JP6998056B2 - A new method for identifying proteins using yeast and a fragmented cDNA library - Google Patents

Description

The present invention relates to, for example, a method for identifying proteins using yeast and a fragmented cDNA library.

It is known that the association or binding of a plurality of molecules inside and outside the cell acts as a biological signal that controls cell proliferation, differentiation, adhesion, and the like. In particular, identification of receptor molecules on the cell membrane is useful for elucidating the physiological actions of various ligand molecules such as hormones. For these purposes, a method of concentrating a protein complex containing a receptor molecule by immunoprecipitation or the like and performing mass spectrometry, a panning method using cultured cells, or the like is becoming popular. However, many "orphan ligands" with unknown receptors are still known. The receptor identification has been attracting attention because it is highly likely to be related to various pathological conditions, but it has not progressed due to the complexity of the screening method and the lack of detection sensitivity.

On the other hand, the yeast two-hybrid method is known as a method for identifying the binding of intracellular (cytoplasmic) molecules (Non-Patent Documents 1 to 3). In the yeast two-hybrid method, a known gene to which a nuclear translocation signal is added using a gene vector and a full-length cDNA chimeric gene to which a nuclear translocation signal is added (usually reverse-transcribed with an oligo dT primer in commercial products) are 2 When expression is performed and a bond between molecules is formed in the nucleus, the bond between molecules is detected by the activity of the reporter gene whose expression is induced, luminescence, or the like.

In the yeast two-hybrid method, when a gene encoding a membrane protein such as a ligand molecule or a receptor is inserted into a vector, the chimeric gene is nucleated by expressing the signal peptide at the 5'end or the transmembrane region near the 3'end. It is difficult to express within, and the conventional yeast two-hybrid method is not suitable for analysis of ligand-receptor binding.

In addition, many modifications of the yeast two-hybrid method have been reported. For example, the cells used may be changed to organisms other than yeast, or the reporter gene portion may be developed for improvement. However, in the past, modifications of the yeast two-hybrid method intended to be expressed in the nucleus as a partial region library of (membrane) proteins and to detect interactions that originally occur extracellularly in the nucleus have been known. There wasn't.

Young, K., Biol. Reprod., 1998, 58 (2), pp. 302-11 Joung, J. et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97 (13), pp. 7382-7 Fields, S. and Song, O., Nature, 1989, 340 (6230), pp. 245-246

As mentioned above, the expression of the signal peptide at the 5'end and the transmembrane region near the 3'end makes the conventional yeast two-hybrid method unsuitable for analysis of ligand-receptor binding. Therefore, it is an object of the present invention to modify the yeast two-hybrid method to provide a method suitable for analysis of a binding between a protein containing a ligand and a receptor.

As a result of diligent research to solve the above problems, unlike the conventional yeast two-hybrid method (preparation by reverse transcription with an oligo dT primer), when preparing a cDNA library, a random primer is used from any location. The fragmented cDNA library is obtained by performing the yeast two-hybrid method using a fragmented cDNA library in which a back-transcribed cDNA or a cDNA that has been intentionally fragmented by cleavage by ultrasonic treatment is inserted into a vector. Contains a signal peptide at the 5'end and a clone lacking the transmembrane region near the 3'end, even if it is a membrane protein, so that it can be stably expressed in the nucleus and extracellularly. The present invention was completed by reproducing the binding between the ligand and the extracellular part of the receptor in the nucleus and finding that binding analysis that could not be performed by the conventional method becomes possible in the process of detecting the binding using the reporter gene. Came to do.

That is, the present invention includes the following.
(1) A method for identifying a protein, which comprises a step of culturing a yeast transformant into which the first and second vectors have been introduced, wherein the first vector is a gene encoding a nuclear translocation signal and a DNA-binding protein. The second vector contains a gene encoding a nuclear translocation signal, a gene encoding a transcriptional activation protein, and a fragmented partial cDNA, which comprises a gene encoding a decoy protein and a gene encoding a decoy protein. The transformed partial cDNA encodes a prey protein lacking a signal peptide, a cell membrane or an intracellular small organ localized sequence or a transmembrane region, and in the nucleus of the yeast transformant, in the nucleus. A fusion protein containing a translocation signal, a DNA-binding protein, and a decoy protein, and a fusion protein containing the nuclear translocation signal, a transcriptional activation protein, and a prey protein encoded by fragmented partial cDNA are expressed, and the decoy protein is expressed. The method for detecting the binding between the protein and the prey protein using the activity of the reporter gene as an index.
(2) The method according to (1), wherein the decoy protein is a ligand and the prey protein is a receptor protein.
(3) The method according to (1), wherein the decoy protein is a protein encoded by a partial cDNA excluding various localized signal sequences.
(4) A cDNA having a length of 100 to 1800 bp obtained by random prime reverse transcription.
(5) The cDNA according to (4), which has a length of 100 to 1000 bp.
(6) A vector containing a gene encoding a nuclear translocation signal, a gene encoding a transcriptional activation protein, and a fragmented partial cDNA, wherein the fragmented partial cDNA is described in (4) or (5). The vector, which is a cDNA.
(7) A reagent kit for identifying a protein according to any one of (1) to (3), which comprises the cDNA according to (4) or (5) or the vector according to (6).

This specification includes the disclosure of Japanese Patent Application No. 2016-112107, which is the basis of the priority of the present application.

According to the present invention, it is possible to contribute to elucidation of signal molecules involved in various diseases by elucidating receptors for orphan ligands and searching and identifying molecules to which membrane proteins bind.

The method for identifying a protein according to the present invention (hereinafter referred to as "the method") comprises a step of culturing a yeast transformant into which the first and second vectors have been introduced, wherein the method comprises the same.
The first vector contains a gene encoding a nuclear translocation signal, a gene encoding a DNA binding protein, and a gene encoding a bait protein.
The second vector contains a gene encoding a nuclear translocation signal, a gene encoding a transcriptional activation protein, and a fragmented partial cDNA, which is a signal peptide, cell membrane or intracellular small organ. It encodes a prey protein lacking a localized sequence or transmembrane region and is
In the nucleus of the yeast transformant, a fusion protein containing the nuclear translocation signal, a DNA-binding protein, and a decoy protein, and a prey encoded by the nuclear translocation signal, a transcriptional activation protein, and fragmented partial cDNA. A fusion protein containing a protein is expressed, and the binding between the decoy protein and the prey protein is detected using the activity of the reporter gene as an index.
The method.

In general, in the conventional yeast two-hybrid method, a fusion protein of a DNA-binding protein and a molecule of interest (decoy protein) and a cDNA library (derived) molecule (prey protein) consisting of a transcriptional activation protein and full-length cDNA are used. The binding of the decoy protein to the library-derived molecule is based on the activity of the reporter gene, which induces expression when the decoy protein is bound to the library-derived molecule by expressing the fusion protein in yeast. Is detected.

The yeast two-hybrid method has become widespread as an inexpensive large-scale screening method using the binding of two molecules as an index. However, when a protein derived from a cDNA library contains a signal peptide, a sequence localized in a cell membrane or various intracellular small organs, the relevant molecule cannot be expressed in the nucleus, and thus a bond with a decoy protein is formed in the nucleus. The expression of the reporter gene cannot be detected. Therefore, the yeast two-hybrid method is generally used as a method for detecting the binding between intracellular proteins. In addition, the library commercially available in the past is a library in which a (almost full-length) cDNA reversely transcribed and synthesized from the 3'end side of mRNA using an oligo dT primer is inserted.

On the other hand, since this method uses a cDNA library in which a fragmented cDNA is intentionally inserted, a partial region of a molecule containing a membrane protein or various localized sequences that was overlooked in the conventional method is expressed in the yeast nucleus. By doing so, the binding to the decoy protein can be detected, and the ligand-receptor binding that normally occurs outside the cell and the association between membrane proteins both inside and outside the cell can be detected in the nucleus of yeast.

In this method, first, the first and second vectors are prepared.

The first vector contains a fusion gene that functionally links a gene encoding a nuclear translocation signal, a gene encoding a DNA binding protein, and a gene encoding decoy proteins. The first vector may be any vector that functions in yeast, and examples thereof include pBTM116, pGBKT7, and pGBT9.

In the first vector, a nuclear localization signal is used to transfer a fusion protein encoded by a fusion gene containing the above-mentioned gene encoding a DNA-binding protein and a gene encoding decoy proteins into the nucleus of yeast. The encoding gene is included or preliminarily added to the fusion gene (for example, Large T antigen residues 47 to 54 (PKKKRKVE: SEQ ID NO: 1), endogenous nuclear localization signal sequence of LexA protein (KRLKK: SEQ ID NO: 2), etc.). Must have been.

Examples of the DNA-binding protein include the DNA-binding domain (DBD) of the Gal4 protein and the DBD of the LexA protein.

Further, as decoy proteins, for example, orphan ligands having unknown receptors, proteins encoded by partial cDNA excluding various localization signals (localization signal sequences to transmembrane domains and organelles, etc.), etc. Can be mentioned.

Thus, in the first vector, for example, in the direction from the N-terminal to the C-terminal, each gene is functionally linked in order to express a fusion protein containing a nuclear translocation signal-DBD-decoy protein. ..

The second vector contains a fusion gene that functionally links a gene encoding a nuclear translocation signal, a gene encoding a transcriptionally activated protein, and a fragmented partial cDNA. The second vector may be any vector that functions in yeast, and examples thereof include pACT2, pGADT7, and pGAD10.

In the second vector, the fusion protein encoded by the fusion gene containing the gene encoding the transcriptional activation protein and the fragmented partial cDNA is transferred into the fusion gene in the nucleus of the yeast. The translocation signal must be endogenous or pre-added (eg, Large T antigen residues 47 to 54 (PKKKRKVE: SEQ ID NO: 1), LexA protein endogenous nuclear localization signal sequence (KRLKK: SEQ ID NO: 2), etc.). It doesn't become.

The transcriptional activation protein includes the DNA-binding protein encoded by the fusion gene contained in the first vector and the transcriptional activity when it is located close to the control sequence (promoter, etc.) of the reporter gene in yeast. It has a function and activates the transcription of a reporter gene, and examples thereof include the activator domain (AD) of the Gal4 protein corresponding to the DBD of the Gal4 protein.

On the other hand, the fragmented partial cDNA is different from the full-length gene cDNA library prepared by using the oligo dT primer, and is commercially available, for example, a known cDNA library preparation (for example, cDNA Library Construction Kit (Takara Bio Co., Ltd.)). In the preparation of a cDNA library using the kit), using mRNA collected from any organism or tissue and a random primer (for example, a random primer consisting of the base sequence shown in SEQ ID NO: 3 used in the examples below). It is a cDNA library reversely transcribed from an arbitrary location, or a cDNA library that is intentionally fragmented by being cleaved by ultrasonic treatment or the like. The cDNA obtained by random prime reverse transcription has, for example, a length of 100 to 1800 bp (preferably 100 to 1000 bp). Fragmented cDNA libraries by cleavage using sonication or the like are prepared, for example, by adding an EcoRI adapter to the end and binding to a large excess vector with an EcoRI stump. The partial cDNA includes a signal peptide, a cell membrane or an intracellular small organ localized sequence, or a prey protein that binds to a ligand or a membrane protein lacking a transmembrane region. Examples of the prey protein include a receptor protein and the like.

Thus, in the second vector, for example, from the N-terminus to the C-terminus, in order to express a fusion protein containing a prey protein encoded by a nuclear translocation signal-Gal4 activator domain (AD) -fragmented cDNA. , Contains each gene in a functionally linked form.

In this method, the first and second vectors are then transformed into yeast to produce yeast transformants expressing the first and second vectors. Examples of the yeast include budding yeast (Saccharomyces cerevisiae) and the like. In the nucleus of yeast, transcription is activated by a DNA-binding protein encoded by the fusion gene contained in the first vector and a transcriptional activation protein encoded by the fusion gene contained in the second vector. There is a control sequence to be converted and a reporter gene located downstream thereof. Examples of the control sequence include a base sequence called UASG (Upstream Activation Sequences for galactose) for Gal4 protein. Examples of the reporter gene include a gene encoding β-galactosidase and HIS3. The control sequence and its downstream reporter gene are present in the yeast nucleus because they are integrated into genomic DNA.

The method for introducing the first and second vectors into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.

Then, the obtained yeast transformant is cultured under viable conditions. In the nucleus of the yeast transformant, a fusion protein containing a nuclear translocation signal, a DNA-binding protein, and a decoy protein, and a prey protein encoded by a nuclear translocation signal, a transcriptional activation protein, and a fragmented partial cDNA. By expressing the fusion protein containing, and interacting with the decoy protein and the prey protein, the DNA-binding protein and the transcriptionally activated protein are in close proximity and bind to a control sequence located upstream of the reporter gene. , The transcription of the reporter gene is promoted. Therefore, using the activity of the reporter gene as an index, the binding between the decoy protein and the prey protein can be confirmed, and the prey protein corresponding to the decoy protein can be identified.

In culturing the yeast transformant, the temperature is set to, for example, 25 to 30 ° C., and the pH of the medium is, for example, pH 5 so that the transformant grows and the reporter protein encoded by the reporter gene is not inactivated. Set to around .8 and continue culturing until the required amount of cells for various assays is obtained.

In yeast transformants in which the activity of the reporter gene has been confirmed, the gene fragment encoding the partial prey protein can be identified by sequencing the fragmented partial cDNA contained in the second vector. .. In addition, by comparing the identified partial prey protein-encoding gene fragment to, for example, a gene sequence registered in a known database, the gene encoding the prey protein (if registered) or the gene concerned. It is also possible to identify the full length prey protein encoded by.

According to this method, it is expected that the detection sensitivity of binding (up to about 1 μM Kd binding can be detected by using the yeast reporter gene system, compared to the panning method in cultured cells. ) And improvement of S / N ratio, speeding up of screening (usually about 14 days are required to determine the binding of about 1 million clones), stability of the gene to be recovered, speeding up of cloning, economic efficiency, etc. Be expected. In addition, the receptor identification of the orphan ligand may be directly linked to drug discovery, and this method can be applied to the receptor identification of the orphan ligand. Furthermore, this method can be widely applied to screening for binding between membrane proteins.

The present invention also relates to a reagent kit for the present method containing the cDNA obtained by the above random prime reverse transcription itself, the second vector itself, the cDNA obtained by the random prime reverse transcription, or the second vector. The reagent kit may further include, for example, a buffer, a container, an instruction manual, etc. used in this method.

Hereinafter, the present invention will be described in more detail with reference to examples, but the technical scope of the present invention is not limited to these examples.
1. 1. Synthesis of fragmented cDNA Fragmented cDNA was synthesized by modifying the method of the cDNA Library Construction Kit (Takara Bio Inc.) with some conditions.

(1) Synthesis of 1st strand cDNA A primer in which 6 nucleotides of 4 types, dATP, dCTP, dGTP, and dTTP, were randomly added to the 3'side of the recognition sequence (5'-CTCGAG-3') of the limiting enzyme XhoI (5'-CTCGAG-3'). Sequence 5'-TAGAACTCGAGNNNNNN-3'(SEQ ID NO: 3); hereinafter referred to as "random primer") (600 μM, 1 μl), 5 μg of mRNA collected from any organism or tissue, and dNTP mixture (dATP). , 5-methyl dCTP, dGTP, dTTP) (10 mM each) 1.2 μl was mixed in a microcentrifuge tube to 10 μl with RNase-free water. This was heated at 65 ° C. for 5 minutes and then quenched on ice for denaturation. To this, 1 μl of reverse transcriptase (PrimeScript RTase Takara Bio Co., Ltd.) (200 U / μl), 1 μl of RNase inhibitor (40 U / μl), and a reaction buffer (250 mM Tris-HCl (pH 8.3)). ), 375 mM KCl, 15 mM MgCl ₂ ) 4 μl and 4 μl of water containing no RNase were added to make 20 μl, and a reverse transcription reaction was carried out at 42 ° C. for 1 hour. After 1 hour, it was cooled on ice for 2 minutes. The cDNA synthesized by this reaction was used as the 1st strand cDNA.

(2) Synthesis of 2nd strand cDNA
4.5 μl of dNTP mixture (dATP, dCTP, dGTP, dTTP), 2 μl of RNase H and DNA ligase mixture, 2 μl of DNA polymerase I (20 U / μl), 30 μl of reaction buffer, RNase on 1st strand cDNA 87.5 μl of water containing no water was added to make 146 μl, and the mixture was reacted at 16 ° C. for 2 hours. After 2 hours, the mixture was allowed to stand at 70 ° C. for 10 minutes and then at room temperature for 5 minutes or longer. The double-stranded cDNA synthesized by this reaction was used as the 2nd strand cDNA.

(3) Smoothing of the ends of 2nd strand cDNA
4 μl of T4 DNA polymerase (1 U / μl) was added to the 2nd strand cDNA and reacted at 37 ° C for 10 minutes to smooth the ends of the 2nd strand cDNA. An equal amount of phenol / chloroform / isoamyl alcohol (25:24: 1 mixture) was added to this, and after mixing well, centrifugation was performed and the upper layer was transferred to a new microcentrifuge tube. An equal amount of chloroform / isoamyl alcohol (24: 1 mixed solution) was added to this, and after mixing well, centrifugation was performed and the upper layer was transferred to a new microcentrifuge tube. To this, 1/10 amount of 3 M sodium acetate buffer (pH 5.2) and 2.5 times amount of ethanol were added, mixed well, and then centrifuged at 15,000 rpm for 30 minutes at room temperature to remove the supernatant. 200 μl of 70% ethanol was added to the precipitate, the precipitate was rinsed, and the precipitate was centrifuged at 15,000 rpm for 3 minutes at room temperature to remove the supernatant. After the precipitate was dried, 12.5 μl of RNase-free water was added to dissolve the precipitate. This was used as the terminal smoothed 2nd strand cDNA.

(4) Addition of EcoRI adapter to terminal smoothed 2nd strand cDNA 12.5 μl of terminal smoothed 2nd strand cDNA solution with EcoRI-SmaI adapter (5'-AATTCCCGGG-3'single strand with dephosphorylated 5'end DNA (SEQ ID NO: 4) and 5'-CCCGGG-3'single-stranded DNA annealed) (0.4 μg / μl) 3.5 μl and T4 DNA ligase (350 U / μl) 2 μl. 2 μl of buffer solution was added to make the total volume 20 μl. This was mixed well and then reacted at 8 ° C. overnight or longer. After the reaction, the mixture was allowed to stand at 70 ° C. for 30 minutes to inactivate T4 DNA ligase, and then allowed to stand at room temperature for another 5 minutes. This was used as the 2nd strand cDNA with an adapter.

(5) Cleavage of 2nd strand cDNA with adapter added XhoI to 20 μl of 2nd strand cDNA solution with adapter, 3 μl of XhoI (10 U / μl) and reaction buffer (500 mM Tris-HCl (pH 7.5)) ), 100 mM MgCl ₂ , 10 mM dithiothreitol, 1 M NaCl) 5 μl, 0.1% BSA (bovine serum albumin) 5 μl, and RNase-free water 17 μl were added to make the total volume 50 μl. This was mixed well and then reacted at 37 ° C. for 3 hours. This reaction cleaves the XhoI recognition sequence in the random primer, but it is not cleaved by XhoI because 5-methyl dCTP is used in the 1st strand cDNA synthesis for the XhoI recognition sequence in the cDNA.

(6) Removal of short-chain DNA Remove unreacted adapters and short-chain DNA of several tens of residues or less using gel filtration or silica-based beads. Xho I cleaved 2nd strand cDNA on a gel filtration column (select the gel type according to the size of the desired cDNA) equilibrated with TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). It was added and centrifuged (conditions depend on the column). By this centrifugation, cDNA of a certain size or larger was obtained, which did not contain an unreacted EcoRI-SmaI adapter or a short DNA fragment cleaved by XhoI. An equal amount of phenol / chloroform / isoamyl alcohol (25:24: 1 mixture) was added to this, and after mixing well, centrifugation was performed and the upper layer was transferred to a new microcentrifuge tube. An equal amount of chloroform / isoamyl alcohol (24: 1 mixed solution) was added to this, and after mixing well, centrifugation was performed and the upper layer was transferred to a new microcentrifuge tube. To this, 1/10 amount of 3 M sodium acetate buffer (pH 5.2) and 2.5 times amount of ethanol were added, mixed well, and then centrifuged at 15,000 rpm for 30 minutes at room temperature to remove the supernatant. 200 μl of 70% ethanol was added to the precipitate, the precipitate was rinsed, and the precipitate was centrifuged at 15,000 rpm for 3 minutes at room temperature to remove the supernatant. After the precipitate was dried, 15 μl of RNase-free water was added to dissolve the precipitate, which was used as EcoRI-XhoI cleavage-treated cDNA.

(7) Binding of EcoRI-XhoI cleaved cDNA to the vector A vector cleaved with EcoRI and XhoI and a cDNA cleaved with EcoRI-XhoI were bound using DNA ligase.

Next, a vector bound to EcoRI-XhoI cleavage-treated cDNA was introduced into Escherichia coli by the electroporation method, and any 10 clones were selected and the size of the fragmented cDNA inserted by the PCR method was analyzed. After confirming that it was 100 to 1000 bases, the cDNA library was recovered from Escherichia coli.

2. 2. Screening of Molecules Derived from Mouse Brain Saccharomyces cerevisiae transformed with pBTM116-HA-KM-hENHO in a library in which cDNA prepared from mRNA collected from mouse brain was inserted into vector pACT2 according to Section 1 above. It was introduced into L40 and seeded in SD-Leu-Trp-His + 3-AT medium (3-AT = 3-amino-1,2,4-triazole). Here, the vector pACT2 in which the cDNA was inserted is a gene encoding Large T antigen residues 47 to 54 (PKKKRKVE: SEQ ID NO: 1) in order from the 5'end to the 3'end, and the activator domain (AD) of the Gal4 protein. It has a gene encoding the above and the prepared cDNA. In addition, the vector pBTM116-HA-KM-hENHO sequentially produces the endogenous nuclear localization signal sequence (KRLKK: SEQ ID NO: 2) and DNA binding domain (DBD) of the LexA protein in the direction from the 5'end to the 3'end. It has a gene encoding and a gene encoding human adropin (hENHO) as a gene encoding a decoy protein. In addition, Saccharomyces cerevisiae L40 has a gene encoding β-galactosidase (reporter gene) and a HIS3 gene under the control downstream of UASG. When the molecules expressed from the two plasmids bind to each other in the cell, the expression of the HIS3 gene enables yeast cells to grow even in a His-deficient medium.

The seeded medium was transferred to an incubator at 30 ° C. and cultured for several days until the colonies were visible. The colonies were transferred to fresh SD-Leu-Trp medium and cultured in an incubator at 30 ° C. A β-galactosidase assay was performed using a part of the grown yeast, and a group of clones positive for β-galactosidase activity was analyzed. As a result, those containing a partial cDNA of a membrane protein were included.

All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (3)

A method for identifying an orphan ligand receptor , which comprises a step of culturing a yeast transformant into which the first and second vectors have been introduced.
The first vector contains a gene encoding a nuclear translocation signal, a gene encoding a DNA binding protein, and a gene encoding a decoy protein.
The second vector contains a gene encoding a nuclear translocation signal, a gene encoding a transcriptional activation protein, and a fragmented partial cDNA, and the fragmented partial cDNA is obtained by reverse transcription using a random primer. A cDNA library prepared to encode a prey protein lacking a signal peptide, cell membrane or intracellular small organ localized sequence or transmembrane region, and containing cDNA having a length of 100-1000 bp. And
The decoy protein is an orphan ligand, and the prey protein is a receptor protein, and
In the nucleus of the yeast transformant, a prey encoded by a fusion protein containing the nuclear translocation signal, a DNA-binding protein, and a decoy protein, and a partial cDNA fragmented with the nuclear translocation signal and a transcriptional activation protein. A fusion protein containing a protein is expressed, and the binding between the decoy protein and the prey protein is detected using the activity of the reporter gene as an index.
The method. The method according to claim 1, wherein the decoy protein is a protein encoded by a partial cDNA excluding various localized signal sequences. The reagent kit for a method for identifying an orphan ligand receptor according to claim 1 or 2 , which comprises a vector containing a gene encoding a nuclear translocation signal, a gene encoding a transcriptional activation protein, and a fragmented partial cDNA. The fragmented partial cDNA is a cDNA library obtained by reverse transcription using a random primer, and is a prey protein lacking a signal peptide, a cell membrane or an intracellular small organ localized sequence, or a transmembrane region. The kit, which is encoded and made to contain cDNA having a length of 100-1000 bp , wherein the prey protein is a receptor protein .