the chemokine family as it has a pattern of Cys-X-Cys (X is optional amino acid).

2~13~~~

Accordingly, the present invention provides a process for constructing a cDNA library comprising:
(1) synthesizing single-stranded DNA complementary to mRNA isolated from subject cells and ligating to the 3'-end of the single-stranded DNA, a DNA oligomer of known sequence;
(2) converting the single-stranded DNA obtained in (1) to a double-stranded DNA using as primer an oligomer ligated to a first restriction enzyme (enzyme I) site, the primer sequence being complementary to the oligomer of known sequence ligated to the single-stranded DNA;
(3) fragmenting the double-stranded DNA obtained in (2), fractionating the fragments obtained by size, ligating linker containing a second restriction enzyme (enzyme II) site, differing from the enzyme I, thereto and fractionating;
(4) amplifying fragments containing the sites of restriction enzymes I and II by a polymerase chain reaction using a first primer containing the enzyme I site and a second primer containing the enzyme II site, digesting the cDNA thus amplified with the restriction enzymes, enzyme I
and enzyme II and fractionating; and (5) ligating the cDNA fragment thus obtained upstream of a gene coding for a secretory protein or membrane protein having deleted therefrom the sequence coding for a signal peptide, integrating the ligated DNA
into a eucaryotic cell expression plasmid vector, and transforming the vector into a host.
The invention further provides a polypeptide having the amino acid shown in SEQ ID. No. 1, in substantially purified form, a homologue thereof or a fragment of the sequence or homologue of a fragment, and DNA encoding such a polypeptide. The polypeptide having the sequence shown in SEQ ID No. 1 has been identified using the process of the invention.

~~~~~~J

Fig. 1 is a conceptional view of the process for constructing a cDNA library according to the present invention.

Fig. 2 is a flow chart for the construction of a plasmid vector pcDL-SRa.

Fig. 3 is a conceptional view of the process for constructing an EcoRI-SacI fragment of hG-CSF.

Fig. 4 is a conceptional view of the process for constructing an SacI-KpnI fragment of hTac cDNA.

Fig. 5 is a flow chart for the construction of pSGT

and pSRT.

Fig. 6 is a conceptional view of the process for constructing an EcoRI-SacI fragment of hRARa.

Fig. 7 is an FAGS histogram showing the expression of a fusion pro tein hG-CSF-hTac on membrane.

Fig. 8 is an FAGS histogram showing the expression of a fusion pro tein hRAR-hTac on membrane.

Fig. 9 is a conceptional view of~the first half of the process for constructing the cDNA library of the Example.

Fig. 1 0 is a conceptional view of the second half of the process or constructing the cDNA library of the Example.

Fig. 11 is a hydrophobicity profile of (a part of) the polypeptide according to the present invention.

The first subject of the present invention is concerned with a process for efficiently constructing a cDNA library of signal peptides.
In one embodiment, the process for constructing a cDNA library of signal peptides according to the present invention comprises the following steps:
(1) synthesizing a single-stranded DNA from mRNA
isolated from the subject cells with the use of a random primer and adding oligo dT to the 3'-end of the single-stranded DNA thus obtained;

(2) synthesizing a double-stranded DNA from the single-stranded DNA obtained in (1) using as a primer a poly A oligomer ligated to a specific restriction enzyme (enzyme I) site;
(3) fragmenting the double-stranded DNA obtained in (2), fractionating the fragments obtained by size, ligating linker containing a specific restriction enzyme (enzyme II) site, differing from the enzyme I, thereto and fractionating again;
(4) performing a PCR using a first primer containing the enzyme I site and a second primer containing the enzyme II site, digesting the cDNA thus amplified with enzyme I
and enzyme II and fractionating; and (5) ligating the cDNA fragment upstream of the gene for a known secretory protein or membrane protein with the deletion of signal peptide and integrating the ligated DNA
into an eucaryotic cell expression plasmid vector, followed by transformation.
Fig. 1 is a conceptional view of the above-mentioned steps.
Now each of these steps will be illustrated in more detail.
In the step (1), if required, the subject cells are stimulated with an appropriate stimulating agent, and then the mRNA is isolated in accordance with known methods as described for example by Okayama H. et al., Methods in Enzymology, 154, 3 (1987).
As the subject cells, any cells may be used, so long as they have a possibility of producing a secretory protein or the like. For example, nerve cells and hematopoietic cells may be cited therefor. A single-stranded cDNA can be synthesized with the use of a random primer by methods known per se. A marketed random primer is available therefor. Subsequently, an oligomer such as oligo dT is added to the 3'-end of the single-stranded cDNA by using a ~.- ~~133~3 _ , _.
terminal deoxytransferase.
In the step (2), a double-stranded cDNA is synthesized by methods known per se. Any restriction enzyme sites may be used as the restriction enzyme (enzyme I) site to be ligated to the oligomer serving as a primer, such as poly A, and the restriction enzyme (enzyme II) site to be used in the next step (3), so long as they differ from each other. It is preferable to use EeoRI and Sacz, as enzyme I and enzyme II respectively.
to In the step (3), the double-stranded DNA is fragmented for example by ultrasonication so as to give an average cDNA length of 300 by and the obtained fragments are fractionated into cDNAs of 200 to 500 by by agarose gel electrophoresis (AGE). Attar blunting with T4DNA
polymerase, enzyme II is ligated and the cDNAt are fractionated into DNAs of 200 to 500 by again by agarose gel electrophoresis. As described above, any enzyme may be used as the enzyme II, so long as It differs from the enzyme I. The procedure in this step increases the likelihood that a cDNA fraymsnt containing a signal peptide exists in the part located between the enzymes I and zI.
In the step (4), PCR is carried out in order to further elevate the possibility that a cDNA fragment containing a signal peptide exists in the part located between tho enzymes I and II. PCR is a wall known technique and automated devices therefore are commercially available. It is sufficient to amplify 25 to 30 times.
The cDNA thus amplified is digested with enzyme I and enzyme II and electrophorased on an agarose gel to thereby fractionate into cDNAs of 200 to 500 bp.
Zn the step (5), a gene for a known secretory protein or the like with the deletion of signal peptide, whicri is called a reporter gene, and a cDNA .fragment obtained in the above (4) are integrated into an euoaryotic cell expression plasmid vector in such a manner that the cDNA fragment is located upstream of the reporter gene. This is followed by ''' ~1133~3 _8_ transformation of the vector into a host.
Various known eucaryotic cell expression plasmid vectors for example, pcDL-SRa and pcEV-4 which are capable of acting in Escherichia coli, are usable in the present invention.
As the reporter gene, genes for mature protein parts of soluble secretory proteins and membrane proteins of any type are usable. The expression of these reporter genes may be confirmed by known methods such as antibody methods.
Human IL-2 receptor a gene is especially suitable as a reporter gene.
A number of E. coli strains are known as hosts for transformation and any of these stains is usable. It is preferable to use DH5 competent cells [described in Gene, 15~ 96, 23 (1990)] therefor. Transformants may be incubated in a conventional manner and thus the cDNA library of the present invention can be obtained.

_ L, _ g _ In the process for constructing a cDNA library according to the present invention, there is a high possibility that gene fragments coding for signal peptides are contained in the library. However, not every clone contains said fragment. Further, not all of the gene fragments code for unknown (novel) signal peptides. It is therefore necessary to screen a gene fragment coding for an unknown signal peptide from said library.
Namely, the cDNA library is divided into small pools of an appropriate size and integrated into an expression system. Examples of the expression system for producing a polypeptide include mammalian cells (for example, monkey COS-7 cells, Chinese hamster CHO cells, mouse L cells, etc.).
Transfection may be performed in accordance with well known methods such as the DEAE-dextran method. After the completion of the incubation, the expression of the reporter gene is examined. It is known that a reporter gene would be expressed even though the signal peptide is the one characteristic to another secretory protein. That is to say, the fact that the reporter gene has been expressed indicates that a signal peptide of some secretory protein has been integrated into the library. Positive pools are further divided into smaller ones and the expression and the judgement are repeated until a single clone is obtained. The expression of the reporter gene can be judged by, for example, fluorescence-labeled antibody assay, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), depending on kinds of the employed reporter gene.
Next, the nucleotide sequence of the isolated positive clone is determined. In the case of a cDNA which is proved to code for an unknown protein, the clone of the full length is isolated with the use of the cDNA as a probe and the full nucleotide sequence can be thus identified. All of these P.W ~ e~ ~I ~'1~~'I ~ ~~ v operations are carried out by methods which are well known by those skilled in the art. For example, the nucleotide sequence may be identified by the Maxam-Gilbert method or the dideoxy terminator method. On the other hand, the full length may be sequenced in accordance with a method described in Molecular Cloning [Sambrook, J., Fritsch, E.F. and Maniatis, T. published by Cold Spring Harbor Laboratory Press in 1989].
The present invention further relates to a novel polypeptide which has been identified using the process of the present invention to construct a cDNA
library and DNA coding for the same. In particular, it relates to:
(1) a polypeptide consisting of an amino acid sequence representing by SEQ ID No. 1;
(2) a DNA coding for the polypeptide described in the above (1);
(3) a DNA having a nucleotide sequence represented by SEQ ID
No. 2; and (4) a DNA having a nucleotide sequence represented by SEQ ID
No. 3.
A polypeptide of Seq. ID No. 1 in substantially purified form will generally comprise the polypeptide in a preparation in which more than 90%, eg.
95%, 98% or 99% of the polypeptide in the preparation is that of the Seq. ID
No. 1.
A polypeptide homologue of the Seq. ID No. 1 will be generally at least 70%, preferably at lest 80 or 90% and more preferably at least 95%
homologous to the polypeptide of Seq. ID No. 1 over a region of at least 20, preferably at least 30, for instance 40, 60 or 100 more-contiguous amino acids. Such polypeptide homologues will be referred to below as a polypeptide according to the invention.
Generally, fragments of Seq. ID No. 1 or its homologues will be at least 10, preferably at least 15, for example 20, 25, 30, 40, 50 or 60 amino acids in length, and are also encompassed by the term "a polypeptide according 2~~33~J
to the invention" as used herein. Particular fragments of the polypeptides of the invention are fragments of which include amino acid residues numbered 1-'70 in Seq ID No. 4 or a homologue thereof.
A DNA cepsble of selectively hybridizing to.the DNA
vg geq. ID No. 2 oz 3 will be generally at least 70~, preferably at least 80 or 90~ and more preferably at least 95~ homologous to the DNA of Seq. iD No. Z or 3 over a region of at least 20, preferably at least 3G, for instance l0 40, 60 or 100 or more contiguous nucleotides. Such DNA
will be encompassed by the term "DNA according to the invention".
particular DNA capable of selectively hybridising to the DNA of SEQ ID No. 2 or 3 is the nucleotide residues i5 numbered 139-348 in SEQ ID No. 4 or a fragment thereof.
DNA according to the invention may be used to produce a primer, cg. a PCR Primer, a probe cg. labelled by conventional means using radioactive or non-radioactive labels, or the DNA may be cloned into a vector. Such 2o primers, probes and other fragments of the DNA of Seq. ID
rto. Z or 3 will be at least 15, preferably at least 20, for example Z5, 30 or 40 nucleotides in lengthy and are also encompassed by the term "DNA according to the invention" as used herein.
25 DNA according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art.
A further embodiment of the invention provides 3o replication and expression vectors comprising DNA according to the invention. Tho vectors may be, for exampl~.
piasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said DNA and optionally a regulator of the promotor.
35 The vector may contain one or more selectable marker genes, for example an ampicillin resistance gene. The vector may be used jn~. for example for the production of RNA
corresponding to the DNA, or used to transform a host cell.
A further embodiment of the invention provides hose "~, 211~3~3 cells transformed or transfected with the vectors for the replication and expression of DNA according to the invention, including the DNA Seq. ID No. 2 or 3 or the open reading frame thereof. The cells will be chosen to be compatible with the vector and may for example be bacterial, yeast, insect or mammalian.
DNA according to the invention may also be inserted into the vectors described above in an antisense orientation in order to provide for the production of antisense RNA (DNA). Antisense RNA (DNA) may also be produced by synthetic means. Such antisense RNA (DNA) may be used in a method of controlling the levels of a polypeptide of the invention in a cell.
A further embodiment of the invention provides a method of producing a polypeptide which comprises culturing host cells of the present invention under conditions effective to express a polypeptide of the invention.
Preferably, in addition, such a method is carried out under conditions in which the polypeptide of the invention is expressed and then secreted from the host cells.
The invention also provides monoclonal or polyclonal antibodies to a polypeptide according to the invention.
The invention further provides a process for the production of monoclonal or polyclonal antibodies to the polypeptides of the invention. Monoclonal antibodies may be prepared by conventional hybridoma technology using a polypeptide of the invention or a fragment thereof, as an immunogen.
Polyclonal antibodies may also be prepared by conventional means which comprise inoculating a host animal, for example a rat or a rabbit, with a polypeptide of the invention and recovering immune serum.
The present invention also provides pharmaceutical compositions containing a polypeptide of the invention, or an antibody thereof, in association with a pharmaceutically acceptable diluent or carrier.
The invention also provides a polypeptide according ~ ~~~33~~

to the invention or an antibody for use in a method of therapy or diagnosis on a human or animal body.
The polypeptides of the present invention include not only those having the amino acid sequence represented by the SEQ ID No. 1 but also those with partial deletion thereof (for example, a polypeptide consisting of the mature protein part alone, or consisting of a part of the mature protein essentially required for the expression of the biological activity), those with partial replacement by other amino acids) (for example, a polypeptide some of amino acids are replaced by those having similar properties) and those with partial addition or insertion of amino acid(s).
It is well known that there are up to six different codons which may code for a single amino acid (for example, one type of codon for Met while six types of - 14 - 21133~'~
_ ~
n for leu). Accordingly, the nucleotide sequence of the DNA can be codo chap ed without altering the amino acid sequence of the poiYPeptide.

The DNA as specif led in ( 2? includes , . .
ii nucleotide sequences coding for the potypeptide represented by a z0 No, 1. Changes in the nucleotide sequence sometimes bring about S q.
an increase in the polypeptide productivity.
The DNA as specified in (3) is an embodiment of the DNA as specified in (2) and represents a natural sequence.
The DNA as specified in '(4} represents a sequence wherein a natural non-translational region is added to the DNA as specified in (~).
A si nal peptide is a highly hydrophobic region located immediately g wnstream of the translation initiation amino acid Met. it is assumed that the do si nal peptide in the polypeptide of the present invention resides in a region g from Met at the 1-position to Ser at the t9-position in the amino acid ranging once represented by Seq ~ xD No. 1. The region essentially sequ r nsible for the expression of the biologicat activity corresponds to the pan espo amino acid sequence of the seq. ~D No. ~ lacking of the .signal of the a tide, i.e., the mature protein part. Thus the signal Peptide never relates to PP
the activity.
The DNA having the nucleotide sequence represented by ge ID No. 3 can be prepared in accordance with the process described as q.
the first subject of the present invention.
Once the nucleotide sequences represented by seq . ID No. 2 nd No. 3 are determined, the DNA of the present invention can be chemically a nthesized. Atternativeiy, the DNA of the present invention can be obtained sy '~"" - 15 _ 2~~~e~3~~
by chemically synthesizing fragments of said nucleotide sequence and hybridizing with the use of the fragments as a probe. Further, the target DNA
can be produced in a desired amount by introducing a vector DNA containing said DNA into an appropriate host and then incubating the host.
Examples of methods for obtaining the polypeptide of the present invention include:
(1 ) isolation and purification from vital tissues or cultured cells;
(2) chemical synthesis of peptides; and (3) _production with the use of gene recombination techniques_ From an industrial viewpoint, the method described in (3) is preferable.
Examples of the expression system (host-vector system) for producing the polypeptide by using gene recombination techniques include those of bacteria, yeasts, insect cells and mammalian cells.
In order to express in E. coli, for example, an initiator codon (ATG) is added to the 5'-end of the DNA coding for the mature protein region. The DNA thus obtained is then ligated to the downstream of an appropriate promoter (for example, trp promoter, lac promoter, ~.p~ promoter, T7 promoter, etc.) and inserted into a vector capable of functioning in E. coli (for example, pBR322, pUClB, pUCl9, etc.), thus constructing an expression vector. Next, an E. coli strain (for example, E. coli DH1, E. coli JM109, E. coli HB101, etc.) transformed with this expression vector is incubated in an appropriate medium. Thus the target polypeptide can be obtained from the incubated cells. Alternately, a bacterial signal peptide (for example, a signal peptide of pelB) may be used and thus the polypeptide can be secreted into the periplasm. Furthermore, a fusion protein together with other polypeptide can be produced.

2~~.~~~3 Expression in mammalian cells can be effected, for example, in the following manner. Namely, a DNA coding for the nucleotide sequence represented by seq. I~ No. 3 is inserted into the downstream of an appropriate promoter (for example, SV40 promoter, LTR promoter, metallothionein promoter, etc.) in an appropriate vector (for example, retrovirus vector, papilloma virus vector, vaccinia virus vector, SV40-series vector, etc.), thus constructing an expression vector. Next, appropriate mammalian cells (for example, monkey COS-7 cells, Chinese hamster CHO
cells, mouse L cells, etc.) are transformed with the expression vector obtained above and the transformant is incubated in an appropriate medium. Thus the target polypeptide can be secreted into the culture medium. The polypeptide thus obtained can be isolated and purified by conventional biochemical methods.
By using the process for constructing a cDNA library which is the first subject of the present invention, a DNA coding for a signal peptide of a secretory protein or a membrane protein can be efficiently selected and, in its turn, an unknown and useful protein can be efficiently found out. The novel polypeptide which is the second subject of the present invention is produced and secreted from a stroma cell line. Therefore, the polypeptide has biological activities relating to the survival and proliferation of hematopoietic stem cells and the proliferation and differentiation of B cells and myeloid cells, and chemoattractant activity of neurophil. Accordingly, the polypeptide of the present invention per se is usable as an agent for preventing or treating, for example, anemia or leukopenia, infections, etc.

~.. _ ~~ _ 211~~~3 In addition, the above-mentioned polypeptide existing in vivo can be assayed by using a polyclonal antibody or a monoclonal antibody for said polypeptide, which is applicable to studies on the relationship between said polypeptide and diseases or to the diagnosis of diseases and the like. The polyclonal antibody and the monoclonal antibody can be prepared by a conventional method with the use of said polypeptide or a fragment thereof as an antigen.
The DNA according to the present invention serves as an important and essential template in the production of the polypeptide of the present invention _ which is expected to be highly useful. Further, the DNA of the present invention is applicable to the diagnosis and treatment of hereditary diseases, i.e., gene therapy, and therapy with ceasing the expression of the polypeptide by using antisense DNA (RNA). Furthermore, a genomic DNA can be isolated by using the DNA of the present invention as a probe. Similarly, a human gene for a related polypeptide being highly homologous with the DNA of the present invention and a gene of an organism other than human for a polypeptide being high homologous with the polypeptide of the present invention can be isolated.
Examples The following Examples and Reference Example are illustrated, but not limit the present invention.
Reference Example 1 Construction and expression of plasmid pcDt_-SRa-h-G-CSF-hTac (pSGT) and plasmid pcDL-SRa-hRARa-hTac (pSRT) A plasmid, wherein a cDNA coding for a fusion protein of hG-CSF
(human granulocyte colony stimulating factor, a typical example of a protein ~- ~8r- X113363 ~"' ~ having a signal peptide) or hRARa (human retinoic acid receptor a, a typical example of a protein having no signal peptide), with hTac (human IL-2 receptor a, used as a reporter gene) was integrated into an eucaryotic cell expression plasmid vector pcDL-SRa having an SRa promoter [described in Mol. Cell. Biol., $, 466 (1988), provided by Dr. Yutaka Takebe, National Institute of Health], was constructed. After transformation, the expression of the reporter protein on the membrane was examined.
(1 ) By employing a plasimd pSP72-hG-CSF, wherein hG-CSF cDNA
had been integrated into the EcoRl site of a plasmid pSP72 (purchased from Promega), as a template and using an SP6 promoter primer (purchased from Takara Shuzo Co., Ltd.) and an hG-CSF specific primer having an Sacl site added thereto, 5' GGeIGATATC GA, GCTCCTCGGGGTGGCACAG 3~
EcoRV ~~Saet 1 ~G~CSr cONI~ antisensa PCR was performed 25 cycles (at 95 °C for one minute, at 48 °C for two minutes and at 72 °C for two minutes). The amplified DNA fragment was digested with SacIrEcoRl and once subcloned into a plasmid pBlue script SK(+) (pBS). After digesting with Sacl-EcoRl again, an EcoRl-Sacl fragment of hG-CSF was obtained. On the other hand, a plasmid pBS-hTac, wherein hTac cDNA had been integrated into the Hindlll site of pBS, was digested with Sacl-Kpnl to thereby give an Sacl-Kpnl fragment of hTac cDNA with the deletion of the signal sequence. These fragments were integrated into the EcoRl-Kpnl site of pcDL-SRa with the deletion of stuffer (Fig. 2) to thereby give a plasmid pcDL-SRa-hG-CSF-hTac (pSGT) (Figs. 3, 4 and 5).
,.~

21~.~3~~

Next, by employing a plasmid pGEM3-hRARa, wherein hRARa cDNA
had been integrated into the EcoRl site of a plasmid pGEM3, as a template and using an SP6 promoter primer and an hRARa specific primer having an Sacl site added thereto, 5' GGA ATAT A T AATGGTGGCTGGGGATG 3' EcoRV Sacl a ' hRARa cDNA antisense PCR was performed. Subsequently, the procedure employed in the above-mentioned case of G-CSF was repeated to thereby give a plasmid pcDt_-SRa-hRARa-hTac (pSRT) (Figs. 6, 4 and 5).
(2) The pSGT or pSRT obtained in the above (1 ) was transfected into COS-7 cells by the DEAE-dextran method [described in detail in Current Protocol in Molecular Biology, chapter 9.2.1.]. After 48 hours, the cells were harvested from a dish and incubated together with mouse anti-Tac IgG
antibody for 20 minutes on ice. After eliminating free antibodies, the mixture was incubated together with goat anti-mouse IgG antibody labeled with fluorescein isothiocyanate (FITC) for 20 minutes on ice. After eliminating free antibodies again, the expression of a fusion protein G-CSF-Tac or RARa-Tac on the membrane was examined with a fluorescence activated cell sorter (Model FACS Can, manufactured by BECTON DICKINSON, hereinafter referred to simply as FACS). Figs. 7 and 8 show the results of the judgement.
As shown in Fig. 7, G-CSF-Tac was expressed on the membrane as well as Tac. As shown in Fig. 8, on the other hand, RARa-Tac was not detected on the membrane but remained within the cells. These results indicate that when a cDNA fragment containing a signal peptide is ligated to the upstream of a reporter gene, said reporter protein is expressed on the cell 211~3~'~
~"'' - 20 -membrane, while when a cDNA fragment having no signal peptide is ligated, the reporter protein is not expressed.
Example 1 Construction of cDNA library having selectivity for signal peptides (Figs. 9 and 10) Total RNA was extracted from a mouse stroma cell line ST2 [cells supporting the survival and proliferation of hematopoietic stem cells and the proliferation and differentiation of B cells and myeloid cells; refer to EMBO
J., 7, 1337 (1988)] by the acid guanidine-phenol-chloroform (AGPC) method [described in detail in "Saibo Kogaku Jikken Protokoru (Protocol in Cellular Engineering Experiments)", published by Shujun-sha, 28 - 31]. Then poly A-RNA was purified by using oligo (dT)-latex (Oligotex-dT30~, marketed from Takara Shuzo Co., Ltd.). By using a random hexamer as a primer, a single-stranded cDNA was synthesized with reverse transcriptase and dT was added to the 3'-end thereof with the use of terminal deoxytransferase. A 17 mer dA
ligated to a restriction enzyme site containing EcoRl 5' GATrC:GGCCGC CTCGAG GAATTC (dA)~~ 3' Notl Xhol EcoRl was annealed and a double-stranded cDNA was synthesized by using the same as a primer. Then the cDNA was fragmentated by ultrasonication so as to give an average length of 300 by and the cDNAs of 200 to 500 by were fractionated by agarose gel electrophoresis. After blunting the ends with T4DNA polymerase, a lone linker containing an Sacl site 5' GAGGTACAAGCTT GATATC GAGCTCGCGG 3' 3~ CATGTTCGAA CTATAG CTCGAGCGCC 5~
Hind III EcoRV Sacl r - ~' [see Nucleic Acids Res., 1$, 4293 (1990)] was li~'a~~ ~rt~d c~NAs of 200 to 500 by were fractionated again by agarose gel electrophoresis. By using a primer (NLC) containing an EcoRi site N LC
5' GATGCGGCCGCCTCGAGGAATTC 3' and another primer (LLHES) containing an Sacl site LLHES
5' GAGGTACAAGCTTGATATCGAGCTCGCGG 3' PCR was performed 25 cycles (at 94 °C for one minute, at 50 °C for two minutes and at 72 °C for two minutes). The amplified cDNA was digested with Sacl and EcoRl and cDNAs of 250 to 500 by were fractionated by agarose gel electrophoresis. The cDNA was ligated to a plasmid obtained by digesting pSRT (prepared in Reference Example 1 ) with Sacl and EcoRl by using T4 DNA ligase. After transformation of an E. colt DHSa strain, a cDNA library having a selectivity for signal peptides was obtained.
Example 2 Screening and analysis of cDNA coding for signal peptide About 1,200 colonies in the library obtained in Example 1 were divided into 24 pools (about 50 colonies/pool). Plasmids of each pools were isolated by the miniprep method and transfected into COS-7 cells by the DEAE-dextran method. After 48 to 72 hours, cell surface-staining for Tac of the transfectant was performed in the same manner as described in Reference Example 1 and 6 positive pools were selected under a fluorescent microscope. Colonies of one pool from among the 6 positive pools were further divided and the same procedure as described above was repeated until a single clone was obtained. Thus a positive clone (pS-TT3) was obtained. Subsequently, by using two synthetic primers 5~ TTTACTTCTAGGCCTGTACG 3 (20 bases upstream from EcoRI cloning'site, for sense) and 5' CCATGGCTTTGAATGTGGCG 3~
(20 bases downstream from Sacl cloning site, for antisense) which were specific for the pcDt_-SRa-Tac vector, the nucleotide sequence of the TT3 insert was determined. An open reading frame following the Tac cDNA with the deletion of the signal sequence in-frame was searched and converted into the deduced amino acid sequence. After performing a hydrophobicity profile, it was confirmed that a hydrophobic region characteristic to a signal peptide was contained therein (Fig. 11 ). Further, the homology with data base on DNA and amino acid levels was examined. As a result, it has been found out that TT3 clone codes for an unknown protein.
Example 3 , Screening of cDNA with the full length and determination of nucleotide sequence A cDNA library was constructed by using Super Script~ Ramda System (marketed from BRL). Next, pS-TT3 was digested with Sacl and EcoRl and a TT3 cDNA fragment was prepared by agarose gel electrophoresis. The library was screened by using an oligo-labeled TT3 cDNA fragment as a probe and thus a number of positive clones were obtained. Among these clones, a TT3-1-6 clone showing the longest insert was selected and an Sall-Notl fragment excised from a ~.gt22A vector was subcloned into pBS to thereby give a plasmid p8S-TT316. By using a T7 primer, the nucleotide sequence of 300 by in the 5'-terminal of TT3-1-6 cDNA was determined. Thus it was confirmed 2~133~3 - ~"..' - 2 3 -that the sequence identical with TT3 of the probe existed in the most 5'-end of TT3-1-6.
Next, a number of pBS-TT316 variant plasmids lacking of the 5'-end or the 3'-end of TT3-1-6 cDNA were constructed by using an Exolll/Mung Bean Deletion Kit (manufactured by Stratagene). By using these variant plasmids, the nucleotida sequence of the full length of the cDNA was determined (sequence No. 3). From the full length cDNA sequence data, an open reading frame was determined and further translated into an amino acid sequence.
Thus the sequence represented by seq. ~n :- No. 1 was obtained. The amino acid sequence at the 30- to 40-positions in the N-terminal of the amino acid sequence thus obtained were compared with known signal peptides.
Thus the signal peptide part of this polypeptide was deduced to thereby give the sequence represented by seq. z~ No. 4.

SEQUENCE LISTING
GENERAL INFORMATION:
APPLICANT:
NAME: Ono Pharmaceuticai Co., Ltd.
STREET: 1-5 Doshomachi 2-chome CITY : Osaka COUNTRY: Japan POSTAL CODE (ZiP): 5a~
TITLE OF THE INVENTION:
Process for constructing cDNA library, and novel polypeptide and DNA coding for the same NUM6ER OF SEQUENCES: 4 SEQUENCE N0: 1 LENGTH: 89 amino acids TYPE: amino acid TOPOLOGY: linear tdOLECULE TYPE: protein SEQUENCE: seq. ID No. 1 Met Asp Ala Lys Val Val Ala Val Leu Ala Lsu Val Leu Ala Ala Leu Cya Its Ssr Asp G1Y LYa Pro Val Ser L~u Ser Ty= Arg Cye Qro Cys '0 25 30 Arg Phe phe Glu Ssr liie Ile Ala Arg Ala Aan Val Lya Hie Lsu Lys ile Leu Asn Thr Fro Asn Cys Ala Leu Gln Ile Val Ala Arg Lsu Lys Asn Asn Asn Arg Gln Val Cys Ile Asp pro Lys Leu Lys Trp Ile Gln .
65 70 ~5 s0 Glu Tyr Lsu Glu Lys Ala Leu Asn Lya - ~ - 25 -SEQUENCE NO: 2 LENGTH: 267 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: cDNA to mRNA
SEQUENCE: Seq. ID No. 2 GCACGGCTGA AGAACAACAA CAGACAAGTG TGCATTGACC. CGAAATTAAA GTGGATCCAA 240 SEQUENCE NO: 3 LENGTH: 1797 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: cDNA to mRNA
SEQUENCE: seq. ID No. 3 ~1~33~3 TTATATGCAC TAGCAATAAA ATGCTAATTG TTTCATGCTG TA,AAAAAAAA AAAAAAA 1797 SEQUENCE NO: 4 LENGTH: 1797 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: cDNA to mRNA
ORIGINAL SOURCE
ORGANISM: Mouse CELL LINE: ST2 FEATU RE
NAME/KEY: CDS
LOCATION: 82 .. 351 IDENTIFICATION METHOD: P

- ~~~~4~'3 NAMEIKEY: sig peptide LOCAT ION : 82 .. ~ 38 IDENTIFICATION METHOD: S
NAMEIKEY: mat peptide LOCATION:139 .. 348 IDENTIFICATION METHOD; S
SEQUENCE: Seq. ID No. 4 GACCACTTTC CCTCTCGGTC CACCTCGGTG TCC?CTTGCT GTCCAGCTCT GCAGCCTCCG 60 ~C,CGCCC?C CCGCCCACGC C ATG GAC GCC AAG GTC GTC GCC G?G CTG GCC 111 Mst Asp Ala Lys Val Val Ala Val ~u Ala ~u Val Leu Ala Ala Irsu Cye Its Ssr Asp Gly Lye Pro Val asr Lou Ser Tyr ArQ Cya Pro Cys Arq ?hs Phs Glu Ssr His Its Ala Arg Ala 15 20 .
AAC GTC AI~G CAT CTG AM ATC CTC AAC ACT CCA AAC TGT GCC CTT CAG 255 Aan Val Lya His Leu Lya I11 Lsu Aan Thr Pro Aen Cys Ala Leu Gln ile v~l Ala Arq Iwu Lys Aan Asn Aan Arg Gln Val Cye Its Aap Pro AAA TTA AAG TGG ATC CAA GAG TAC CTG G1~G AAA GC? TTA AAC AAG T1~ 351 Lys leu Lya T=P Ile Gln Glu Tyr Lsu Glu Lya Ala Lsu Aen Lya GCACAACAGC CCAA1~GGACT TTCCAGTAGA CCCCCGAGGA AGGCTGACAT CCGTGGGAGA 411 ACTGJ~CTGGG GTCATGCTAA GGTTTGCCAG CATAAAGACA CTCCGCCATA GCATATGG:A 531 CGI~TATTGCA GCTTATATTC ATCCCTGCCC TCGCCCGTGC ACAATGGAGC TTTTATRAC? 591 CCTCATCTTC ATTTTAAAAA GCAGTGATTA CTTCAAGGGC TG?ATTCAGT T?GCTTTGGA ~~1 GCTTCTCTT? GCCCTGGGGC CTCTGGGCAC AGTTATAGAC GGTGGCTT?G CAGGGAGCCC 771 TAGAGAGAAA CCT?CCACCA GAGCAGAGTC CGAGGAACGC TGCAGGGCTT GTCCTGCAGG 831 GGGCGCTCC? CGACAGATGC CTTGTCCTGA GTCAACACAA GATCCGGCAG AGGGAGGCTC 891 ACTGTGACAT TATATGCACT AGCAATAAAA TGCTAATTGT TTCATGCTGT Pu4AAAAAAAA 1791 AAAAAA

Claims

-29-

1. A process for constructing a cDNA library enriched for cDNAs coding for signal peptides comprising:
(1) synthesizing single-stranded DNA complementary to mRNA isolated from subject cells and ligating to the 3'-end of the single-stranded DNA, a DNA oligomer of known sequence;
(2) converting the single-stranded DNA obtained in (1) to a double-stranded DNA using as primer an oligomer ligated to a first restriction enzyme (enzyme I) site, the primer sequence being complementary to the oligomer of known sequence ligated to the single-stranded DNA;
(3) fragmenting the double-stranded DNA obtained in (2), fractionating the fragments obtained by size and ligating linker containing a second restriction enzyme (enzyme II) site, differing from the enzyme I, to fragments of 200 to 500 base pairs;
(4) amplifying fragments containing the sites of restriction enzymes I and II by polymerase chain reaction using a first primer containing the enzyme I site and a second primer containing the enzyme II site, digesting the cDNA thus amplified with the restriction enzymes, enzyme I and enzyme II, and fractionating by size; and (5) ligating the cDNA fragments of 204 to 500 base pairs thus obtained upstream of a gene coding for a secretory protein or membrane protein having deleted therefrom the sequence coding for a signal peptide, integrating the ligated DNA into a eucaryotic cell expression plasmid vector, and transforming a host cell with the vector.

2. A process according to claim 1 wherein, in step (1), the single-stranded DNA is synthesised from mRNA isolated from the subject cells with the use of a random primer and the DNA oligomer of known sequence is oligo dT and, in step (2), the primer is a polyA oligomer ligated to the restriction enzyme (enzyme I) site.

3. A process according to claim 1 or 2, wherein EcoRI is used as the enzyme I, SacI is used as the enzyme II, and human IL-2 receptor .alpha. gene is used as a knows secretory protein or membrane protein gene with the deletion of signal peptide.