JP2008169119A - New method for eluting ligand-bonding molecule - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for eluting a ligand-bonding molecule. <P>SOLUTION: This method for eluting the ligand-bonding molecule is provided by performing the bonding reaction of a ligand with the ligand-bonding molecule, subjecting the complex of the ligand with the ligand-bonding molecule under an enzymatic decomposition for cleaving, isolating and eluting the ligand-bonding molecule specifically bonded with the ligand from the ligand, or performing the bonding reaction of a ligand-carrying carrier linking the ligand with the carrier through a linker capable of being decomposed by an enzyme, with the ligand-bonding molecule, subjecting the complex of the ligand-carrying carrier with the ligand-bonding molecule under an enzymatic decomposition for cleaving, isolating the complex of the ligand with ligand-bonding molecule from the carrier and eluting the ligand-bonding molecule bonded specifically with the ligand. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for eluting molecules that bind to a ligand.

<Conventional elution method for ligand-binding molecules>
In the identification of proteins and phage display methods, the conventional ligand binding molecule elution method uses SDS, urea, guanidine HCl, low pH, etc. to recover phages and proteins that bind to the ligand relatively strongly. Conditions are selected.

<Problems of conventional ligand binding molecule elution method>
The ligand is not used as a ligand-only carrier but is used in a state of being supported on some resin carrier. In general, the phage display method is (1) a phage population in which various peptide sequences or proteins are displayed on the surface of phage particles, that is, a library is mixed with a ligand-carrying carrier to perform a binding reaction, and (2) is washed under appropriate conditions. Thus, non-specifically bound phages are reduced, and (3) phage clones having affinity for the target ligand are eluted. Phage that binds strongly to the ligand, although it varies depending on the resin carrier, is eluted under strong conditions such as SDS, urea, guanidine HCl, and low pH. As a problem of elution under these strong conditions, some phages are damaged by these treatments. At this time, not only the ligand but also the carrier has a lot of non-specific adsorption, and the elution under strong conditions causes the phage that was non-specifically adsorbed to the carrier and not the ligand to be eluted together with noise. It becomes.

  As described above, in the conventional ligand-binding molecule elution method, due to elution under strong conditions, for example, in the phage display method, a phage that displays a peptide that is a ligand-binding molecule may be damaged. In addition, there is a problem that a ligand-binding molecule is adsorbed nonspecifically to a carrier, not a ligand, resulting in noise. That is, in affinity selection from the display library (a process of selecting clones using the binding activity to the ligand from the library as an index), when elution is performed under strong conditions, the clone is damaged, and it binds to the carrier instead of the ligand. There was a problem that clones were eluted as noise.

  Accordingly, an object of the present invention is to provide a novel method for eluting a ligand-binding molecule in view of the above situation.

  As a result of diligent research to solve the above problems, a ligand and a ligand-binding molecule are subjected to a binding reaction, and a complex of the ligand and the ligand-binding molecule is subjected to enzymatic degradation and cleaved. It was found that a ligand-binding molecule bound to can be released and eluted. In addition, a ligand-carrying carrier and a ligand-binding molecule in which the ligand and the carrier are linked via an enzyme-decomposable linker are bound to each other, and the complex of the ligand-carrying carrier and the ligand-binding molecule is subjected to enzymatic degradation and cleaved. Thus, it was found that a complex of a ligand and a ligand-binding molecule was released from the carrier, and the ligand-binding molecule specifically bound to the ligand could be eluted, and the present invention was completed.

The present invention includes the following.
(1) a step of binding reaction between a ligand and a ligand binding molecule, a step of subjecting a complex of the ligand and the ligand binding molecule to enzymatic degradation, and a step of eluting the ligand binding molecule, A method for eluting a ligand-binding molecule, which comprises releasing a ligand-binding molecule from a ligand.
(2) The method according to (1), wherein the ligand is supported on a carrier.
(3) The method according to (2), wherein the carrier is a magnetic bead.
(4) The method according to (1), wherein the ligand-binding molecule is a display library.
(5) A step of reacting a ligand-carrying carrier and a ligand-binding molecule in which the ligand and the carrier are linked via an enzyme-decomposable linker, and subjecting the complex of the ligand-carrying carrier and the ligand-binding molecule to enzymatic degradation And a step of eluting the ligand-binding molecule, wherein the complex of the ligand and the ligand-binding molecule is released from the carrier by the enzymatic degradation.
(6) The method according to (5), wherein the carrier is a magnetic bead.
(7) The method according to (5), wherein the enzyme-decomposable linker is selected from the group consisting of DNA, RNA and peptide.
(8) The method according to (5), wherein the ligand-binding molecule is a display library.

  According to the ligand binding molecule elution method of the present invention, only the ligand binding molecule bound to the ligand can be specifically eluted. In addition, in the affinity selection from the display library, clones that bind to the ligand can be selectively eluted as compared with the conventional general elution method, so that weakly bound clones can be recovered by reducing the number of washings. . Furthermore, the time required for cleaning is shortened, and the number of panning operations can be reduced because noise is reduced.

Hereinafter, the present invention will be described in detail.
The method for eluting a ligand-binding molecule according to the present invention (Reaping Elution method; hereinafter referred to as “RE method”) binds a ligand and a ligand-binding molecule, and uses the complex of the ligand and the ligand-binding molecule for enzymatic degradation. In this method, the ligand-binding molecule specifically bound to the ligand is released from the ligand by cleaving and eluted (hereinafter referred to as “first embodiment of RE method”). In the RE method, a ligand-carrying carrier and a ligand-binding molecule in which a ligand and a carrier are linked via an enzyme-decomposable linker are subjected to a binding reaction, and the complex of the ligand-carrying carrier and the ligand-binding molecule is subjected to enzymatic degradation. In this method, the complex of the ligand and the ligand-binding molecule is released from the carrier by cleaving, and the ligand-binding molecule specifically bound to the ligand is eluted (hereinafter referred to as “the second embodiment of the RE method”). Called).

  In the conventional method for eluting ligand-binding molecules (FIG. 1A), for example, in a phage display method, a magnetic bead carrying a biopolymer (ligand) and a phage (presenting a ligand-binding molecule) are subjected to a binding reaction. By elution (for example, use of a strong eluent), phages are damaged, or clones bound to a carrier (in this case, magnetic beads) instead of a ligand are eluted as noise. On the other hand, according to the RE method (FIG. 1B), a ligand binding molecule or a ligand and a ligand can be obtained by subjecting the ligand itself or an enzymatically degradable linker to enzymatic degradation by a hydrolase (shown as “scissors” in FIG. 1B). The complex with the binding molecule can be released from the carrier and the ligand binding molecule can be eluted. Thus, according to the RE method, only the ligand-binding molecule bound to the ligand can be specifically eluted.

  Here, the ligand broadly refers to a molecule that binds to a specific molecule (for example, protein or peptide) with a certain binding affinity. On the other hand, the ligand binding molecule means a molecule that binds to the ligand. That is, when two molecules interacting (binding) are defined as a ligand, the other is a ligand-binding molecule.

  In the first embodiment of the RE method, the ligand is subject to enzymatic degradation. Accordingly, the ligand may be any molecule that undergoes enzymatic degradation. For example, amylose, cellulose, xylan used to collect a gene that recognizes a biopolymer (or a protein or peptide encoded by the gene). , Biopolymers such as chitin, mannan, galactan, DNA, RNA, peptide, polypeptide and the like. The ligand may be a multimer such as an oligomer. Furthermore, the enzyme used in the first embodiment of the RE method is an enzyme using each ligand as a substrate. For example, amylase is amylase, cellulose is cellulase, xylan is xylanase, chitin is chitinase, mannan. Include mannase, galactan includes galactosidase, DNA includes DNase or restriction enzyme, RNA includes RNase, and peptides and polypeptides include peptidases.

  In the first embodiment of the RE method, when the ligand is insoluble, it can be used as it is as a solid phase. In other cases, the ligand is supported on a carrier. Examples of the carrier include beads such as magnetic beads, agarose beads, immunobeads and hydrophilic beads, and plates for enzyme immunoassay (EIA). For example, the ligand is covalently bonded by chemical cross-linking using a functional group such as an amino group, a carboxyl group, a hydroxyl group, an epoxy group, and a tosyl group disposed on the surface of the carrier, and is bonded to the carrier.

  In the first embodiment of the RE method, for example, a display library can be used as the ligand-binding molecule. A display library refers to a collection of amplifiable units in which various expressed proteins and genes encoding them are physically linked or associated in a corresponding state. In this specification, each unit is called a clone. The target clone can be selected from these using the binding activity as an index, and the same clone can be amplified. A typical example is a phage display library in which the unit is a phage. Other units include cells such as E. coli (Lu Z. et al., Biotechnology 13 (4): 366 (1995)) and yeast (Shustra, EV et al., J. Mol. Biol. 292 (5): 949 (1999)). There are also cell surface displays that can be used. Ribosome display method (Mattheakis, LC et al., Proc. Natl. Acad. Sci. USA. 91) using in vitro expressed proteins using PCR-amplifiable RNA or DNA as a unit without using other phages or cells. : 9022 (1994), and Lipovsek, D. and Pluckthun. A., J. Immun. Methods, 290: 51 (2004)), mRNA display method (Keefe, AH and Szostack, JW, Nature 410: 715 (2001) ) And in vitro compartmentalization (IVC) method (Mastrobattista, E. et al., Chem. Biol. 12: 1291 (2005)).

  In the first embodiment of the RE method, a ligand (or a ligand-carrying carrier) and a ligand-binding molecule are first subjected to a binding reaction. Here, the binding reaction means a state in which a complex of a ligand and a ligand binding molecule is generated by affinity binding between the ligand and the ligand binding molecule. The binding reaction conditions can be appropriately selected according to the ligand to be used and the ligand binding molecule. For example, when a phage display library is used as a screening target for obtaining a ligand-binding molecule, the binding reaction can be performed under the following binding reaction conditions.

The binding reaction solution composition, eg, PBS conventionally used well Experimental Biochemistry (8g NaCl, 0.2g KCl, 2.68g Na 2 HPO 4 · 7H 2 O, 2.4g KH 2 PO 4 / L, pH7.4) Ya A solution obtained by adding 0.1% Tween-20 as a surfactant to a phosphate buffer near neutral pH such as TBS (10 mM Tris-HCl, 150 mM NaCl pH 7.5) may be used. In general, the more the pH deviates from near neutral, the less bound phage. In order to narrow down the conditions, a buffer in which the pH of the binding reaction solution is in the range of pH 3 to pH 10 can be selected. In general, the higher the salt concentration, the fewer phage clones that bind. In order to narrow down the conditions, the salt concentration can be selected in the range of 0M to 3M. For blocking purposes, for example, 0.1% -5% BSA (bovine serum albumin), 0.1% -5% skim milk, 0.1% -10% FBS (fetal bovine serum), or 1 mg / ml-100 mg / ml nucleic acid A solution may be added. In the binding reaction, for example, the temperature may be room temperature and mixed for 1 hour to 12 hours. The temperature of the binding reaction may be selected in the range from 0 ° C. to 50 ° C., for example, for the purpose of narrowing down candidates. T7 phage is lethal at 50 ° C or higher, but only one round of selection is performed. When DNA is recovered and used, lethal 50 ° C or higher can be used as a binding reaction condition. For example, in the case of a propeller type inversion type tube mixer, the stirring rate may be 60 rpm to 180 rpm, and in the case of a tube stand type mixer, mixing may be carried out for about 1 minute once every 5 minutes so that the ligand-carrying carrier does not precipitate. The density of the ligand-carrying carrier in the binding reaction may be about 10 ⁹ / ml or 1 mg / ml, for example, for φ1 μm beads. Increasing the density of the ligand-carrying carrier can recover a phage clone complex having a low affinity, and decreasing the density reduces the complex of a phage clone having a low affinity.

  Then, washing can be performed to remove free phage that is not bound to the ligand-carrying support after the binding reaction. Washing can be performed using, for example, a solution obtained by adding 0.1% Tween-20 to PBS or TBS, similarly to the buffer used for the binding reaction. What is necessary is just to use the capacity | capacitance of a washing | cleaning solution several times or more of a coupling | bonding reaction liquid volume, for example. For example, the washing time may be changed 5 times at a time, and the washing solution may be exchanged three times. The cleaning process can be omitted. Omission of washing makes it easy to obtain clones with low affinity or clones with high complex formation rate and high dissociation rate even with high affinity. On the other hand, the cleaning time and frequency can be further increased. Extending the washing time makes it easier to obtain clones with high affinity and a slow complex dissociation rate.

  Next, in the first embodiment of the RE method, the obtained complex of a ligand and a ligand-binding molecule is subjected to enzymatic degradation targeting the ligand and cleaved (decomposed). Enzymatic degradation conditions (eg, amount of enzyme, temperature, pH, etc.) can be appropriately determined according to the enzyme used.

  The elution of the ligand binding molecule is desirably performed using a solution having the same composition as the washing solution. For example, when a phage display library is used as a screening target for obtaining a ligand-binding molecule, a change in the buffer composition may cause the release of noise phages bound to the phage outline other than the display protein. . For the same reason, it is desirable that the temperature of the elution conditions is the same as the washing operation. The elution time is preferably as gentle and short as possible (for example, about 5 minutes) as long as the ligand-binding clone (ligand-binding molecule) can be recovered by enzymatic degradation. This is because noise phages are liberated due to the extended time. In this way, the ligand binding molecule bound to the ligand can be released from the ligand (or ligand and carrier).

  On the other hand, in the second embodiment of the RE method, examples of the ligand include antigens, antibodies, cytokines, cytokine receptors, enzyme inhibitors, inhibited enzymes, coenzymes, enzymes, enzyme substrates, and the like. Since there are binding proteins for almost any compound, almost any compound can be a ligand. Further, in the first embodiment of the RE method described above, biopolymers listed as ligands (for example, amylose, cellulose, xylan, chitin, mannan, galactan, DNA, RNA, peptide, polypeptide, etc.) or xylose can be used as a ligand. Good. Furthermore, examples of the ligand-binding molecule include a display library, as in the first embodiment of the RE method.

  Examples of the enzyme (linker / enzyme) corresponding to the enzyme-decomposable linker include amylose / amylase, cellulose / cellulase, xylan / xylanase, chitin / chitinase, mannan / mannase, galactan / galactosidase, DNA / DNase or Restriction enzymes, RNA / RNases, and peptides, polypeptides or proteins / peptidases (eg, Factor Xa, enterokinase, thrombin, prolyl endopeptidase, V8 protease). When DNA or RNA is used as a linker capable of enzymatic degradation, it may be single-stranded or double-stranded.

  Furthermore, examples of the carrier used in the second embodiment of the RE method include those listed in the first embodiment of the RE method described above.

  In the second embodiment of the RE method, first, a ligand and a carrier are linked via an enzyme-decomposable linker to produce a ligand-carrying carrier. Binding between the ligand and the enzyme-decomposable linker and between the carrier and the enzyme-decomposable linker can be performed according to the binding method between the ligand and the carrier in the first embodiment of the RE method.

  Next, in the second embodiment of the RE method, according to the first embodiment of the RE method, the ligand-carrying carrier and the ligand-binding molecule are subjected to a binding reaction, and then washed, and the ligand and the ligand-binding molecule on the ligand-carrying carrier are washed. To obtain a complex produced by binding to. Next, the obtained complex is subjected to enzymatic degradation using a linker capable of enzymatic degradation, and eluted to release a complex of a ligand and a ligand-binding molecule. Furthermore, the ligand binding molecule is recovered by solid-liquid separation.

Specifically, in FIG. 1C, as a generalization of the RE method (second embodiment), a bait ligand (for example, a high affinity inhibitor) as a ligand, synthetic DNA as an enzymatically degradable linker, and epoxy magnetic beads as a carrier The case where is used is shown. The synthetic DNA is aminated at one end to link with an epoxy magnetic bead and has an SH ₂ group at the other end to link to a bait ligand. These bait ligands, synthetic DNA and epoxy magnetic beads are then ligated. Furthermore, a bait ligand-carrying magnetic bead linked via the synthetic DNA obtained is bound to a target enzyme or protein that is a ligand-binding molecule, and the target enzyme or protein is bound to the bait ligand on the magnetic bead. Furthermore, by cleaving synthetic DNA with DNase, the target enzyme or protein bound to the bait ligand can be released from the magnetic beads and eluted.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.

[Example 1] Collection of amylose-binding clones (first embodiment of RE method)
1-1. Panning with amylose beads A phage display library (FRTL17) using metagenome as a source was prepared using DNA (metagenome) extracted and purified from forest soil and T7SELCT 10-3b (NOVAGEN) (International Publication No. 2005/121336). Issue pamphlet).

  Immediately before use, 50 μl of FRTL17 P2 (double amplified) lysate was infected to host E. coli BLT5615 (5 ml) prepared by induction with IPTG according to the manual, and cultured at 37 ° C. for 2 hours in L medium. I let you.

  The centrifuged lysate supernatant was washed 3 times with 4 ml of PBST (PBS + 0.1% Tween20) using an ultrafiltration centrifuge unit, Amicon YM-100, and the medium was removed and concentrated 5 times. 100 μl of this phage solution (ligand binding molecule) in 100 μg of amylose magnetic beads (New England Biolabs No. E8035S) (ligand-supported carrier: in the amylose magnetic beads, amylose (ligand) and magnetic beads (carrier) are cleaved by alpha amylase. Possible) and mixed for 16 hours at 4 ° C. The supernatant was discarded and washed 4 times with 400 μl PBST and once with 400 μl PBS. To this bead, 100 μl of PBS containing 2.2 μg of Bacillus licheniformis alpha amylase (Sigma No. A4551) was added and mixed, and left at room temperature for 5 minutes. After standing, the supernatant was recovered and used as the first round eluate.

  Next, 50 μl of the first round eluate was infected with host E. coli BLT5615 (1 ml) prepared by induction with IPTG, and cultured at 37 ° C. for lysis. After lysis, the supernatant medium collected by centrifugation was replaced with PBST and concentrated 5 times as described above (one round ended). Such an operation was repeated 4 rounds.

Phage titer (concentration) was measured according to the manual of Novagen. The eluate phage titer increased from 10 ⁵ / ml in the first round to 10 ⁸ / ml in the fourth round.

1-2. Monitoring of enriched DNA 25 μl of 10% SDS was added to 100 μl of 5 times concentrated lysate after each round amplification obtained in 1-1 above, and incubated at 70 ° C. for 10 minutes. Next, 100 μl of TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA) saturated phenol and 25 μl of chloroform were added and mixed for 1 minute.

  After centrifugation at 15000 rpm for 1 minute, the aqueous layer was collected, and 1/10 volume of sodium acetate and 2 μl of 20 mg / ml glycogen were added and mixed. Further, 2.5 volumes of ethanol was added, and the DNA precipitate was collected by centrifugation at 15000 rpm for 10 minutes and dissolved in 20 μl of TE. This was used as a template DNA for PCR at 1 μl per 20-30 μl of reaction solution.

  For PCR, Ex-Taq (TAKARA) was used for the enzyme and the reaction solution, and T7UP and T7Down (Novagen) were used at 0.2 μM as primers. The program was 96 cycles at 2 minutes for 1 minute, 94 ° C for 30 seconds, 55 ° C for 30 seconds and 72 ° C for 90 seconds, and 72 ° C for 5 minutes in 1 cycle.

  After completion of PCR, 7 μl of the reaction solution was analyzed by agarose electrophoresis. The results are shown in FIG. FIG. 2 shows the results of insert PCR analysis (agarose electrophoresis pattern) after each round of amplification when amylose magnetic beads are used in the RE method. The numbers indicate the number of rounds, and M is a size marker.

  As shown in FIG. 2, it was found that a specific DNA fragment was enriched (an increase in S / N ratio) as the round progressed. When the number of washings during panning was 3 times, a pattern in which more DNA fragments were enriched was obtained.

1-3. Analysis of collected DNA sequence The fourth round eluate obtained in 1-1 above was appropriately diluted and plated on host bacteria and soft agar according to the manual of Novagen to form plaques. Next, plaque PCR was performed from more than 20 plaques, the sequence of the amplified DNA fragment was examined, and the homology analysis of the encoded amino acid sequence was performed. The results are shown in Table 1 and FIGS. Table 1 shows the results of homology analysis of amino acid sequences encoded by DNA fragments recovered by the RE method with amylose magnetic beads. 3A to E show the alignment of the results of homology analysis of the amino acid sequences (SEQ ID NOs: 1 to 5) of the 5 clones in Table 1. FIG. 3A to 3E, the amino acid sequence encoded by the DNA fragment from which the Query sequence is obtained, while the Sbjct sequence is the amino acid sequence of the protein hit.

  As a result, clear homology was recognized with enzymes that hydrolyze and modify sugars as shown in Table 1 and FIGS. From the above results, enzyme gene fragments that catalyze the hydrolysis or transfer of sugar with low noise could be collected by the RE method.

[Example 2] Comparison of enrichment efficiency of biotin enzyme fragment clones (RE method second embodiment)
2-1. Binding of DNA Spacer (Enzyme-Decomposable Linker) to Beads (Carrier) A complementary 24-mer 5′-terminal aminated oligo DNA and 5′-terminal biotinylated oligo DNA having the following sequences were annealed under the following conditions.
5 ′ terminal aminated oligo DNA: 5′NH ₂ -GCTGCAAGCTTTGAATTCGGATCC (SigmaGenosys: SEQ ID NO: 6)
5 'terminal biotinylated oligo DNA: 5' Biotin-GGATCCGAATTCAAAGCTTGCAGC (SigmaGenosys: SEQ ID NO: 7)

  Each oligo DNA was dissolved in PBS so as to have a concentration of 0.2 mM, mixed in an equal amount, then placed at 95 ° C. for 5 minutes, left uncooled, and gradually cooled to 30 ° C. over 20 hours. A part was subjected to polyacrylamide electrophoresis, and 90% or more was judged to be a double chain due to the mobility delay.

Next, 60 μl of each of the annealed oligo DNA solution, 3M (NH ₄ ) ₂ SO ₄ and 40 mM carbonate buffer (pH 10.8) was mixed, and further 16 mg at 37 ° C. with 3 mg of magnetic beads (Dnynabeads M270-Eppoxy (Dynal)). Mixed for hours. To block unreacted epoxy groups, 400 μl of 1M Tris-HCl (pH 8.0) was added and mixed at 37 ° C. for 1 hour.

  After mixing for 1 hour, the supernatant was removed by separation with a magnet, and the beads were washed 3 times with 400 μl of TE. The biotinylated DNA beads were stored at 4 ° C. in TE until use.

2-2. Preparation of streptavidin beads (ligand-supporting carrier) Add 1 μl of biotinylated DNA beads obtained in 2-1 above to 180 μl (1 mg / ml PBS) of streptavidin (PIERCE) (ligand) and mix at 4 ° C overnight. did. After overnight mixing, the supernatant was removed and the beads were washed 3 times with 400 μl PBST (PBS + 0.1% Tween20) and stored at 4 ° C. in PBST until use.

2-3. Enrichment factor measurement
The T7 phage clone 1SASD34 has a biotin enzyme fragment consisting of approximately 76 amino acids in the display portion and binds to streptavidin (Japanese Patent Application No. 2005-283992).

Therefore, 200 μl (PBST) of a mixture of 1 × 10 ⁹ wild-type T7 phage and this 1/250 amount of 1SASD34 phage (biotin enzyme gene: ligand binding molecule) was added to 200 μg of the beads obtained in 2-2 above, Mix for 1 hour at 23 ° C.

After mixing for 1 hour, the supernatant was removed and washed 3 times for 5 minutes with 400 μl PBST. During the third washing, 400 μl of the washing solution (bead suspension) was divided into two equal parts and transferred to another tube, and the supernatant was removed. One side was added with 100 μl of 1% SDS, and the other side was added with 100 μl DNase I solution (TKARA DNase I 700 U / ml PBS + 5 mM MnCl ₂ ) and left at 23 ° C. for 5 minutes, and then the supernatant was collected.

As measured in the Novagen manual at each phage titer of soft earth Gar method, 1% SDS eluate 0 / ml, DNaseI eluate was 1.5 × 10 ⁴ / ml. Under the former condition, it is considered that the phage was damaged by SDS. As a result of affinity staining of the latter plate with alkaline phosphatase-labeled streptavidin (Japanese Patent Application No. 2005-283992), 60 plaques (85%) out of 70 plaques were positive. The enrichment factor (concentration rate) under these conditions was 212 (0.4% → 85%).

  As described above, by the RE method, bound phages could be recovered more efficiently than elution with 1% SDS.

[Example 3] Recovery of biotin enzyme gene fragment from soil metagenome by RE method (second embodiment of RE method)
250 μl of 5 × FRTL17P2 / PBST (ligand binding molecule) and 100 μg of the beads (ligand-supporting carrier) obtained in Example 2-2 above were mixed overnight at 4 ° C., and 5 μL with 400 μl of PBST in the same manner as in Example 1. Washing 3 times per minute. The phage recovered by treating these beads with 100 μl of DNaseI solution for 5 minutes was infected with E. coli BLT5615 and amplified, and two rounds of panning were performed.

  The eluate collected in the second round was plated to form plaques and affinity stained with alkaline phosphatase-labeled streptavidin (Japanese Patent Application No. 2005-283992). The results are shown in FIG. FIG. 4 shows the positive plaque staining pattern in the second round.

  As a result, 8.4% of plaques were stained as shown in FIG. Furthermore, the corresponding plaques were collected with a chip and subjected to plaque PCR according to the manual of Novagen, and the base sequence of the amplified DNA fragment was examined. FIG. 5 shows an alignment of amino acid sequences encoded by the base sequence of the amplified DNA fragment in each positive clone. In FIG. 5, “B” surrounded by a square represents a biotin-binding amino acid position.

  As shown in FIG. 5, it was found that all have a biotin binding site of a novel biotin enzyme gene fragment. That is, more than 10 kinds of new biotin enzyme fragments could be easily collected by only two screenings.

  From the above results, the target gene fragment was efficiently concentrated and recovered from the metagenome by the RE method.

  The RE method can be used for inhibitors, enzyme gene cloning, and drug target identification by phage display method or in vitro display method.

FIG. 1A shows a conventional elution method. FIG. 1B shows the RE method. FIG. 1C shows the generalization of the RE method. FIG. 2 shows the results of insert PCR analysis (agarose electrophoresis pattern) after each round of amplification when amylose magnetic beads are used in the RE method. FIG. 3A shows an alignment of the results of homology analysis of the amino acid sequence (SEQ ID NO: 1) of the clone (No. 1) in Table 1. FIG. 3B shows an alignment of the results of homology analysis of the amino acid sequence (SEQ ID NO: 2) of the clone (No. 2) in Table 1. FIG. 3C shows an alignment of the results of homology analysis of the amino acid sequence (SEQ ID NO: 3) of the clone (No. 3) in Table 1. FIG. 3D shows an alignment of the results of homology analysis of the amino acid sequence (SEQ ID NO: 4) of the clone (No. 4) in Table 1. FIG. 3E shows an alignment of the results of homology analysis of the amino acid sequence (SEQ ID NO: 5) of the clone (No. 5) in Table 1. FIG. 4 shows the biopanning for obtaining biotin enzyme gene fragments from the soil metagenome by the RE method, and the staining pattern of alkaline phosphatase-labeled streptavidin in the second round of positive plaques. FIG. 5 shows an alignment of amino acid sequences encoded by the base sequence of the amplified DNA fragment in each positive clone obtained in FIG.

Claims (8)

Binding a ligand and a ligand-binding molecule;
Subjecting the complex of the ligand and ligand binding molecule to enzymatic degradation;
Eluting the ligand binding molecule;
And elution of the ligand binding molecule, wherein the ligand binding molecule is released from the ligand by the enzymatic degradation.   The method according to claim 1, wherein the ligand is supported on a carrier.   The method according to claim 2, wherein the carrier is a magnetic bead.   The method of claim 1, wherein the ligand binding molecule is a display library. A step of binding reaction between a ligand-carrying carrier and a ligand-binding molecule in which a ligand and a carrier are linked via an enzymatically decomposable linker;
Subjecting the complex of the ligand-carrying carrier and ligand-binding molecule to enzymatic degradation;
Eluting the ligand binding molecule;
And releasing a complex of the ligand and the ligand-binding molecule from the carrier by the enzymatic degradation.   6. A method according to claim 5, characterized in that the carrier is a magnetic bead.   6. The method of claim 5, wherein the enzymatically degradable linker is selected from the group consisting of DNA, RNA and peptides.   6. The method of claim 5, wherein the ligand binding molecule is a display library.

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