JPH11290076A - Production of structure-discriminating protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently producing a structure- discriminating protein which discriminates a native protein from a denatured one. SOLUTION: This is a method for producing a structure-discriminating protein which recognizes a protein 1 having normal structure to bind, but does not recognize a protein having abnormal structure. The structure-discriminating protein is isolated by (1) applying a phase display library containing a phase which displays a structure-discriminating protein to affinity chromatography using gel 6a which was coupled to the protein 1 having normal structure, (2) eluting a fraction 5a which adsorbed onto gel 6a, (3) applying the fraction 5a to affinity chromatography using gel which was coupled to the protein having abnormal structure, (4) selecting a phase having an activity to bind the protein 1 having normal structure from passed fractions, (5) releasing the structure- discriminating protein from the phase, followed by (6) selecting the structure- discriminating protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a protein having a normal structure (hereinafter referred to as "natural protein") and a protein having an abnormal structure (hereinafter referred to as "denatured protein").
The present invention relates to a method for producing a structure-identifying protein for identifying the structure of a protein.

[0002]

2. Description of the Related Art As is well known, there are various binding proteins in nature, that is, proteins that recognize and bind to a specific protein. One example is an antibody protein. In the immune system, this antibody protein strictly discriminates itself from foreign substances. The protein that recognizes this foreign substance is called an immunoglobulin or an antibody, and the foreign substance is called an antigen (non-self antigen).

The genes of immunoglobulins have been elucidated by Tonegawa et al. [Maki, R. et al., Proc. Natl. Acad. Scc.
i., USA, 77, 2138-2142 (1980)], revealed the mechanism of gaining diversity for its structural genes and antigens at the genetic level.

As shown in FIG. 2, the antibody (8) is composed of a tetramer consisting of two H chains (heavy chains) (10) and two L chains (light chains) (11). I have. About half of the L chain (11) and about 3/4 of the H chain (10) are called constant (C) regions because their amino acid sequences hardly change even when the type of antigen changes, and the L chain (11) ), C of H chain (10)
The regions are called C _L and C _H , respectively. The remaining portion is called a variable (V) region because the amino acid sequence varies depending on the type of antigen, and the L chain (11) and the H chain (1
V region of 0) are respectively referred to as V _L, V _H. Furthermore, in the V region, a hypervariable region (CDR, complementarity de-
termining region), which are CDR1,
They are called CDR2 and CDR3.

[0005] The gene of the antibody (8) generates the diversity of the antibody (8), that is, the activity of specifically recognizing and binding to each foreign substance by rearranging the DNA. In the antibody gene obtained from the rearranged B cells, the L chain gene assembles three gene fragments of V, J (joining) and C, and the H chain gene as V and D (diversity).
y), J and C are assembled together. That is, the manner in which these gene fragments are linked changes, resulting in diversity in the amino acid sequence encoded by the gene.

For example, the mouse H chain (10) gene has about 100 V fragments, about 20 D fragments, and 4 J fragments.
A fragment, and a combination of one C fragment,
The rearrangement, ie the selection of genes from each fragment and their ligation, gives rise to a great deal of diversity. In addition, CDR1
And CDR2 are encoded by a V fragment, and CDR3 is encoded by a V fragment.
It is encoded by the 3 'end region of the fragment, the 5' end region of the D and J fragments. Therefore, in the CDR,
The amino acid sequence of CDR3 is the most varied.

Here, it has not been reported that such an antibody (8) generally recognizes both a native protein and a denatured protein and discriminates between them. However, recently, it has been revealed that denatured proteins such as prion protein that causes mad cow disease and denatured human lysozyme protein that causes amyloidosis cause various diseases in vivo. (Prusiner, SB, TIBS, 2
1, 482-487 (1996); Pepys, MB, et al., Nature, 5
53-557 (1993)].

[0008] In addition, techniques for expressing proteins of many higher organisms, including humans, in large amounts in various hosts by recombinant DNA technology have been developed. Many of these expressed proteins are intracellular. Has been shown to accumulate as insoluble inclusion bodies. In order to recover the native protein from these inclusions, it is necessary to treat the inclusions with a denaturing agent such as urea or guanidine hydrochloride and to unwind the solution to obtain the native protein. This unwinding process is generally extremely inefficient, and the unwound solution always contains unwound natural protein and unwound denatured protein. A means for efficiently separating these native proteins from denatured proteins has not been known yet, which is one of the drawbacks of the recombinant DNA technology.

[0009] Therefore, it is extremely useful to develop a technique for rapidly detecting, discriminating, and separating a native protein and a denatured protein. For example, an anti-human ICAM-1 antibody (manufactured by Takara Shuzo Co., Ltd.) is known as a conventional structure identification protein for identifying the structure of such a protein.

[0010]

However, since the above-mentioned anti-human ICAM-1 antibody was collected by accident, there is a problem that a method for efficiently producing it has not yet been established.

The present invention has been made in view of the above problems, and has as its object to provide a method for efficiently producing a structure discriminating protein that discriminates the structure between a native protein and a denatured protein. I do.

[0012]

Means for attaining the above object are as follows. First, a method for producing a structure discriminating protein which recognizes and binds to a native protein but does not recognize a denatured protein. The solution containing the structure-identifying protein is subjected to affinity chromatography using a gel to which the natural protein is coupled, and the fraction adsorbed to the gel is eluted. And subjecting the denatured protein to affinity chromatography using a gel to which the denatured protein has been coupled, collecting the passing fraction, and isolating the structure-identifying protein contained in the passing fraction.

Second, there is provided a method for producing a structure discriminating protein which recognizes and binds to a denatured protein but does not recognize a native protein.
After subjecting to affinity chromatography using the gel to which the denatured protein is coupled, and eluting the fraction adsorbed to the gel, the eluted fraction is used as the gel to which the natural protein is coupled. The objective is to isolate the structure-identifying protein contained in the passed fraction by subjecting it to affinity chromatography and collecting the passed fraction.

Third, there is provided a method for producing a structure-recognition protein which recognizes and binds to a native protein but does not recognize a denatured protein, wherein a phage display library comprising a phage displaying the structure-recognition protein is provided. After subjecting to affinity chromatography using a gel to which the natural protein has been coupled and eluting a fraction adsorbed to the gel, the eluted fraction is subjected to a gel coupled to the denatured protein. The objective of the present invention is to provide an affinity chromatography to be used, select a phage having a binding activity with the natural protein from the flow-through fraction, and isolate and discriminate the structure discriminating protein from the phage.

Fourth, the eluting fraction is subjected to at least panning using the natural protein to thereby purify the fraction in advance.

Fifth, there is provided a method for producing a structure-recognition protein which recognizes and binds to a denatured protein but does not recognize a native protein, and comprises a phage display library containing a phage displaying the structure-recognition protein. After subjecting the fraction adsorbed to the gel to affinity chromatography using a gel to which the denatured protein has been coupled to elute, the eluted fraction is subjected to the gel coupled to the natural protein. The objective of the present invention is to isolate the phage having binding activity to the denatured protein from the flow-through fraction by subjecting the phage to affinity chromatography to be used, isolating the phage discriminating protein from the phage, and selecting the phage.

Sixth, after growing at least one of the phage display library, the elution fraction, the passage fraction, and the phage having an activity of binding to the native protein or the denatured protein. It is to move on to each subsequent operation.

[0018]

Embodiments of the present invention will be described below with reference to the drawings. Note that the same components as those of the above-described conventional technology are denoted by the same reference numerals and description thereof is omitted.

As shown in FIGS. 1 to 7, in this embodiment, for example, native egg white lysozyme [natural protein (protein having a normal structure)] (1) is recognized and bound to it, A method for producing a single-chain antibody (structure discriminating protein) (3) that does not recognize egg white lysozyme [denatured protein (protein having an abnormal structure)] (2) will be described.

That is, this production method can be performed, for example, using the phage fd
(Phage) After growing a phage display library containing a phage antibody (5) in which a single-chain antibody (3) is displayed on (4), use the gel (6a) to which the natural egg white lysozyme (1) is coupled. The fraction (5a) adsorbed on the gel (6a) is eluted and proliferated, and the eluted fraction (5a) is, if necessary, mixed with the natural egg white lysozyme (1). Provide for the panning to be used, and elute the fraction (5
After b) is propagated, it is further subjected to affinity chromatography of the natural egg white lysozyme (1), if necessary,
After growing the eluted fraction (5c), the eluted fraction (5c) is subjected to affinity chromatography using a gel (6b) coupled with the denatured egg white lysozyme (2) to grow the flow-through fraction (5d). From the fraction (5d), a phage (5e) having an activity of binding to the natural type egg white lysozyme (1) is selected and grown, and the single-chain antibody (3)
Is isolated from the phage (5e) by selection.

As shown in FIG. 1, the phage antibody (5) was prepared from Escheric (Escheric) having a circular single-stranded DNA (7).
The single-chain antibody (3) is expressed (displayed) on a phage fd (4) of H. coli through a coat protein expressed on its surface, for example, g3p (gene 3 protein).

As shown in FIG. 1 and FIG. 2, the single-chain antibody (3) is, for example, a single-chain Fv in which V _L is connected via a linker (9) downstream of V _H of the antibody (8). Fragment (hereinafter, referred to as “scFv”).
This single-chain antibody (3) such as scFv is used, for example, in a phage display library.
(Winter, G. and Milstein, C. Nature, 349, 293-299
(1991)], a known library created by Nissim et al. [Nissim, A., et al., EMBO J., 13, 6].
92-698 (1994)], etc. through a predetermined operation.

The structure-recognition protein is not limited to the single-chain antibody (3) as in this embodiment, but may be, for example, V _L , C _L , V _H , C _{H of} the antibody (8).
Various proteins such as a _Fab fragment, which is a heterodimer composed of _H1, and a synthetic protein can be used.

In this embodiment, a phage display library is used, but the present invention is not limited to this. Instead of this phage display library, the structure identification of the single-chain antibody (3) or the like is used. Various solutions containing proteins may be used. In this case, there is no need to remove the structure discriminating protein from the phage, and thus there is an advantage that the protein can be easily recovered and isolated.

The solution containing the structure-identifying protein may be, for example, an extract from animal or plant tissues such as animal organs or plant roots, leaves or stems, or an extract from animals or plants or cells of microorganisms. And a culture solution in which a structure-recognition protein is generated using various gene library DNAs as a template and various hosts such as Escherichia coli, and a culture solution of various cells.

The phage display library can be suitably used particularly for selecting a protein that recognizes self and / or a low molecular weight antigen. Here, when the phage fd (4) or the like is used as in this embodiment, the g
By connecting the target peptide or protein to the N-terminus of the coat protein such as 3p and expressing (displaying) these on the surface of the phage fd (4), the function can be examined.

Therefore, if E. coli or the like is used, the phage fd
The single chain antibody (3) such as the scFv or the _Fab fragment or the like can be expressed on the surface of (4) [Hoogenboom, HR, et al., Nucleic Acids Res.,
19, 4133-4137 (1991)].

As in the latter, V _H and C _{H1 of the heavy} chain (10)
Is expressed on the surface of phage fd (4),
The portion of the V _L and C _L L chain (11) is simultaneously secretory expression can be a F _ab rebuild both in the periplasm of E. coli.

On the other hand, if an amber mutation is inserted between the single-chain antibody (3) such as the scFv and a coat protein such as g3p and the host is properly used, the fragment of the single-chain antibody (3) can be dissolved. Can be recovered as a protein. Therefore, in vivo [Marks, JD, et al., J. Mo
l. Biol., 222, 581-597 (1991)] or in vitro [H
oogenboom, HR and Winter, G., J. Mol. Biol., 22
7, 381-388 (1992)], the use of the V gene rearranged in order to produce a more versatile combination makes it possible to use a single chain having a different binding ability from the same library regardless of the method of immunizing the animal. Antibody (3) and the like can be isolated.

Thus, from these libraries, it is possible to select various structural recognition proteins such as a single-chain antibody (3) that recognizes an antigen including a foreign antigen and a self antigen.

The different V _H and V _L gene combinations are derived from the number of lymphocytes amplified by the polymerase chain reaction (PCR) and are formed by the random fragment pairing. By repeating selection several times from the library thus prepared, a phage antibody (5) having a binding ability to a target substance is selected.

That is, mRNA is extracted from human peripheral blood lymphocytes by a known method to prepare a complementary DNA, and the V gene of an antibody rearranged artificially by PCR is amplified to prepare its repertoire. . By constructing a library in which the V regions of the H chain (10) and L chain (11) are expressed on the surface of a linear phage, a desired antibody or the like can be obtained from various combinations.

The library is, as shown in FIG.
An artificial CDR3 was constructed using PCR primers designed to introduce a random amino acid sequence consisting of 4 to 12 residues, and the resulting CDR3
_VH gene having a region [Hoogenboom, HR and Wint
er, G., J. Mol. Biol., 227, 381-388 (1992)].

This library contains various combinations of the human V _H gene consisting of 50 human germ cell V _H genes rearranged in vitro and human Vλ3 as the light chain (11).
Genes were combined and expressed on the phage fd (4) as the previously described scFv. Each of the 50 germline _VH fragments is composed of _VH family-based primers VHBACKSfi and HSLP4-HSLP.
12 [Marks, JD, et al., J. Mol. Biol., 222, 581
-597 (1991)].
nboom, HR and Winter, G., J. Mol. Biol., 227, 3
81-388 (1992)].

Human V_HArrangement of CDR1 and CDR2 of fragment
Row diversity is approximately 50 V from germ cells._HOn the type of fragment
(Tomlinson, I.M., et al., J.
 Mol. Biol., 227, 776-798 (1992)]. In addition, CDR3
Of the germ cell derived V _HFragment sequence, and about 3
Since it is generated by a combination of 0 D fragments and 6 J fragments,
(Ichihara, Y., et al., EMBO J., 7, 4141-4150 (198
8)], while being extremely variable,
The numbers also differ from 4 to 25 residues. Therefore, the HSLP program
Lymer consists of J fragment and 4-12 amino acids
Random sequence [(A / C, N, N) n, (n is 4 to 1
An integer of 2 and N represents a base A, T, G or C)]
It is designed so that a DNA fragment to be introduced can be introduced. Change
In addition, the 3 'terminal part of the primer is V_HFR3 of fragment
(Genes flanked by regions encoding CDR2 and CDR3
(Area on the wafer).
Here, the sequence of the HSLP is “5′-GAC CAG GGT ACC TTG
 GCC CCA [(A / C) NN] n TCT TGC ACA GTA ATA CAC GGC CG
T GTC ”. The mold is_HSegment
10 infected with the encoding recombinant M13 phage
⁶Supplied as E. coli.

The first PCR is performed, for example, by treating the DNA at 94 ° C. for 1 minute to form a single-stranded DNA with VHBACKSfi and HS.
Anneal at 55 ° C. for 1 minute in the presence of LP primer. Subsequently, D was added at 72 ° C. for 1.5 minutes in the presence of four types of deoxyribonucleotide triphosphates (dNTP).
React with NA polymerase. This reaction is repeated 25 times to amplify the DNA.

In the second PCR, a restriction enzyme cleavage site for cloning, for example, at the 3 ′ end of the JH fragment, for example,
Introduce SalI site. In this case, the above-mentioned VHBACKSfi and JHSAL [Nissim, A.,
et al., EMBO J., 13, 692-698 (1994)]
The DNA is amplified by repeating the PCR reaction 15 more times.
Here, the sequence of JHSAL is “5′-GCC TGA ACC GCC T
CC ACC AGT CGA CAC GGT GAC CAG GGT ACC TTG GCC CCA
(SEQ ID NO: 3) ", and the underlined portion is the SalI site.

450 (50 germ cell-derived V _H fragments and 4 to 12 CDR3s of nine different lengths
After checking the size of the band of the sample (total of CR products) by agarose electrophoresis, a fragment obtained by treatment with restriction enzymes NcoI and SalI was used to obtain a fragment obtained by pHEN1-Vλ3 [Ho
ogenboom, HR and Winter, G., J. Mol. Biol., 22
7, 381-388 (1992)]. It should be noted that a gene encoding Vλ3 (V _L ) has already been incorporated into this vector. Also, t in FIG.
The portion designated as ag contains the sequence encoding the c-myc peptide.

The resulting single-chain antibody (3) is shown in FIGS.
And expressed on phage fd (4) in the form of scFv shown in As a result, nine types of phagemid libraries each containing at least 10 ⁷ different clones can be constructed. Each library can be extracted as a phage by using, for example, a helper phage VSC-M13 (Stratagenes) or the like. These libraries are stored by infecting E. coli with phagemid.

For the growth of the library, about 10 ⁸ of the above-mentioned recombinant Escherichia coli were cultured at 37 ° C., for example, in a 50 mL medium containing ampicillin at 600 nm turbidity (OD).
And shake culture until the value becomes 0.5. Next, the 10m
L to VSC-M13 or M13-KO7 (Strata
gene (manufactured by Gene Co., Ltd.)
Add to 0 times, and incubate at 37 ° C for 30 minutes while standing still. Next, the cells were collected by centrifugation at 4000 rpm for 10 minutes, and suspended in 30 mL of a medium containing ampicillin and kanamycin. The medium was further added to 300 mL, followed by shaking at 30 ° C. overnight. I do.

In preparing the pHEN1 phagemid particles, the above culture solution may be centrifuged at, for example, 8,000 rpm for 10 minutes, and phage may be collected according to a conventional method. That is, a 1/5 amount of a polyethylene glycol solution (containing 20% polyethylene glycol 6000 and 2.5 M NaCl) was added to the obtained supernatant and mixed well.
Leave at ℃ for 1 hour. After suspending the pellet obtained by centrifugation at 4000 rpm for 30 minutes in 40 mL of water,
Add 8 mL of the above polyethylene glycol solution and add 4 ° C
And leave for 20 minutes. This solution was prepared at 4000 rpm for 10 minutes.
Centrifuge for 10 minutes to remove the supernatant. The pellet thus obtained was suspended in 2.5 mL of water, and further suspended in 4000 mL.
Centrifuge at rpm for 10 minutes to remove residual cell debris and store at 4 ° C. 1 × 10 ^{13 to} 5
X10 ¹³ PFU phage are obtained.

In order to select the phage antibody (5), first, a target protein having a normal structure that is recognized and bound by the single-chain antibody (3) is gelated according to, for example, a Pharmacia instruction manual (procedure). Coupling to (6a). The gel (6a) includes, for example, CNBr-act
In addition to ivated Sepharose 4B gel (Pharmacia), conventionally known appropriate ones can be used. The target protein may be any protein, for example, various proteins other than egg white lysozyme.

In order to carry out the coupling treatment, first, the gel (6a) is expanded in 5 mL of 1 mM HCl for 15 minutes under ice cooling. Next, the gel (6a) was transferred to a glass filter (G3), and washing and swelling were repeated while slowly filtering with 1 mM HCl.
Transfer into a M carbonate buffer (pH 8.3) to form a gel suspension.

This gel suspension was combined with 0.5 M NaCl-
A solution in which, for example, natural egg white lysozyme (1) or the like is dissolved as the target protein in a 0.1 M carbonate buffer (pH 8.3) is mixed and reacted while shaking at 4 ° C. overnight. After the completion of the reaction, the gel (6a) was filtered with a glass filter, and 0.5 M NaCl-0.1 M carbonate buffer (pH
Wash several times in 8.3) to remove excess natural egg white lysozyme (1). The gel (6a) was reacted in 0.2 M glycine (pH 8.0) at 4 ° C. for 16 hours to block the remaining active groups.
Carbonate buffer (pH 8.3) and 0.5M NaCl-0.
The operation of alternately washing with a 1 M carbonate buffer (pH 4.0) is repeated five times. Lastly, after washing with a 0.5 M NaCl-0.1 M carbonate buffer (pH 8.3), the cells are suspended in a phosphate buffer containing salt (hereinafter, referred to as “PBS”), filled in a column, and then mixed with a natural type. Egg white lysozyme (1) is subjected to affinity chromatography.

On the other hand, natural egg white lysozyme (1) coupled to gel (6a) by the above method was
A denatured egg white lysozyme (2) -coupled gel (6b) was prepared by stirring and denaturing in a 6M urea solution containing mercaptoethanol at 37 ° C. for 30 minutes. After washing with twice the volume of PBS and packing the column, denatured egg white lysozyme (2)
For affinity chromatography.

As shown in FIG. 5, the phage solution described above was applied to the affinity column (12) of the natural egg white lysozyme (1), and the fraction (5a) adsorbed on the gel (6a) was removed. After elution with 100 mM triethylamine, the eluted fraction (5a) is expanded.

As shown in FIG. 6, a natural egg white lysozyme (1) was separately placed in a tube (Nunc-imm) for panning.
unotube, Nunc) (13) and leave at room temperature overnight. The next day, the tube (13) is washed three times with PBS, then blocked using, for example, PBS containing skim milk (14), and further washed three times with PBS.

Next, as shown in FIG. 6, if necessary, PBS containing skim milk (14) in tube (13)
And the above-mentioned elution fraction (5a) are added and reacted at room temperature. Thereafter, the tube (13) was washed 20 times with PBS containing 0.1% Tween 20 and further 20 times with PBS.
Remove the surfactant.

The bound fraction (5b) is, for example, 100 m
M triethylamine may be added and eluted by spinning up and down for 10 minutes. The eluted fraction (5b) must be immediately neutralized. The eluted fraction (5b) after the neutralization is 4 ° C.
To save. The phage thus obtained is infected with, for example, Escherichia coli TG1 strain and propagated in the same manner as the above-described library.

Next, the grown E. coli colonies are transferred to a 96-well plate into which 100 μL of a medium containing ampicillin has been dispensed, and grown at 37 ° C. overnight with shaking. Then, a small amount is transferred from each well to a 96-well plate containing 200 μL of the same medium, and cultured at 37 ° C. for 1 hour with shaking. 10 ⁹ PFU of helper phage V in each well
After adding CS-M13 and leaving at 37 ° C. for 30 minutes, 3
Shake at 7 ° C for 1 hour and centrifuge at 4000 rpm for 10 minutes or under similar conditions to remove the supernatant. The pellet is suspended in 200 μL of a medium containing ampicillin and kanamycin, and cultured by shaking at 30 ° C. overnight. The culture is centrifuged in the same manner, and the supernatant is used for phage ELISA. By repeating such a process at least once, preferably a plurality of times, the target phage (5b) can be concentrated.

As shown in FIG. 7, the phage (5b) selected in this manner is again applied to an affinity column (12) to which natural egg white lysozyme (1) has been coupled, if necessary. The fraction (5c) adsorbed on the gel (6a) is eluted by the above method using, for example, triethylamine, and immediately neutralized.

Next, the phage solution is applied to an affinity column (15) to which denatured egg white lysozyme (2) is coupled, and a flow-through fraction (5d) not adsorbed on the gel (6b) is selected. This natural egg white lysozyme (1)
Phage (5e) that recognizes and binds to native egg white lysozyme (1) but does not recognize denatured egg white lysozyme (2) if affinity column chromatography is performed at least once with denatured egg white lysozyme (2) Can be selected.

Thus, the eluted fraction (5a) is subjected to panning using a natural protein such as the natural egg white lysozyme (1) if necessary, and further to affinity chromatography of the natural protein if necessary. If the phage is purified in advance, the purity of the phage such as the phage antibody (5) can be increased, so that there is an advantage that the subsequent operation can be performed more efficiently.

In this embodiment, the affinity chromatography of natural egg white lysozyme (1) and denatured egg white lysozyme (2) shown in FIG. 7 is performed only once, but the present invention is not limited to this. However, if necessary, the fraction (5d) passed through the affinity chromatography of denatured egg white lysozyme (2) may be further subjected to the affinity chromatography one or more times, or The fraction (5d) may be subjected to affinity chromatography on natural egg white lysozyme (1), and these two types of affinity chromatography may be repeated.

Next, the phage (5
d) is expressed as a soluble protein. That is, first,
10 ⁵ PFU of phage (5d) was transformed into E. coli HB2151.
At 37 ° C. for 30 minutes. This is diluted, spread on a medium containing ampicillin, and cultured overnight at 37 ° C. The resulting colony is transferred to a 96-well plate into which 100 μL of a medium containing ampicillin has been dispensed, and cultured at 37 ° C. overnight. Again, the cells are transferred to a 96-well plate containing the same medium, and further cultured by shaking at 37 ° C. until the turbidity (OD) at 600 nm becomes about 0.9. When the turbidity reaches the target value, isopropylthio-β-D-galactoside (hereinafter abbreviated as “IPTG”) is added and shaken for an additional 16 to 24 hours to induce expression of the gene.
After centrifugation at 4000 rpm for 10 minutes, 100 μL thereof is subjected to ELISA.

After the scFv light chain (V _L ), c-my
Since the peptide of c is linked, an antibody 9E10 recognizing the peptide [Clackson, T., et al., Nature, 352, 624]
-628 (1991)] can be used to detect the expressed scFv.

That is, first, natural egg white lysozyme (1) is coated on each well of a 96-well plate according to a conventional method. The culture supernatant containing the above scFv is added to each well and allowed to adsorb, and then 9E10 (Boehringer Mannheim) is added and reacted. Next, an anti-mouse antibody conjugated with peroxidase (manufactured by Sigma) was added to add 9E10
And then washed. ABTS [2,2'-a
zino bis (3-ethylbenzthiazoline-6-sulphonic acid) d
iammonium salt] and aqueous hydrogen peroxide and left for 20 to 30 minutes, then add sodium fluoride and read the absorbance at 405 nm to quantify the amount of scFv [Nissim, A., et al. ., EMBO J., 13, 692-698 (1994)
].

As described above, after the affinity chromatography of native egg white lysozyme (1) and denatured egg white lysozyme (2) is performed using the phage display library, the natural type egg white lysozyme (2) is subjected to the affinity chromatography from the passed fraction (5d). Phage having binding activity to egg white lysozyme (1) (5
e), and only the structure discriminating protein such as the single-chain antibody (3) is removed and selected from the phage (5e). Thus, it is possible to produce efficiently and various structural discriminating proteins. There are advantages.

As in this embodiment, the phage display library and the eluted fractions (5a, 5
b, 5c), the flow-through fraction (5d), and / or the phage (5e) having a binding activity with a natural protein such as the natural egg white lysozyme (1). If the operation is shifted to the above operation, the yield after each operation can be increased. Therefore, there is an advantage that each operation can be performed more easily.

As described above, in this embodiment, the case of producing a single-chain antibody (3) or the like that recognizes only the native type egg white lysozyme (1) has been described. By performing the same operation by reversing the treatment for lysozyme (2) and natural egg white lysozyme (1), a single-chain antibody (3) or the like that recognizes only denatured egg white lysozyme (2) is produced. You can also.

[0061]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in more detail with reference to embodiments, but the present invention is not limited to these embodiments.

A known library of Nissim et al. [Nissim,
A., et al., EMBO J., 13, 692-698 (1994)]
As shown in the flowchart of FIG. 8, production of an anti-naturally occurring egg white lysozyme antibody (scFv fragment) as a structure discriminating protein and various analyzes were performed.

[1. Culture of library] That is, 50 μL of the above library (about 10 ⁸ cells) was added to 100 μg
/ ML ampicillin (Sigma) and 50 mL of 2 × TY containing 1% glucose (containing 16 g of tryptone, 10 g of yeast extract, and 5 g of NaCl in 1 L)
Was added to a 200 mL triangular Meyer, and cultured at 37 ° C. with stirring. When the absorbance at a wavelength of 600 nm reaches 0.5, 10 mL of the culture solution is taken and helper phage V
CS-M13 (Stratagene) was used for E. coli (Escheric).
hia coli) TG1 strain and helper phage in a ratio of 1: 2
0 was added.

On the other hand, the remaining 40 mL of the culture solution was 2500
Centrifuge at 15 rpm for 15 minutes to remove about 0.5 mL of the precipitate.
Of TYE containing 100 μg / mL ampicillin and 1% glucose (1 in 1 L).
0 g tryptone, 5 g yeast extract, 8 g NaC
l, including 15g of Bacto agar) Plate and spread 3
Cultured at 7 ° C overnight. Then, all of them were replaced with an appropriate amount of 2 × T
Y (2 to 5 mL), and glycerol was added to a final concentration of 20% for cryopreservation at -80 ° C.

The culture solution (10 mL) to which the helper phage VCS-M13 was added was cultured in a water bath at 37 ° C. for 30 minutes, and then centrifuged at 2500 rpm for 15 minutes to remove the supernatant. The precipitate was suspended in 30 mL of 2 × TY containing 100 μg / mL of ampicillin and 25 μg / mL of kanamycin (manufactured by Wako Pure Chemical Industries, Ltd.).
100 μg / mL ampicillin and 2
270 mL of 2 × T containing 5 μg / mL kanamycin
After adding to Y, the cells were cultured at 30 ° C. overnight with stirring.

[2. Preparation of Phage Antibody] The solution cultured overnight at 4 ° C. was centrifuged at 8,000 rpm for 10 minutes to remove the precipitate, and then the PEG / NaC was added to the culture supernatant.
1 solution (20% polyethylene glycol 6000 and 2.5
M NaCl) (1/5 volume) was added, and the mixture was stirred well and allowed to stand at 4 ° C for 1 hour. Then, 4 ° C, 8000r
The supernatant was removed by centrifugation at pm for 30 minutes, the precipitate was suspended in 40 mL of sterile water, 8 mL of the above PEG / NaCl solution was added, and the mixture was stirred and allowed to stand at 4 ° C. for 20 minutes.

After centrifugation at 4000 rpm for 30 minutes, the supernatant was removed by decantation, and then PEG / NaC
again, at 4000 rpm to completely remove the solution.
For 10 minutes, and the supernatant was removed with an aspirator. The precipitate is then suspended in 2.5 mL of sterile water and
After centrifugation at 00 rpm for 10 minutes, the supernatant was transferred to a new tube to obtain a phage antibody solution. The titer of phage antibodies in the solution (titer), measured E. coli strain TG1 as host, 1 × 10 ¹² P
FU.

[3. Preparation of affinity column] [3-1. Preparation of affinity column in which natural egg white lysozyme is coupled to gel] According to the instruction manual of Pharmacia, CNBr-activated Sepharose 4B gel (hereinafter simply referred to as “gel”) (manufactured by Pharmacia) (natural egg white lysozyme). Was coupled.

That is, first, 1 g of a gel was weighed, and cooled under ice-cooling for 5 g.
Swelled in mL of 1 mM HCl for 15 minutes. Next, the gel was transferred to a glass filter (G3), and washing and swelling were repeated while slowly filtering with about 300 mL of 1 mM HCl. Then, a 0.5 M NaCl-0.1 M carbonate buffer (p
H8.3) Transferred to 9 mL to make a gel suspension.

This gel suspension (1 mL) was mixed with 0.5 M NaC
A solution in which natural egg white lysozyme was dissolved at a rate of 10 mg / mL in a 1-0.1 M carbonate buffer (pH 8.3) was mixed, and reacted at 4 ° C. while shaking overnight. After completion of the reaction, the gel was filtered with a glass filter, and 0.5M Na
Extra natural egg white lysozyme was removed by washing several times with a Cl-0.1 M carbonate buffer (pH 8.3). The gel was reacted in 0.2 M glycine (pH 8.0) at 4 ° C. for 16 hours to block the remaining active groups.
Cl-0.1 M carbonate buffer (pH 8.3) and 0.5 M
The operation of alternately washing with NaCl-0.1 M carbonate buffer (pH 4.0) was repeated five times. Finally, 0.5M Na
After washing with a Cl-0.1 M carbonate buffer (pH 8.3), the cells were suspended in PBS (phosphate buffer containing salt). Half of this was packed in a column (0.7 cmΦ × 4 cm) to prepare an affinity column of natural egg white lysozyme.

[3-2. Preparation of affinity column in which denatured egg white lysozyme was coupled to gel)
The gel to which natural egg white lysozyme is coupled by the above method is denatured by stirring at 37 ° C. for 30 minutes in a 6 M urea solution containing 1 M β-mercaptoethanol, followed by washing with 100 times the volume of PBS of the gel. Column (0.7cm
Φ4 cm) to prepare an affinity column of denatured egg white lysozyme.

[4. Selection of Phage Antibody Recognizing Natural Egg White Lysozyme by Affinity Chromatography] After washing the affinity column of natural egg white lysozyme prepared in step 2 with PBS, 10 ¹² PFU of the phage antibody prepared in 1 above was applied to this column.
Then, PBS, 0.5 M NaCl-50 mM Tris
After washing the column with 10 mL each of -HCl (pH 7.5, 8.5, 9.5), the phage antibody adsorbed on the gel was eluted with 5 mL of trimethylamine. In order to immediately neutralize the eluted phage antibody solution, the tube from which the eluate was collected was previously filled with 1M Tris-HCl (p
H7.4) 2.5 mL was dispensed. Table 1 shows the results.

[0073]

[Table 1]

The obtained phage antibody solution was added to 9 mL of the E. coli TG1 strain which had been cultured until the absorbance at a wavelength of 600 nm reached 0.5, and cultured at 37 ° C. for 30 minutes. The culture was centrifuged at 2500 rpm for 15 minutes at 4 ° C., and the obtained precipitate was suspended in a small amount of 2 × TY, and then plated on a TYE plate containing 100 μg / mL ampicillin and 1% glucose. . 37
After overnight culture at 2 ° C., all cells were
The suspension was suspended in Y, glycerol was added thereto, and the mixture was stored frozen at -80 ° C.

[5. Selection of Phage Antibody Recognizing Native Egg White Lysozyme by Panning Method] About 50 μg / mL
Was added to a tube (manufactured by Nunc) having a high protein binding ability and left overnight at room temperature. The next day, after removing the solution containing natural egg white lysozyme, PBS was injected and poured, followed by decantation for washing three times by removing the washing solution. Next, a tube containing 2% skim milk (hereinafter abbreviated as “2% MPBS”) is filled up to the edge of the tube, sealed with parafilm and left at 37 ° C. for 2 hours.
Washed twice. In addition, 4. 10 ¹² PFU prepared in
Was added and a 2% MPBS solution containing phage was added, and the mixture was left at room temperature for 2 hours. Next, the plate was washed 15 times with PBS containing 0.1% Tween 20, and then also washed 15 times with PBS.

In order to elute the phage antibody solution, 10
1 mL of 0 mM triethylamine was added to the tube and the tube was spun for 10 minutes. The phage antibody solution eluted in this manner is mixed with 1M Tris-HCl (pH
7.4) The mixture was added to a tube containing 0.5 mL and immediately neutralized. Table 2 shows the results.

[0077]

[Table 2]

Next, 1 mL of a phage antibody solution was added to 9 mL of the E. coli TG1 strain, which had been cultured until the absorbance at a wavelength of 600 nm reached 0.5, and cultured at 37 ° C. for 30 minutes. Thereafter, serial dilutions in a 10 ¹ to 10 ⁴ prepared by 200μL respectively, were plated by 100μL of which the plates of TYE containing 100 [mu] g / mL ampicillin and 1% glucose. Also,
The remaining culture solution was centrifuged at 2500 rpm for 20 minutes, and the precipitate was suspended in 1 mL of 2 × TY.
Plated on TYE containing μg / mL ampicillin and 1% glucose. The resulting Escherichia coli was assumed to contain a phage (phage antibody) displaying an anti-natural egg white lysozyme antibody, and all plates were cultured at 37 ° C. overnight.

[6. Selection of a phage displaying an anti-natural egg white lysozyme antibody that recognizes native egg white lysozyme but does not recognize denatured egg white lysozyme] 2% MP containing 10 ¹² PFU of phage antibody obtained in
The BS solution was mixed with the 3-1. The affinity column of natural egg white lysozyme prepared in the same manner as in 4. Phage antibodies recognizing native egg white lysozyme were eluted from the gel and collected in the same manner as described above.

Next, the eluted phage antibody was used as described in 1. above. And 2. The 10 ⁸ PFU phage antibody recognizing natural egg white lysozyme obtained by amplification or the like according to the method described in 3-2. The modified egg white lysozyme prepared in the above was applied to an affinity column.

The fractions that passed without adsorption were collected, and the fractions were further applied to the above-mentioned denatured egg white lysozyme column twice to collect phage antibodies that did not recognize denatured egg white lysozyme. Table 3 shows the results.

[0082]

[Table 3]

The fraction passing through the column in this way was
mL was added to 5 mL of E. coli TG1 strain previously cultured until the absorbance at 600 nm became 0.5, and the mixture was added at 37 ° C. for 3 hours.
Incubated for 0 minutes. 100 μL of this culture solution was added to 100 μg
/ ML containing ampicillin and 1% glucose
After plating on the plate of E, the colonies obtained by culturing at 37 ° C. overnight were subjected to the following 7. The operation was performed.

[7. Detection of phage displaying anti-native egg white lysozyme antibody by ELISA] Approximately 200 colonies generated by the phage displaying the anti-natural egg white lysozyme antibody selected in the above were used for a 96-well microtiter plate (100 μL / 100 μg / mL ampicillin and 1% glucose dispensed in 100 μL / well). Corning)
The cells were cultured overnight at 37 ° C. with stirring. The next day, culture 3
0 μL was transferred to a 96-well microtiter plate into which 200 μL of 2 × TY containing 100 μg / mL ampicillin and 1% glucose was dispensed, and cultured at 37 ° C. for 1 hour with stirring. Then add 10 ^{9 to} each well
PFU helper phage VCS-M13, 100 μl
2 containing g / mL ampicillin and 1% glucose
× TY and 25 μL each, and allowed to stand at 37 ° C. for 30 minutes, further stirred for 1 hour, and then stirred at 4 ° C. and 2000 rpm for 3 minutes.
The supernatant was removed by centrifugation for 0 minutes. 10 wells per well
200 μL each of 2 × TY containing 0 μg / mL ampicillin and 1% glucose was added to suspend the precipitate, and the suspension was cultured overnight at 30 ° C. with stirring.
After centrifugation at pm for 30 minutes, the supernatant was used as a phage solution in the next ELISA.

To a 96-well microtiter plate (Coastar), 50 μL of 10 μg / mL natural egg white lysozyme was added, and allowed to stand at room temperature overnight.
Wash the plate once with S, then add 200 μL of 2% M
PBS was added to each well and left at room temperature for 2 hours to block. Thereafter, 50 μL of the phage solution prepared as described above was added to each well, and the mixture was allowed to stand at room temperature for 2 hours, and then once with PBS containing 0.05% Tween 20.
Further, the plate was washed twice with PBS. In this well, horseradish peroxidase (hereinafter abbreviated as “HRP”) conjugated anti-M13 phage antibody (Pharmaci
a) (50 μL each) and left at room temperature for 1 hour.
Meanwhile, 2,2′-azinobis (3-ethylbenzothiazoline) -6 sulfonic acid diammonium salt (hereinafter, referred to as “diammonium salt”)
Abbreviated as "ABTS". ) 10 mg was added to 20 mL of 50 mM citric acid (pH 4.5) to prepare a color developing solution. Plates were washed twice with PBS containing 0.05% Tween20, and
After washing twice with BS, 36 μL of a hydrogen peroxide solution was added to the above-mentioned coloring solution and mixed well.
0 μL was added. After leaving at room temperature for 20-60 minutes,
Using a microplate immunoreader, wavelength 405
The absorbance at nm was measured, and the binding ability to native egg white lysozyme was examined. As a result, out of 400 colonies, 23 colonies having strong binding activity were obtained.
These clones were replaced by the following 8. The operation was performed.

[8. Competition inhibition of phage antibodies displaying anti-natural egg white lysozyme that recognizes only natural egg white lysozyme with natural egg white lysozyme] Among the phage antibodies that displayed the anti-natural egg white lysozyme antibody that recognizes only natural egg white lysozyme confirmed in the above, a competitive inhibition experiment was performed on eight clones.

That is, natural egg white lysozyme was dissolved in PBS, and a dilution series up to 1 to 10 ⁻⁵ mg / mL was prepared.
Each μL was prepared. 50 μL of 10 μg / mL natural egg white lysozyme was added to 96-well microtiter plates by the amount required for each well, and the plate was allowed to stand at room temperature overnight. After washing the plate once with PBS, 200 μL
Was added to each well and left at room temperature for 2 hours to perform blocking. Then, add 4% MPB to each well
S was added in 25 μL portions. Then 10 per well
20 μL of a phage antibody solution prepared to contain ¹⁰ PFU / 50 μL and 25 μL of a PBS solution prepared to contain 5 μL of native egg white lysozyme were added.
As a positive control, 5 μL of PBS was added to each well to which no natural egg white lysozyme was added.

After leaving at room temperature for 3 hours, 0.05% Twee
Washing was performed once with PBS containing n20, and twice with PBS. To this well, 50 μL of an HRP-conjugated anti-M13 phage antibody was added, and left at room temperature for 1 hour. Meanwhile, 10 mg of ABTS was added to 50 mM citric acid (pH
4.5) In addition to 20 mL, a coloring solution was obtained. Plate
After washing twice with PBS containing 0.05% Tween20 and then twice with PBS, the above-mentioned coloring solution was added with 36 μl of hydrogen peroxide solution.
L and mix well, then add 50 μL to each well
Was added. After leaving at room temperature for 20 to 60 minutes, the absorbance at a wavelength of 405 nm was measured using a microplate immunoreader to examine the binding ability to natural egg white lysozyme. Since similar results were obtained for all clones, the results for clone 2A3 are shown in FIG. The vertical axis in FIG. 9 represents the absorbance at a wavelength of 405 nm, and the horizontal axis represents the amount of natural egg white lysozyme. As is clear from FIG. 9, the binding activity of clone 2A3 to native egg white lysozyme was reduced depending on the amount of native egg white lysozyme, which means that clone 2A3 was an anti-natural egg white lysozyme antibody. Is shown.

[9. Detection of scFv fragment] [9-1. Preparation of scFv fragment] In the above process, clones confirmed to bind to native egg white lysozyme but not to denatured egg white lysozyme were replaced with sc
It was examined whether it works as an Fv fragment.

That is, 7. A part of the phage antibody prepared in the above was taken and inoculated into E. coli host HB2151. 37
After culturing at 30 ° C. for 30 minutes, the cells were plated on a TYE plate containing 100 μg / mL ampicillin and 1% glucose, and the colonies produced by culturing at 37 ° C. overnight were removed from the plate, and 100 μg / mL ampicillin and 1% Was transferred to 3 mL of 2 × TY containing glucose and cultured overnight at 37 ° C. with stirring. The next day, 50 μL of the culture solution was taken, added to a 500 mL Meyer containing 30 mL of 2 × TY containing 100 μg / mL ampicillin and 1% glucose, and cultured at 37 ° C. When the value of the absorbance at a wavelength of 600 nm reached 0.9, IPTG (isopropylthio-β-D-galactoside) was added to a final concentration of 1 mM, and the mixture was cultured at 30 ° C. for 16 hours with stirring. Thereafter, the cells were removed by centrifugation at 4 ° C. and 2500 rpm, the supernatant was filtered, transferred to another container, and concentrated about 10-fold to obtain a scFv fragment solution. A portion of the remaining culture was added with an equal volume of 80% glycerol,
Stored at 80 ° C.

[9-2. ELI of scFv fragment
Detection by SA] 7. In the same manner as in the case of detection by ELISA using a phage antibody solution of Example 1, blocking was performed after coating with natural egg white lysozyme. Thereafter, 50 μL of a scFv fragment solution containing 2% skim milk was added to each well. 2 at room temperature
After standing for a period of time, the wells were washed with P containing 0.05% Tween20
Wash once with BS and twice more with PBS, 4 μg / mL
Anti-mouse c-myc antibody (9E10) (Boehringer M
annheim) (50 μL each) and left at room temperature for 90 minutes. Wells were washed 2 times with PBS containing 0.05% Tween20.
After washing twice with PBS and twice with PBS, 50 μL of an HRP-conjugated anti-mouse antibody (Sigma) was added, and
It was left at room temperature for 90 minutes. Meanwhile, ABTS 10mg
Was dissolved in 20 mL of 50 mM citric acid (pH 4.5) to prepare an ABTS solution. After washing twice with PBS containing 0.05% Tween20 and twice with PBS, 36 μL of a hydrogen peroxide solution was added to the above ABTS solution to prepare a color developing solution, and 50 μL of this was added to each well. 20-6
After standing at room temperature for 0 minutes, the absorbance at a wavelength of 405 nm was measured, and the binding ability of the antibody fragment to native egg white lysozyme was examined.

When the measurement was performed on eight clones, four clones (2A3, 2G2, 2F3, and 2F12) having an absorbance at a wavelength of 405 nm of 0.5 or more were obtained. Therefore, these were selected as single-chain antibodies that specifically bind to native egg white lysozyme.

[10. Detection of Protein Recognizing Native Egg White Lysozyme by Western Blotting] And 9. 2A3, 2G2, 2F3, and 2F12 clones showing binding activity by ELISA were subjected to Western blotting to further examine the binding activity.

That is, about 5 μg of natural egg white lysozyme was applied per lane, and the method of Laemmli et al. [Laemmli,
U., Nature, 227, 680-685 (1970)].
SDS-PAGE was performed using 18% SDS polyacrylamide gel at 18 mA. Then, Hybond-E
A voltage of 7 was applied to the CL membrane (Amersham) by the semi-dry method.
V, electroblotting for 90 minutes, blocking was performed with 10% MPBS at room temperature for 2 hours, and the membrane was washed twice with PBS containing 0.2% Tween20 for 5 minutes, and 0.2% Tween20 and 7. containing 2% skim milk; Phage solution (10 ¹¹ PFU / m
L) 1 mL was added to the hybrid bag containing the membrane. After shaking at 4 ° C overnight, the membrane is
After washing twice with PBS containing Tween20 for 5 minutes each,
Sheep anti-M13 antibody conjugated to RP was
The mixture was diluted 5000-fold with 2% MPBS containing 2% Tween20, and shaken at room temperature for 2 hours. As a control,
Phage antibodies recognizing both native and denatured egg white lysozyme were prepared by the same method and subjected to the same operation.

Thereafter, a diaminobenzidine solution (0.5
(mg / mL) 30 mL was transferred to a staining container, and immediately before use, 10 mL of hydrogen peroxide solution was added, and a membrane was put therein. After shaking at room temperature for about 10 minutes (depending on the degree of staining), the membrane was transferred to distilled water to stop the reaction. After washing well with distilled water and air-drying and observation, as shown in FIG. 10, the presence of egg white lysozyme was confirmed by CBB staining in all lanes (see the left side of FIG. 10).
In Western blotting, binding of the control to denatured egg white lysozyme was observed, but binding activity was not observed in the four clones (see the right side of FIG. 10). This result and the above 9-2. ELIS
The result of A shows that the obtained phage antibody recognizes native egg white lysozyme but not denatured egg white lysozyme.

[11. Determination of base sequence of single-chain antibody recognizing only native egg white lysozyme] Among clones having activity confirmed by ELISA, base sequences of 2A3, 2G2, 2F3 and 2F12 were determined, and CDRs were determined.
Three regions and the germline were examined.

The nucleotide sequence was determined by the method of Sanger et al. [Sanger,
F., Nicklen, S. and Coulson, AR, Proc. Natl. Ac
ad. Sci. USA, 74, 5463-5467 (1977)]
Using a PRISM ready terminator kit that can determine the base sequence by a terminator method using dideoxy NTP to which a fluorescent dye has been bound, using a 373A DNA sequencer manufactured by KK. For western blotting or the like, a part of the phage prepared in a large amount is taken out and a single chain (s
The phage DNA of s) was prepared and used as a template.
The primer had an 18-mer sequence immediately after the CDR3 region [CAGGGTACCTTGGCCCCA (SEQ ID NO: 4), see Sequence Listing].
And an 18-mer sequence in the V region of the L chain [GGCCACAGAC
ACAGCAGG (SEQ ID NO: 5), see Sequence Listing]. Since the template is ssDNA, 0.8 pmol of the primer [3.2 pmo for double-stranded (ds) DNA]
l] was used.

The primer, template, and premix (ABI) were mixed according to the method of Sanger et al.
A cycle sequence reaction was performed at 96 ° C. for 30 seconds, 50 ° C. for 15 seconds, and 60 ° C. for 4 minutes. Thereafter, the DNA was purified using the CTAB precipitation method according to the protocol.

After completion of the PCR, first, the reaction solution was transferred to an Eppendorf tube, and 2 μL of 3M sodium acetate and 95%
Add 50 μL of ethanol, stir vigorously and cool on ice.
After standing for 0 minutes, the mixture was centrifuged at 14,000 rpm at 4 ° C. for 30 minutes. The supernatant was removed, 250 μL of 70% ethanol was added to the precipitate, and the mixture was vigorously stirred for 30 seconds.
The supernatant was removed by centrifugation at 14000 rpm for 5 minutes. Thereafter, the precipitate was air-dried, and 4 mL of a sample buffer (deionized formaldehyde + EDTA + blue dextran) was added to obtain a sample solution. This sample solution was applied to a DNA sequencer to determine the nucleotide sequence. Table 4 shows the characteristics of clone 2A3.

[0100]

[Table 4]

The nucleotide sequence encoding clone 2A3 and the amino acid sequence deduced therefrom are shown in SEQ ID NO: 2 (see Sequence Listing) and FIG. In FIG. 11, amino acid number 31
-35 is the CDR1 region, amino acids 50-66 are the CD
The R2 region, amino acids 99-105, correspond to the CDR3 region. The other clones 2G2, 2F3, and 2F12 that were active in the ELISA were also sequenced, and were found to have the same nucleotide sequence as the above 2A3.

[0102]

As described above, according to the first aspect of the present invention, a native protein and a denatured protein are subjected to affinity chromatography using a solution containing the structure discriminating protein, and then a flow-through is performed. Since it is only necessary to collect the components, there is an advantage that a structure discriminating protein that recognizes only the native protein can be easily and efficiently produced, and various structure discriminating proteins can be produced.

According to the second aspect of the present invention, it is only necessary to carry out affinity chromatography of a denatured protein and a native protein using a solution containing the structure discriminating protein, and then to collect the flow-through fraction. Therefore, there is an advantage that a structure identification protein that recognizes only a denatured protein can be easily and efficiently produced, and various structure identification proteins can be produced.

According to the third aspect of the present invention, after performing affinity chromatography of a native protein and a denatured protein using a phage display library, the binding activity to the native protein is determined from the flow-through fraction. Since a phage having the structure is selected, and a structure discriminating protein such as the single-chain antibody is eliminated and selected from the phage, a structure discriminating protein that recognizes only the natural protein can be efficiently produced, as in the case of claim 1. There is an advantage that various structural identification proteins can be produced. In addition, since a phage display library is used, there is an advantage that phage can be cultured and propagated in each operation.

According to the fourth aspect of the present invention, the eluted fraction is previously purified by subjecting it to at least panning using the native protein, so that the purity of the phage displaying the structure discriminating protein can be reduced. Can be kept high. Therefore, there is an advantage that the subsequent operation can be performed more efficiently.

According to the invention of claim 5, after the affinity chromatography of the denatured protein and the native protein is performed using the phage display library, the binding activity of the denatured protein to the denatured protein is determined from the flow-through fraction. Since a phage having a phage is selected and the structure discriminating protein is eliminated and selected from the phage, a structure discriminating protein that recognizes only the denatured protein can be efficiently produced,
There is an advantage that various structure discriminating proteins can be produced.
In addition, similar to the effect of claim 3, since a phage display library is used, there is an advantage that phage can be cultured and propagated in each operation.

According to the invention of claim 6, at least one of the phage display library, the elution fraction, the passage fraction, and a phage having an activity of binding to the native protein or the denatured protein. Are propagated and then proceed to subsequent operations, so that the yield after each operation can be increased. Therefore, there is an advantage that each operation can be performed more easily.

[0108]

[Sequence list]

SEQ ID NO: 1 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Origin Direct origin Library name: Nissim library Clone name: 2A3

SEQ ID NO: 2 Sequence length: 112 Sequence type: amino acid Topology: Linear Sequence type: Peptide Origin Direct origin Library name: Nissim library Clone name: 2A3 Sequence: The sequence of FIG. Translated into amino acids

SEQ ID NO: 3 Sequence length: 51 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA Sequence: GCCTGAACCG CCTCCACCAG TCGACACGGT GACCAGGGTA CCTTGGCCCC A 51

SEQ ID NO: 4 Sequence length: 18 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA Sequence: CAGGGTACCT TGGCCCCA 18

SEQ ID NO: 5 Sequence length: 18 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA Sequence: GGCCACAGAC ACAGCAGG 18

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a phage antibody displaying a single-chain antibody (scFv).

FIG. 2 is a schematic diagram of an antibody molecule.

FIG. 3 is an explanatory diagram showing the structure of the H chain V region gene of the library.

FIG. 4 is a schematic diagram of pHEN1-Vλ3.

FIG. 5 is an explanatory diagram showing a mode (I) of selecting phage by affinity chromatography of native egg white lysozyme.

FIG. 6: Selection of phage by panning method (I
Explanatory drawing showing I).

FIG. 7: After performing affinity columnography of native egg white lysozyme, eluted fraction adsorbed on the gel is grown, and the eluted fraction after the growth is subjected to affinity chromatography of denatured egg white lysozyme (III) )
FIG.

FIG. 8 is a flowchart showing the procedure for selecting an anti-naturally occurring egg white lysozyme antibody and determining its amino acid sequence in Examples.

FIG. 9 is a graph showing competitive inhibition of phage displaying clone 2A3.

FIG. 10 is an explanatory diagram showing the results of Western blotting using a phage that recognizes only native egg white lysozyme.

FIG. 11 is an explanatory diagram showing the sequence of the H chain V region that recognizes native egg white lysozyme.

[Description of Signs] 1 natural egg white lysozyme [natural protein (protein having normal structure)] 2 denatured egg white lysozyme [denatured protein (protein having abnormal structure)] 3 single-chain antibody (structure discriminating protein) 4 phage fd (phage) 5a Elution fraction after affinity chromatography of native egg white lysozyme 5d Passage fraction after affinity chromatography of denatured egg white lysozyme 5e Phage 6a, 6b gel

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // (C12P 21/08 C12R 1:19)

Claims (6)

[Claims] 1. A method for producing a structure discriminating protein which recognizes and binds to a protein having a normal structure but does not recognize a protein having an abnormal structure, comprising: After subjecting to the affinity chromatography using the gel to which the protein having the protein is coupled to elute the fraction adsorbed to the gel, the eluted fraction is used for the gel to which the protein having the abnormal structure is coupled. A method for producing a structure-recognition protein, characterized in that the structure-recognition protein contained in the flow-through fraction is isolated by subjecting it to affinity chromatography to recover the flow-through fraction. 2. A method for producing a structure discriminating protein which recognizes and binds to a protein having an abnormal structure but does not recognize a protein having a normal structure, comprising: After subjecting to affinity chromatography using a gel to which the protein having a protein is coupled, and eluting a fraction adsorbed to the gel, the eluted fraction is used as a gel to which the protein having the normal structure is coupled. A method for producing a structure-recognition protein, characterized in that the structure-recognition protein contained in the flow-through fraction is isolated by subjecting it to affinity chromatography to recover the flow-through fraction. 3. A method for producing a structure-recognition protein which recognizes and binds to a protein having a normal structure but does not recognize a protein having an abnormal structure, wherein the phage display live comprises a phage displaying the structure-recognition protein. The rally is subjected to affinity chromatography using a gel to which the protein having the normal structure is coupled, and the fraction adsorbed on the gel is eluted. By subjecting the coupled gel to affinity chromatography using the gel, selecting a phage having a binding activity with the protein having the normal structure from the flow-through fraction, removing the structure-discriminating protein from the phage, and selecting the phage. A method for producing a structure discriminating protein, which comprises isolating. 4. The method according to claim 3, wherein the eluted fraction is subjected to at least panning using the protein having the normal structure to thereby purify the fraction in advance. 5. A method for producing a structure discriminating protein which recognizes and binds to a protein having an abnormal structure but does not recognize a protein having a normal structure, wherein the phage display live comprises a phage displaying the structure discriminating protein. The rally is subjected to affinity chromatography using a gel to which the protein having the abnormal structure is coupled, and the fraction adsorbed on the gel is eluted. Subjected to affinity chromatography using the coupled gel, a phage having a binding activity with the protein having the abnormal structure is selected from the passing fraction, and the structure discriminating protein is eliminated and selected from the phage. A method for producing a structure discriminating protein, which comprises isolating. 6. Proliferating at least one of the phage display library, the eluted fraction, the passage fraction, and a phage having a binding activity to the protein having the normal structure or the protein having the abnormal structure. 6. The method for producing a structure-identifying protein according to any one of claims 3 to 5, wherein each of the steps is followed by a subsequent operation.