JPH0923885A - Gene expression library and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject library capable of selecting useful substances by substituting a DNA to which a uniforming manipulation is performed in a cDNA clone containing a translation region of a protein for or inserting it into the part of a coat protein gene of a filamentous bacteriophage. SOLUTION: This gene expression library containing all of cDNA clones at a same ratio and useful as a source for finding substances which can become new drugs by a screening with its affinity, etc., is obtained by separating a single chain cDNA and a double chain cDNA in a cDNA clone containing a protein translation region as a uniforming manipulation, then amplifying the single chain cDNA by a polymerase chain reaction (PCR method) using a lone linker, performing the degeneration and reassociation of the double chain cDNA and introducing the obtained DNA by substituting for or inserting into the part of the coat protein gene of a filamentous bacteriophage for the preparasion the library.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to gene libraries, and in particular, messenger RNAs that exist with various probabilities.
(MRNA) homogenized complementary DNA (cDN
A) relates to an expression library in which a fusion protein or a single protein is expressed on the surface of a phage vector.

[0002]

2. Description of the Related Art Various preparation techniques are known for gene libraries. Further, it has been attempted to catalog cDNA by producing a cDNA with a reverse transcriptase that complementarily converts expressed mRNA into DNA, targeting a gene encoding a structural gene. Regarding library formation, the target of means for selecting clones from the library is DNA, RNA or expressed protein,
Vectors are selected for each.

Attempts have been made in recent years to homogenize the proportion of mRNA and form a library. For example, Mino
ru SHKo is a mouse fibroblast-like cell Ltk ^- cell to mRN
After obtaining A and converting it into cDNA, separation of single-stranded cDNA and double-stranded cDNA, which is called homogenization cycle, and single-stranded cD
By the denaturation / re-association of the double-stranded cDNA by the PCR method of NA, the subtraction of the mRNA of the cell (subtracting the overlapping mRNA molecules) is performed to obtain the cDNA homogenized library DNA, Polymerase chain reaction (PCR method) using loan linker
Amplification is carried out according to the procedure described above, and the library is constructed by introducing it into a plasmid vector. (Nucleic Acids Res., 18 p570
5-5711,1990)

[0004]

[Problems to be Solved by the Invention] As a method for finding an unknown protein, first, a cDNA library in which all expressed mRNAs are cataloged is used as the source of the substance, and further the functional aspect of the expressed protein is investigated. Constructed as something that can be screened,
From this, there is no one that can select a substance that can be used as a medicine by various screening methods, particularly by looking at the interaction with a protein or the like in vivo.

The above-mentioned Minoru SHKo library contains cDNA limited to the 3'side from the viewpoint of preventing duplication of mRNA, and does not include all structural gene portions. The expressed protein cannot be screened in a state close to that in vivo.

In the present invention, it is possible to see the interaction with a protein or the like in vivo, that is, it is possible to perform the screening in a state close to the natural state, and further, it is easy to screen and all the screening targets are lined up. It is to provide an expression cDNA homogenized library.

[0007]

[Means for Solving the Problem] As a method for finding an unknown protein, the following means are considered.

(1) A method of using a screening method that reflects a certain physiological function, selecting a substance of a possible substance origin, purifying a substance that affects the physiological function, and finally isolating the substance. (2) A method of identifying substances showing affinity for a certain substance (receptor, ligand, enzyme, antibody, etc.) by using biochemical / physicochemical means. (3) A method of determining a family closely related to a protein by utilizing the homology of the amino acid of a known protein and the gene sequence encoding the same.

The present invention is to provide a library which can be a screening target and is particularly effective in performing the screening of (2). Furthermore, it is to provide a homogenized library of expressed cDNA in which all screening targets are arranged, not just a population of substances.

The gene of an animal individual encoding a protein is
It is said that there are 50,000 to 100,000 kinds, and its mRNA is known to exist with various probabilities. For example, the actin gene mRNA, which forms the cytoskeletal system, is always present in most cells in relatively large amounts due to constitutive expression. In addition, cells that are responsible for the production of proteins such as albumin that are required in large quantities at all times are
Many A are duplicated. On the other hand, physiological functions of the living body are regulated by the interaction of factors that maintain homeostasis, and some of the factors are expressed by the mRN encoded by them.
Some are only trace amounts of A and cannot exist unless induced.

Many of the cDNA libraries prepared so far reflect the amount ratio of mRNA of cells from which mRNA originates, or are treated with induction to increase the number of molecules of the target protein, that is, mRNA. It was made from the cells.

[0012] In many of these cDNA libraries, it is difficult to identify a gene even if its function is clear due to the difference in existence probability when cloning a gene, and the scale of screening is determined by the difference in induction of the desired product. was there.

In order to overcome this drawback, the above-mentioned Mi
A homogenized cDNA library of noru SHKo has been created. This homogenized cDNA library has as its purpose the dramatic simplification of the gene isolation method by cataloging the genes expressed in individual organisms and the low probability of genes (eg, few copies) being present. / M of cell
RNA).

However, in this library, mRNA duplication is subjected to a uniform cycle treatment (PCR method and double-stranded cDNA).
Subtraction of mRNA is performed by denaturing and reassociating ), The gene is constructed with a 3'-side sequence whose characteristics are clear, that is, a cDNA sequence in the untranslated region that does not include the structural gene. Even if the target gene is cloned once in this library, it is necessary to screen the gene library containing the structural gene again with the cloned clone to obtain the full-length one. Furthermore, the protein of the structural gene portion could not be the target of screening.

In order to screen a target substance using the affinity for a protein as an index, a cDNA containing a structural gene portion is used.
A must be expressed. Moreover, in order to express the homogenized library, it is considered that the conventional vector for the purpose of efficient expression of the specific gene may cause the bias of the expression and is not suitable.

As a system capable of uniformly expressing many kinds of genes, there is a protein expression system by filamentous phage reported by GSSmith. (Science, 228 p13
15-1317, 1985) This system inserts another gene into the pIII protein region, which is a part of the minor coat protein of filamentous phage, and expresses it as a fusion protein. This fusion protein not only maintains the ability of pIII protein to initiate infection in E. coli, but is also expressed on the phage body surface and retains the binding ability (affinity) peculiar to the protein. Was shown. (SFParmley and GPSmith, G
(ene, 73 p305-318) Furthermore, by taking advantage of this excellent property, J. J. et al., by inserting a synthetic DNA mixture encoding a random amino acid sequence.
K. Scott and GPSmith have created a random peptide library. (Science, 249 p386-390, 1990) For the homogenized expression cDNA library of the present invention, a protein expression system using a filamentous phage having a structural gene portion inserted can be used. This phage contains an example of antibody gene expression (JD Marks et al., J. Mol. Biol., 222 p.
581-597, 1991), a gene having a length of 800 to 1,000 bases can be inserted.

The base length of 800 to 1,000 corresponds to about 270 to 330 as the number coding for amino acids. It is thought that there are around 50,000 types of all genes in humans and animals.
Many NAs have a length of 1,000 to 2,000 or more. In other words, it is considered impossible to insert the structural gene as it is into the filamentous phage.

However, to search for protein interactions, the structure of a certain structural gene as it is is not necessary. M.Go envisions a structure called a module that can be identified by physicochemical calculation as a functional unit of a protein in order to elucidate the systematic evolution of the function of the protein. Then, it is shown that a specific affinity appears by the combination of this module. (Michiko Go, Protein / Nucleic Acid / Enzyme 39 p2449-2456, 199
4) That is, protein interactions showing affinity can be searched for as long as they include a protein portion, for example, a module.

A water-soluble protein is generally formed by connecting spherical domains. The globular domain is usually a cluster of about 50 to 200 amino acid residues, but as an exception, a large domain of 300 residues or more is also known. The globular domain is a combination of modules. A module is a compact structural unit composed of consecutive amino acid residues of about 10 to 40 residues. (Michiko Go, Experimental Method for Molecular Evolution, Chapter 18, edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin, 1993) Since the module is considered as a target for affinity search, the length of the DNA encoded from that amino acid is Thirty
~ 120 base length. Since the filamentous phage expression system described above can insert a length of 800 to 1,000 bases, a modular structure of about 7 to 33 can be expressed.

Considering the length of mRNA, when targeting homogenized cDNA, those containing a structural gene portion are identified as upstream, middle, and midstream of the open reading frame.
It can be used as an affinity search library by converting it into a library as a cDNA fragment corresponding to the downstream. Furthermore, the genes of animal individuals that encode proteins are 5 to 10
Since it is said that there are many kinds, it is sufficient to screen phages 1 × 10 ^{5 to} 1 × 10 ⁶ as a homogenized library. The number of screenings is a number that can be cataloged by numbering or the like.

That is, a library containing a homogenized cDNA and containing a structural gene portion can be screened by affinity with the module structure contained therein.

For screening this library,
By using a plate coated with a specific protein, phages that specifically bind can be selected by a panning method or the like. The protein to be coated is not particularly limited, but a protein that is considered to be involved in specific tissues, cells, and receptors and physiological phenomena is preferable. After confirming the affinity, the nucleotide sequence of the inserted cDNA fragment of the selected phage is determined, and if it is different from a known one, a factor or the like involved in a novel physiological activity can be found.

[0023] Further, it is possible to construct a screening system for inhibitors and the like, by constructing an experimental system that expresses the found factor and inhibits its affinity or exerts an influence.

If the one showing the affinity has the relationship between the antibody and the antigen, it can be obtained by screening if either one is known. The resulting affinity analysis can be done via a modular structure. This provides the material for a faster analytical tool.

The yeast two-hybrid system (two-hybrid system) has been developed as a system closer to that in vivo rather than the affinity test. This is between two fusion proteins (two-h
This is an experimental system for detecting the interaction (binding activity) of the ybrid) with the activation of yeast intracellular transcription as an index. (Fields, S. and
Song, O., Nature, 340 p245-246, 1989) In the two-hybrid system, a gene was constructed by fusing the target protein to the binding region of transcription factor SRF or GAL4 on a yeast expression vector, -Introducing into the indicator yeast strain (62L) carrying the galactosidase gene (LacZ) as the reporter gene. On the other hand, cDNA
Library, a DNA having restriction enzyme cleavage sites at both ends so that a homogenized expression library can be constructed in the present invention
Which is an activator of transcription on the yeast expression vector
It is constructed downstream of the activator domain of P16 (or GAL4) and is similarly introduced. Colonies are formed on a nylon membrane, expression from both plasmids is induced by a galactose medium, and then X-gal is used as a substrate, and the blue color of the colonies is used as an index for screening. As a reporter gene, one using the yeast gene HIS3 is also known.

[0026]

That is, the present invention relates to an expression library containing all cDNA clones in the same proportion.

Further, regarding a DNA having a restriction enzyme cleavage site at both ends of the DNA, which can construct an expression library containing all cDNA clones at the same ratio, the restriction enzyme cleavage site mentioned here is not particularly limited, Preferably, a restriction enzyme site that can be inserted into the expression vector is desirable. Also, it may be attached to both ends or one end as a multi-cloning site.

An expression library containing all the cDNA clones at the same ratio is produced by a phage, and the phage may be a phage capable of expressing the inserted gene, and preferably lambda phage or filamentous. Menthus phage is mentioned.

Furthermore, the present invention relates to an expression library in which all the cDNA clones are contained in the same ratio, and which is produced by the filamentous phage.

The present invention is a cDNA containing a protein translation region.
The clone A is homogenized, and the obtained DNA is replaced with a part of the minor coat protein gene of filamentous phage or introduced by insertion to construct a library. All cDNA clones are contained at the same ratio. The present invention relates to a method for producing an expression library. Furthermore, homogenization operation is performed on the cDNA clone containing the translation region of the protein by homogenization cycle treatment using a loan linker,
The present invention relates to a method for producing an expression library in which all cDNA clones are contained in the same ratio, in which the obtained DNA is replaced with a part of the coat protein gene of filamentous phage or introduced by insertion.

The homogenization cycle treatment using a loan linker utilizes the phenomenon that complementary DNA or RNA hybridizes to self-subtraction of cDNA described by Ko, MSH et al. In order to achieve homogenization of cDNA and to recover the decrease in the amount of DNA after self-subtraction, use a linker sequence called non-adhesive cohesive end and blunt end, which is carried out under the conditions considered. That is, amplification of cDNA as a population by the Uncard PCR method. (Ko, MSH et al., Nucleic Acids Res.,
18 p4293-4294, 1990) Furthermore, the present invention relates to a method for selecting a protein having a high affinity for a certain protein based on the affinity for the protein from an expression library containing all cDNA clones at the same ratio.

Affinity screening is carried out by selecting a filamentous phage as a vector and searching for one having affinity for a certain protein after expression.
Using a plate coated with a specific protein, it is possible to select phages that specifically bind by the panning method, etc., and by using the yeast two-hybrid system, it is possible to screen more in vivo. .

The present invention also relates to a method for selecting a desired antigen by affinity, wherein a certain protein is an antibody in affinity screening.

Further, the method of selecting by affinity with a certain protein relates to an affinity screening method which is a yeast two-hybrid system.

The present invention relates to a gene product obtained by an affinity screening method from an expression library containing all cDNA clones at the same ratio, and this gene product serves as a probe for obtaining a complete structural gene, In addition, it can be a material for which the affinity can be examined by three-dimensional structural analysis in module units. If a lineup of various proteins with high affinity to low affinity for target tissues, cells, receptors, or various factors can be found, there is a possibility that new physiological activity can be found, and It is possible to provide a material whose molecular biology can be explained quickly.

Furthermore, the present invention relates to a drug comprising a gene product obtained from an expression library in which all cDNA clones are contained in the same proportion.

Even if attention is paid only to the affinity, there is a possibility that the agonist or antagonist of the receptor can be easily extracted only by the module structure contributing to the important affinity, and thus it becomes a drug which has not been known until now. There is a nature.

Each clone of the homogenized expression cDNA library can be cataloged by numbering (tag), and 1 × 10 ⁵ to 10 ⁶ individual clones can be subjected to various assay systems to obtain a database whose function has been clarified. Can be converted. In the future, it will be possible to provide a protein origin that can be applied to the development of a drug that was unknown at all, by combining or modifying regulatory proteins for more moderate regulation or combining functions.

The origin of mRNA to be converted into cDNA is not particularly limited, but human-derived cells and organs are preferable from the viewpoint of pharmaceutical purposes. Methods for extracting mRNA from cells or organs are generally known, and examples thereof include the guanidine hydrochloride method and the lithium chloride method. The separation of poly (A) ⁺ RNA is also generally known,
The oligo dT column method may be used.

The obtained poly (A) ⁺ RNA may be synthesized from the poly A chain. For example, oligo dT-NotI primer (5'-AATTCGCGCGCCGCTTTT).
2 with reverse transcriptase using TTTTTTTTTTTT-3 ')
This is a single-stranded cDNA. At this time, if the synthesis conditions are selected, 200
CDNA of several Kb of base pairs can be synthesized from the base pairs. The obtained cDNA is subjected to size fractionation by agarose gel electrophoresis or the like so as to obtain a desired population of base pairs. After fractionation,
Recovered by elution, ethanol precipitation, etc.
Both ends of A are aligned with T4 DNA polymerase treatment.

A Loan linker is connected to the obtained cDNA using a ligase. Examples of loan linkers include LL-SalI and LL-SalIA.
(5'-ATTGACGTCGACTATCCAGG-
3 ') and LL-SalIB (5'-CCTGGATA
GTCGACGTC-3 ') can be used.
(Ko, MSH et al., Nucleic Acids Res., 18 , p4293-4294,
1990) LL-SalIA, B is phosphorylated and annealed. The annealed LL-SalI cDNA as a blunt end is ligated with T4 DNA ligase.

The obtained cDNA-lawn linker product is amplified by the PCR method. A hybrid is formed by denaturing and re-associating the amplified cDNA-lawn linker product. Only single strands that do not form hybrids are collected by a hydroxyapatide column (65 ° C incubation).

The single-stranded cDNA is amplified by the PCR method to obtain double-stranded cDNA.

At the time of denaturation and reassociation treatment, the midstream / upstream region of the structural gene can be obtained as a single strand by adding the downstream cDNA of the structural gene, and the midstream and downstream cDNA of the structural gene can be added. The upstream region of the structural gene is obtained as a single strand.

By amplifying each single-stranded population by the PCR method, a cDNA population capable of constructing each upstream, middle and downstream structural gene library as a protein module library can be obtained.

When the desired cDNA population is obtained, various linkers may be prepared and linked in consideration of insertion into a vector for which affinity is searched.

[0047]

EFFECTS OF THE INVENTION An expression library containing all constructed cDNA clones at the same ratio serves as a substance origin obtained by screening a substance that can be a novel drug with affinity and the like. Further, it is possible to provide a material for clarifying a physiological phenomenon based on affinity.

[Brief description of drawings]

FIG. 1 shows a procedure for preparing a homogenized cDNA expression library.

FIG. 2 shows an example of the use of a homogenized cDNA expression library.

Claims (11)

[Claims] 1. An expression library containing the same ratio of all cDNA clones. 2. A DNA having restriction enzyme cleavage sites at both ends of DNA capable of constructing an expression library containing all cDNA clones at the same ratio. 3. The expression library according to claim 1, which is produced by a phage. 4. The expression library according to claim 3, wherein the phage is a filamentous phage. 5. A cDNA clone containing a protein translation region is homogenized, and the obtained DNA is replaced with a part of the coat protein gene of filamentous phage or introduced by insertion to construct a library. For producing an expression library containing the same proportion of cDNA clones 6. A method for producing an expression library containing all the cDNA clones according to claim 5 at the same ratio, wherein the homogenization operation is carried out by homogenization cycle treatment using a loan linker. 7. A method for selecting a protein having a high affinity for a certain protein from an expression library containing all the cDNA clones in the same proportion based on the affinity for the protein. 8. The method according to claim 7, wherein the certain protein is an antibody, and a desired antigen is selected by affinity. 9. The yeast two-hybrid system is used as a method of selecting by affinity with a certain protein.
Or the method according to claim 8. 10. An expression library containing all the cDNA clones in the same proportion, wherein the expression library comprises the expression library of claim 7 or 8.
Alternatively, the gene product obtained by the method according to claim 9. 11. A drug comprising a gene product obtained from an expression library containing all cDNA clones in the same proportion.