JP2013039060A - Linker for evolving protein with enzyme-like activity, and method for screening such protein using the linker - Google Patents

Abstract

PROBLEM TO BE SOLVED: To create a linker for evolving use, capable of efficiently screening a protein with enzyme-like activity in a cDNA display method.SOLUTION: The linker for evolving protein with enzyme-like activity is provided, comprising: a main chain having a side chain linkage site in close proximity to the 3' terminal end side; a side chain with its one end linked to the side chain linkage site; and a substrate unit bound via crosslinks to the main chain; wherein, the main chain has an mRNA binding site and a crosslink-forming site forming crosslinks to the substrate unit; the substrate unit has a substrate unit-sided crosslinking site, a solid phase binding site, a substrate linkage site, and a RNA ligase recognition sequence; and the side chain has a protein binding site; in the substrate unit, the substrate linkage site is situated between the substrate-sided crosslinking site and the solid phase binding site and at a position where a protein with enzyme-like activity bound to the side chain can be bound.

Description

  The present invention relates to a protein evolution linker having enzyme-like activity and a method for screening a protein having enzyme-like activity using the linker.

Conventionally, in the evolutionary engineering of enzymes, for example, a specific gene is selected from a gene library, this is cloned, the cloned gene is incorporated into a cell, the cell is transformed, screening using an agarose gel plate, microtiter Various screening methods such as screening using a plate, cell-in-droplet, and cell surface display have been used to select and further improve a protein having a desired function.
In such a method, a specific strain is selected by amplifying a gene, integrating it into a plasmid or the like, and growing cells transfected with the recombinant plasmid on a plate containing a specific drug. Thereafter, in order to grow the obtained strain and obtain the expressed protein, further purification such as cell disruption and separation by electrophoretic movement is required, which requires enormous time and labor.

On the other hand, in evolution engineering, display technologies such as phage display that can be handled if the library size is about 10 ⁸ have been developed for the purpose of obtaining affinity molecules such as antibodies. However, in the phage display technology using cells, there are limitations such as inability to obtain peptides that are toxic to cells, and thus it is not always possible to handle libraries with sufficient diversity and size.
In order to overcome these limitations, cell-free translation display technology has been developed, and ribosome display technology (see Non-Patent Documents 1 and 2) and mRNA display (In vitro virus) technology (see Non-Patent Documents 3 and 4). It has been developed. These have been mainly aimed at obtaining affinity molecules as described above, but in recent years there have been examples of use for the purpose of enzyme evolution.
Specifically, ribosome display technology (see Non-Patent Document 5, hereinafter referred to as “Conventional Technology 1”), mRNA display technology (see Non-Patent Document 6, hereinafter referred to as “Conventional Technology 2”), and cDNA display. Technology (see Patent Document 1).

  Protein screening using the ribosome display technology of Prior Art 1 is performed according to the following procedure. First, a mixture of active and inactive T4 DNA ligase gene and mRNA encoding spacer peptide is prepared, cDNA having cohesive ends and mRNA are hybridized, in vitro translation is performed, and mRNA-cDNA- A ribosome-enzyme complex is formed. The displayed active T4 DNA ligase enzyme binds other dsDNA probes modified with biotin into this complex. The complex is dissociated and the mRNA encoding the active T4 DNA ligase gene is labeled with biotin. Selection of mRNA modified with biotin on magnetic beads is performed using streptavidin. The obtained mRNA is collected, amplified by RT-PCR, and selected by performing in vitro translation. The prior art 1 is to repeat this cycle.

  In the screening of proteins using the mRNA display technique of Conventional Technique 2, a certain DNA is transcribed into mRNA, and puromycin is bound to it and translated. A protein is bound to the puromycin for display, and this is reverse transcribed using a modified primer, and the cDNA bound to the primer is selected and amplified by PCR. What repeats this is the technique of Conventional Example 2.

Japanese Patent No. 4318721

Mattheakis et sl., Proc Natl Acad Sci U S A. (1994) 91, 9022-9026 Hanes J, Pluckthun A. Proc Natl Acad Sci U S A. (1997) 94, 4937-4942 Nemoto N, et al. FEBS Lett. (1997) 414, 405-408 Roberts RW, Szostak JW. Proc Natl Acad Sci U S A. (1997) 94, 12297-12302 Takahashi et ai., BBRC, 336 (2005) 987-993 Seeling, et al., Nature vol. 448, 16 Aug. 2007, 824-832

Prior art 1 is excellent in that the concentration efficiency can be increased without using cells. However, there is a problem in that it lacks stability due to the use of a ligated structure utilizing non-covalent bonds in which T4 DNA ligase is displayed on the ribosome via a spacer.
Moreover, since the prior art 2 is a display technology using a linked structure using a covalent bond, it is superior to the prior art 1 in terms of stability. However, because the nucleic acid molecule covalently linked to the protein is mRNA, not cDNA, its stability is incomplete.

On the other hand, in the screening method for selecting a protein having enzyme-like activity, it is preferable to select a protein having various activities regardless of whether it is toxic to cells or not. .
Furthermore, in order to efficiently select proteins having various activities, a display method capable of processing a large amount of sample in a short time with a smaller system is required. In order to meet such a demand for high throughput, it is preferable that the displayed protein can be stably recovered and purified by a simple method.
From the above, it is a linker that is compatible with the cDNA display method, and it is possible to evaluate and select a large amount of the activity of a desired protein in a short time, and to evolve such a protein to have a desired function. There was a high social demand for a linker for protein evolution.

The present invention has been completed under the above circumstances.
In the first aspect of the present invention, a main chain having a side chain linking site in the vicinity of the 3 ′ end side, a side chain having one end connected to the side chain linking site, and a bridge to the main chain via a bridge. The main chain is located on the 3 ′ end side of the linker and functions as a primer for reverse transcription, and the 5 ′ end side of the linker. A cross-linking site that forms a cross-link with the substrate unit; and the substrate unit has a cross-linking site on the side of the substrate unit for forming a cross-link with the main chain, and a solid phase. A cross-link is formed between the main chain and a solid phase binding site having a predetermined molecule that forms a bond with the substrate, a substrate linking site to which a substrate capable of acting on the protein having the enzyme-like activity is linked, and the main chain One end forms an RNA ligase recognition site And an RNA ligase recognition sequence whose other end is linked to the substrate linking site; the substrate linking site is between the substrate side cross-linking site and the solid phase binding site and the side chain A protein evolution linker having an enzyme-like activity, wherein the side chain has a protein-binding site for binding a protein having an enzyme-like activity. It is.

Here, the main chain preferably has a cross-linking site that forms a cross-linkage with the substrate unit on the 5 ′ side of the side chain binding site.
The substrate unit is bonded to the main chain by bonding a bridge between the main chain cross-linking site and the substrate unit side cross-linking site. The crosslink formed here is preferably any one selected from the group consisting of photocrosslinking, chemical crosslinking, and enzymatic crosslinking.
In addition, one end of this substrate unit is configured to form an RNA ligase recognition site after a bridge is formed between the main chain and becomes a double strand.

  A second aspect of the present invention is a linker having a main chain having a side chain linking site in the vicinity of the 3 ′ end, a side chain having one end linked to the side chain linking site, and a substrate unit. The main chain is located on the 3 ′ end side of the RNA ligase recognition site, is located on the 5 ′ side of the RNA binding site that functions as a primer for reverse transcription, and the RNA ligase recognition site; A main chain side cross-linking site that forms a cross-link with the substrate unit, an RNA ligase recognition site, and a double stranded formation region for forming the RNA ligase recognition site; the other end of the side chain The portion comprises a protein binding site for binding a protein having enzyme-like activity; the substrate unit comprises a solid phase binding site having a predetermined molecule that forms a bond with a solid phase; and the enzyme-like site. Produced by active protein A substrate linking site to which a possible substrate is linked, and a cross-linking site where one end forms a bridge with the main chain and the other end is linked to the substrate linking site. A protein evolution having an enzyme-like activity, wherein the substrate linking site is located between the cross-linking site and the solid-phase binding site and is capable of binding a protein having an enzyme-like activity bound to the side chain; It is a linker.

Here, the main chain preferably has a cross-linking site that forms a cross-linkage with the substrate unit on the 5 ′ side of the side chain binding site.
The substrate unit is bonded to the main chain by bonding a bridge between the main chain cross-linking site and the substrate unit side cross-linking site. The crosslink formed here is preferably any one selected from the group consisting of photocrosslinking, chemical crosslinking, and enzymatic crosslinking.
In the second embodiment, the substrate unit forms a cross-link with the cross-linking site in the double-stranded part formed on the 3 ′ side of the main chain.

  Note that, in each of the substrate units constituting the linker of the first and second aspects of the present invention, the solid-phase binding site on the 3 ′ end side is composed of biotin; an amino group, a thiol group, and a carboxyl group. Any one selected from the group consisting of a functional group; an antigen in an antigen-antibody reaction; a histidine tag, a streptag, GST (glutathione S-transferase, hereinafter abbreviated as “GST”), and a purification tag comprising polydeoxyadenosine It is preferable that a substrate capable of acting on the protein having the enzyme-like activity is linked to the substrate linking site.

  Here, the combination of a protein having an enzyme-like activity bound to the protein binding site of the side chain and a substrate is a polypeptide having an activity equivalent to that of a peptide-degrading enzyme and an arbitrary peptide sequence; A polypeptide having an equivalent activity and an arbitrary sugar chain; a polypeptide having an activity equivalent to a nucleolytic enzyme and an arbitrary nucleic acid sequence; a polypeptide having an activity equivalent to a lipolytic enzyme and an arbitrary lipid; and a synthetic polymer. It is preferably any one selected from the group consisting of a polypeptide having an activity of degrading and any synthetic polymer.

A polypeptide having an activity equivalent to that of a nucleic acid ligase and an arbitrary nucleic acid sequence; a polypeptide having an activity equivalent to that of a peptide-linked enzyme and an arbitrary peptide sequence; a polypeptide having an activity equivalent to that of a peptide transferase and an arbitrary peptide A polypeptide having an activity equivalent to that of a molecule-ligating enzyme that links a molecule to a polypeptide or peptide, and any peptide sequence and any transfer molecule substrate; having an activity equivalent to that of a molecule-linking enzyme that links a molecule to a nucleic acid Polypeptide, any nucleic acid sequence and any transfer molecule substrate; polypeptide having activity equivalent to glycosyltransferase; any sugar chain and a substrate to be modified with any sugar chain; and catalyzing a novel ligation reaction It is preferably any one selected from the group consisting of a polypeptide having an activity and an arbitrary substrate.

  A third aspect of the present invention is an mRNA-linker, wherein the above-described linker for protein evolution and mRNA having a sequence complementary to a part of the main chain of the linker are bound by RNA ligase at the mRNA binding site. A conjugate generating step; a conjugate that synthesizes a protein having enzyme-like activity using the mRNA as a template, and obtains an mRNA-linker-protein conjugate in which the protein is bound to a protein binding site on the side chain of the linker A production step; a cDNA-linker-protein conjugate purification step that synthesizes a cDNA chain by reverse transcription using the mRNA as a template; a substrate in which the protein having the enzyme-like activity is linked to a substrate linkage site; The above-mentioned cDNA-linker-protein conjugate is obtained by linking these by acting on a desired substrate bound on the phase. An immobilization step of immobilizing on the solid phase; a first washing step of washing the solid phase to which the cDNA-linker-protein conjugate is bound with a first buffer; and the immobilized cDNA-linker-protein A method for screening a protein having enzyme-like activity, comprising: a recovery step of recovering a conjugate; and an amplification step of amplifying the recovered cDNA-linker-protein conjugate.

  The fourth aspect of the present invention is an mRNA-, wherein the above-described linker for protein evolution and mRNA having a sequence complementary to a part of the main chain of the linker are bound by RNA ligase at the mRNA binding site. Linker conjugate production step; synthesis of a protein having enzyme-like activity using the mRNA as a template, and obtaining an mRNA-linker-protein conjugate in which the protein is bound to a protein binding site on the side chain of the linker A cDNA-linker-protein conjugate purification step for synthesizing a cDNA chain by reverse transcription reaction using mRNA as a template; and the cDNA-linker-protein conjugate via a solid-phase binding site. A solid phase binding step of binding to the cDNA; and the solid phase to which the cDNA-linker-protein conjugate is bound as a first buffer. Washing with a first washing step; causing the protein having an enzyme-like activity bound to a protein binding site on the side chain of the linker to act on the substrate linked to the substrate linking site, and the cDNA-linker-protein A cleaving step for cleaving the conjugate from the solid phase; a recovery step for recovering the cleaved cDNA-linker-protein conjugate; and an amplification step for amplifying the cDNA of the recovered cDNA-linker-protein conjugate; A method for screening a protein having enzyme-like activity.

According to the present invention, a linker for protein evolution can be efficiently generated. Further, by using this linker, a protein having a desired enzyme-like activity can be efficiently screened.
Furthermore, by changing the combination of a protein having enzyme-like activity and a substrate, it is possible to obtain a protein having completely novel activity.

FIG. 1A schematically shows the structure of the A-type linker of the present invention. FIG. 1B schematically shows the structure of the B-type linker of the present invention. FIG. 2 is a diagram schematically showing the puromycin segment constituting the linker of the present invention. FIG. 2 is a diagram schematically showing amino segments constituting the linker of the present invention. FIG. 4 is a diagram showing the results of gel electrophoresis of an azide segment and a cross-linked product (F). FIG. 5 is a diagram schematically showing a substrate unit constituting the linker of the present invention. FIG. 6 is a diagram showing gel electrophoresis results of a psoralen-amino segment, a puromycin segment, and a cross-linked product (E) obtained by cross-linking them.

FIG. 7 is a diagram showing the results of gel electrophoresis of the A-type linker (crosslinked product (G)) of the invention in which the crosslinked product (E) and the substrate unit are crosslinked. FIG. 8 is a chromatogram when the psoralen-amino segment and the puromycin segment are cross-linked and then the unreacted psoralen-amino segment and cross-linked product (E) are separated by HPLC. FIG. 9 is a diagram showing a gel electrophoresis result when the cross-linked product (F) is cleaved with an enzyme (subtilisin) displayed on puromycin. FIG. 10 shows the results of gel electrophoresis when the substrate was cleaved with the enzyme (RNase T1) displayed on puromycin of the cross-linked product (F).

Hereinafter, the present invention will be described in more detail with reference to FIGS.
As shown in FIG. 1A, the protein evolution linker of the first aspect of the present invention (hereinafter sometimes referred to as “A-type” linker) includes (a) a side chain linking site in the vicinity of the 3 ′ end. A linker having a main chain, (b) a side chain having one end connected to the side chain linking site, and (c) a substrate unit bonded to the main chain via a bridge.
Here, the main chain includes (a1) an mRNA binding site and (a2) a cross-linking site. The mRNA binding site is located on the 3 ′ end side of the linker and functions as a primer for reverse transcription. In addition, a compound or a substituent for forming a crosslink is disposed at the crosslink formation site, and this crosslink formation site is located on the 5 ′ end side of the linker. And a bridge | crosslinking is formed between the substrate unit side bridge | crosslinking parts mentioned later, and a principal chain and a substrate unit are combined.

The side chain includes (b1) one end and (b2) the other end, and is connected to the side chain connecting portion of the main chain at the one end, and the other end. Has a protein binding site for binding a protein having enzyme-like activity.
The substrate unit further comprises (c1) a solid phase binding site, (c2) a substrate linking site, (c3) a substrate unit side cross-linking site, and (c4) an RNA ligase recognition sequence. This substrate unit has a structure in which an RNA ligase recognition sequence is arranged at one end and a solid-phase binding site is arranged on the other side with a substrate on which the protein having the enzyme-like activity can act. ing.
Here, the substrate unit side cross-linked site is located on the 5 ′ side of the substrate unit, and at this cross-linked site, a cross-link with the main chain is formed and bonded to the main chain. Since the one side end has a sequence complementary to the main chain except for several bases at the 5 ′ end, a double strand is formed by removing these several bases. Then, several bases at the 5 ′ end that do not form a double strand are RNA ligase recognition sites.

As shown in FIG. 1B, the protein evolution linker of the second aspect of the present invention (hereinafter sometimes referred to as “B-type linker”) has (a ′) a side chain linking site in the vicinity of the 3 ′ end. A linker having a main chain provided, (b) a side chain having one end connected to the side chain linking site, and (c ′) a substrate unit bonded to the main chain via a bridge.
Here, the main chain includes (a1) an mRNA binding site, (a2 ′) a crosslinking site, (a3 ′) an RNA ligase recognition site, and (a4 ′) a double-stranded forming region. This double-stranded forming region has a sequence complementary to the corresponding region of the main chain and forms the RNA ligase recognition site, but several bases located at the 5 ′ end thereof are complementary to the main chain. Since it does not have a sequence, it does not form a main strand and a double strand. This part becomes the RNA ligase recognition site of (a3 ′).
Thereafter, the RNA ligase recognizes the RNA ligase recognition site formed as described above and links the mRNA and the linker.

  Here, the mRNA binding region (a1) is a region that functions as a primer for reverse transcription when reverse transcription is performed on the linker. This region preferably consists of about 1 to 15 bases, particularly 3 to 5 bases. If the number of bases exceeds 15, the protein binding efficiency as a linker is deteriorated, so that the number of bases described above is preferable from the viewpoint of the binding efficiency with a protein and the reaction efficiency as a primer.

The substrate unit (c ′) of the second aspect includes (c1) a solid phase binding site, (c2) a substrate linking site, and (c3 ′) a cross-linking site. Here, the solid phase binding site and the substrate linking site are the same as the substrate unit of the first aspect.
Here, the substrate unit has a cross-linking site (c ′) that forms a cross-link with the main chain at one end, and a solid-phase bond that forms a bond with a solid phase at the other end. Each has a site.
And this bridge | crosslinking formation site | part is located in the arbitrary places of the said substrate unit, A bridge | crosslinking with a principal chain is formed in this bridge | crosslinking formation site | part, and it couple | bonds with a principal chain. The position of the cross-linking site can be arbitrarily set according to the substrate recognition / cleavage site.

The solid phase binding site contained in the linker of the present invention refers to a site capable of forming a bond with a specific molecule immobilized on the solid phase. For example, when streptavidin is immobilized on the solid phase, biotin hits this. In addition, when the substrate is immobilized on the solid phase via streptavidin and the substrate immobilized on the solid phase and the substrate coupled to the substrate coupling site in the substrate unit are bound by the ligation enzyme, It will contain such a substrate immobilized via avidin.
The solid phase binding site of the substrate unit used in the A-type linker and B-type linker of the present invention is constituted by a predetermined molecule or functional group.
More specifically, each of the substrate units constituting the linker of the first aspect and the second aspect of the present invention has biotin; amino group, thiol group at the solid phase binding site on the 3 ′ end side thereof. And an antigen in an antigen-antibody reaction; any one selected from the group consisting of a histidine tag, a streptotag, GST (glutathione-S-transferase), and a purification tag consisting of polydeoxyadenosine It is preferable that a substrate capable of acting on the protein having the enzyme-like activity is linked to the substrate linking site.

The solid phase binding site of the substrate unit used in the A-type linker and B-type linker of the present invention is composed of predetermined molecules. Examples of such a predetermined molecule include biotin when avidin and streptavidin are bound to the solid phase, maltose when maltose binding protein is bound, and G protein when bound. When guanine nucleotide, polyhistidine peptide is bound, metal such as Ni or Co, when glutathione-S-transferase is bound, glutathione, sequence-specific DNA or RNA binding protein is bound In some cases, DNA or RNA having a specific sequence thereof, antigen or epitope peptide when antibody or aptamer is bound, calmodulin binding peptide or ATP binding protein when calmodulin is bound ATP, estradiol receptor tamper When the substance is bound, estradiol and the like can be mentioned.
Among these, it is preferable to use a metal such as biotin, maltose, Ni or Co, glutathione, an antigen molecule or an epitope peptide, and it is more preferable to use biotin from the viewpoint of easy linker synthesis.

In addition, the crosslink formation site arranged in the main chain in the A type linker and in the substrate unit in the B type linker has a great influence on the structure and function of the linker, such as photocrosslinking, chemical crosslinking, and enzymatic crosslinking. There is no particular limitation as long as it can form a crosslink under no conditions. Specifically, for example, psoralen (furo [3,2-g] chromen-7-one) is bonded to the cross-linking site, so that the main-chain cross-linking site and the substrate unit are cross-linked by light irradiation. It is possible to form a cross-link between a site or a cross-link formation site of the main chain and a cross-linking site on the substrate unit side, and bond these.
The side chains of the A-type linker and B-type linker of the present invention can be expressed as follows, for example, when the side chain linking site of the main chain is composed of, for example, Amino-Modifier C6 dT. .

  5 '-(S) -TC (F)-(Spacer) n-CC- (Puro) -3'

In this formula, (S) represents a thiol modifier, (F) represents a fluorescent dye, and (Puro) represents puromycin or an analog thereof described later. n represents an integer of 1 to 8.
In this case, for example, the 5 ′ end of the side chain is 5′-Thiol-Modifier C6 (S-Trityl-6-mercapto hexyl-1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite. ) Can be crosslinked using EMCS (N- (6-Maleimidocaproyloxy) succinimide) to connect the main chain and the side chain.
Thiol modifiers include Thiol-Modifier C6 SS (1-O-Dimethoxytrityl-hexyl-disulfide, 1 '-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite), T5'-Thiol-Modifier C6 (S-Trityl-6-mercapto hexyl-1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite). It is preferable to use Thiol-Modifier C6 SS from the viewpoint of easy deprotection of the thiol group.

  Since the side chain has a fluorescent group between the protein linking site and the side chain linking site, the presence or absence of a linker can be easily detected in each step of the cDNA display method described later. . Examples of the fluorescent group used here include a free functional group such as a carboxyl group that can be converted into an active ester, a hydroxyl group that can be converted into a phosphoramidide, or an amino group. It is preferred to use fluorescent compounds that can be linked. Examples of such fluorescent compounds include fluorescein isothiocyanate (FITC), phycobiliprotein, rare earth metal chelates, dansyl chloride, tetramethylrhodamine isothiocyanate, Fluorescein-dT (5'-Dimethoxytrityloxy-5- [N-(( 3 ', 6'-di pivaloylfluoresceinyl) -aminohexyl) -3-acryimido] -2'-deoxyUridine-3'-succinoyl-long chainalkylamino)). Among these, it is preferable to use Fluorescein-dT used as a compound for molecular labeling because synthesis is easy.

  For (Spacer), a commercially available product that can be used as a spacer can be used.For example, Spacer Phosphoramidite 18 ((18-0-Dimethoxytritylhexaethyleneglycol, 1-[(2-cyanoethyl)-(N, N-diisopropyl) ] -phosphoramidite), Spacer Phosphoramidite 9 (9-O-Dimethoxytrityl-triethylene glycol, 1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite), Spacer C12 CE Phosphoramidite (12- (4,4 '-Dimethoxytrityl oxy) dodecyl-1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite), Spacer Phosphoramidite 9 (9-O-Dimethoxytrityl-triethylene glycol, 1-[(2-cyanoethyl)- (N, N-diisopropyl)]-phosphoramidite) etc. Among these, the use of Spacer Phosphoramidite 18 is flexible, low in steric hindrance and hydrophilic. Can be preferably used.

  When the side chain is short, steric hindrance occurs when the puromycin analog is incorporated into the ribosome, so that Spacer Phosphoramidite 18 (hereinafter sometimes referred to as “Spacer 18” or “Spacer 18”) is 1. It is preferable to have a structure (n = 1 to 8) of about 8 pieces. In view of the balance between the synthesis efficiency of the linker and the formation efficiency of each of the above-mentioned linkages, it is more preferable that the structure has about 4 such Phosphoramidite molecules (n = 4).

Here, the above-mentioned side chain protein binding site (Puro) is composed of puromycin, ribocytidylpuromycin (rCpPur), deoxydyl whose 3 'end of the nucleotide sequence and amino acid form an amide bond. In addition to puromycin analogues such as puromycin (dCpPur) and deoxyuridylpuromycin (dUpPur), 3'-N-aminoacylpuromycin aminonucleoside (PANS-amino acid), 3'-N-aminoacyl adenosine aminonucleoside (AANS) -Amino acid).
Examples of PANS-amino acids include PANS-Gly, PANS-Val, and PANS-Ala. Examples of AANS-amino acids include AANS-Gly, AANS-Val, and AANS-Ala. In addition, those in which a nucleoside and an amino acid are ester-bonded can be used, but it is particularly preferable to use puromycin because of the high stability of protein ligation at the protein ligation site.

MRNA binds to the mRNA binding site that functions as a primer for reverse transcription, and a complex of mRNA complementary to the main chain DNA is generated. From this mRNA, the corresponding protein is generated, and the side chain protein described above. Bound to the binding site.
In any of the linkers of the present invention, the length of the side chain and the main chain is such that the substrate linked to the substrate binding site of the substrate unit exists within the range where the protein bound to the protein binding site of the side chain can work well. Has been adjusted. For this reason, it is possible to determine what activity these proteins have when these proteins bound to the protein binding site act on the substrate in the substrate unit.

For example, when such a protein bound to a protein binding site on the side chain has an activity of cleaving the substrate, the linker of the present invention is bound to a solid phase, and the cleaved linker is recovered, Measure the amount of protein bound to the linker. By comparing the measured activity with a standard enzyme having a similar activity, the specific activity of the obtained protein can be known.
Conversely, when such a protein has an activity to bind a substrate, a free linker is bound to a solid phase and the amount of the free linker is measured. By comparing the measured activity with a standard enzyme having a similar activity, the specific activity of the obtained protein can be known.
In particular, if fluorescein isothiocyanate (FITC) or Fluorescein-dT as described above is incorporated in the side chain, there is an advantage that the amount of the recovered linker can be easily measured.

  The combination of the protein having the enzyme-like activity and the substrate is: a polypeptide having an activity equivalent to that of a peptide-degrading enzyme and an arbitrary peptide sequence; a polypeptide having an activity equivalent to that of a glycolytic enzyme and an arbitrary sugar chain; A polypeptide having an activity equivalent to a nucleolytic enzyme and an arbitrary nucleic acid sequence; a polypeptide having an activity equivalent to a lipolytic enzyme and an arbitrary lipid; and a polypeptide having an activity to degrade a synthetic polymer and an arbitrary synthetic polymer; It is preferably any one selected from the group consisting of

Examples of the combination of the protein having the enzyme-like activity and the substrate include the following.
Examples of a polypeptide having an activity equivalent to that of a peptide-degrading enzyme and an arbitrary peptide sequence include a polypeptide having an activity equivalent to that of a protease, peptidase, etc., and an arbitrary peptide sequence that can cleave them.
Examples of a polypeptide having an activity equivalent to that of a sugar chain degrading enzyme and an arbitrary sugar chain include a polypeptide having an activity equivalent to that of glycosylase, amylase, and the like, and an arbitrary sugar chain capable of cleaving these.

Examples of a polypeptide having an activity equivalent to that of a nucleolytic enzyme and an arbitrary nucleic acid sequence include a polypeptide having an activity equivalent to that of a ribonuclease, deoxyribonuclease, restriction enzyme, etc., and any single strand or two that can be cleaved by these. For example, a nucleic acid sequence of this strand.
Examples of a polypeptide having an activity equivalent to that of a lipolytic enzyme and an arbitrary lipid include a polypeptide having an activity equivalent to that of a lipase, phospholipase, or the like, and any lipid that can be cleaved by these.
Examples of polypeptides having the activity of degrading synthetic polymers include nylon-degrading enzymes, polyester-degrading enzymes, polyurethane-degrading enzymes, polyethylene-degrading enzymes, polypeptides having the same activity as polyvinyl alcohol-degrading enzymes, and these are cleaved. Any synthetic polymer that can be mentioned.

  The combination of the protein having the enzyme-like activity and the substrate comprises a polypeptide having an activity equivalent to that of a nucleic acid ligase and an arbitrary nucleic acid sequence; a polypeptide having an activity equivalent to that of a peptide-linked enzyme and an arbitrary peptide sequence; A polypeptide having an activity equivalent to that of an enzyme and an arbitrary peptide sequence; a polypeptide having an activity equivalent to that of a molecule-linked enzyme for linking a molecule to the polypeptide peptide, an arbitrary peptide sequence and an optional transfer molecule substrate; a molecule to a nucleic acid A polypeptide having an activity equivalent to that of a molecular ligation enzyme for ligating a nucleic acid, an arbitrary nucleic acid sequence and an arbitrary transfer molecule substrate; a polypeptide having an activity equivalent to a glycosyltransferase, an arbitrary sugar chain, and the aforementioned arbitrary sugar chain And a substrate having an activity catalyzing a novel ligation and an arbitrary substrate. Is, it is preferable that either.

Examples of the combination of the protein having the enzyme-like activity and the substrate include the following.
Examples of a polypeptide having an activity equivalent to that of a nucleic acid ligase and an arbitrary nucleic acid sequence include a polypeptide having an activity equivalent to that of a DNA ligase, RNA ligase, etc., and an arbitrary nucleic acid sequence peptide such as a DNA or RNA to which these can be ligated Is mentioned.
Examples of a polypeptide having an activity equivalent to that of a peptide-linked enzyme and an arbitrary peptide sequence can be ligated with a polypeptide having an activity equivalent to that of an intein, sortase, transglutaminase, etc. Any peptide sequence may be mentioned.
Examples of a polypeptide having an activity equivalent to that of a polypeptide or a molecule linking enzyme for linking a molecule to a peptide, and any peptide sequence and any transfer molecule substrate include biotin ligase, ubiquitin ligase, ghrelin-O-acyltransferase, etc. And any peptide sequence having the same activity as that of, and any transfer molecule substrate such as biotin, ubiquitin, and fatty acid.

A polypeptide having an activity equivalent to that of a molecule linking enzyme for linking a molecule to a nucleic acid, and an example of an arbitrary nucleic acid sequence and an arbitrary transfer molecule substrate, such as a polypeptide having an activity equivalent to an aminoacyltransferase, T4 polynucleotide kinase, etc. And any nucleic acid sequence and any transfer molecule substrate such as amino acids, phosphates and the like.
Examples of a polypeptide having an activity equivalent to glycosyltransferase, an arbitrary sugar chain and a substrate to be modified with the arbitrary sugar chain include α1,3-N-acetylgalactosaminyltransferase, β1,4-N- Activity equivalent to acetylgalactosaminyltransferase-III, β1,3-N-acetylgalactosaminyltransferase-II, β1,3-N-acetylgalactosaminyltransferase-VI, α1,2-fucosyltransferase-II, etc. And any of the sugar chains or sugars on which they can act, and any substrate on which other sugar chains are desired to be modified.

Below, the manufacturing method of the linker of this invention is demonstrated. The linker of the present invention is synthesized by dividing into four segments: a segment constituting a side chain having a protein binding site, a segment constituting a main chain, and two segments constituting a substrate unit. After that, each is manufactured by crosslinking.
In the following description, puromycin is used as a protein binding site on the side chain, spacer 18 is used as a spacer, and fluorescein-dT is incorporated as a fluorescent group. Hereinafter, the segment constituting the side chain is referred to as “puromycin segment”. These reagents can be purchased from Glen Research.
Moreover, although the crosslink formation site | part is provided in the segment which comprises a principal chain, psoralen shall be used for bridge | crosslinking. Hereinafter, these are referred to as “soralen-amino segments”.

Further, the two segments constituting the substrate unit are an “azide segment” having an azide and an “alkyne segment having an alkyne”. Of these two segments, the azide segment is a segment corresponding to an RNA recognition sequence that forms an RNA ligase recognition site when bound to the main chain by crosslinking, and the alkyne segment comprises a substrate linking site and a solid phase binding site. It is a segment that has.
In addition, subtilicin is used as the enzyme that binds to puromycin, and the substrate linked to the substrate linking site is a peptide having an arbitrary sequence.
The above segment used in the present invention may be synthesized according to a conventional method, and a puromycin segment having the following structure is synthesized. The number of Spacers 18 can be appropriately increased or decreased between 1 and 8.

  5 '-(S) -TC (F)-(Spacer18)-(Spacer18)-(Spacer18)-(Spacer18) -CC- (Puro) -3'

The puromycin segment can be synthesized by using the above-described reagents and reacting according to the phosphoramidite method using an automatic nucleic acid synthesizer.
A psoralen-amino segment is then synthesized. In designing the amino segment, coding sequences of various mRNAs can be referred to. For example, mRNA encoding various receptor proteins with known sequences, mRNA encoding various antibodies or fragments thereof, mRNA encoding various enzymes, mRNA of unknown sequence transcribed from DNA in various gene libraries, organic MRNA with random sequence transcribed from DNA with a sequence synthesized at random by synthesis, mRNA transcribed from DNA that encodes a protein with unknown sequence by insertion of random mutation using PCR method, etc. And any mRNA that does not contain a termination codon can be selected.

(SEQ ID NO: 1)
5 '-(psoralen) -TACGACGATCTCGAACGAACCACCCCCGCCGCCCCCCG- (T-NH2) -CCT-3'

When synthesizing the above psoralen-amino segment, the side chain binding site (T-NH2) includes, for example, Amino-Modifier C6 dT (5'-Dimethoxytrityl-5- [N- (trifluoroacetylaminohexyl)- 3-acrylimido] -2'-deoxyUridine, 3 '-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite).
Next, for example, an azide segment having the following structure is synthesized.

(SEQ ID NO: 2)
5'-CCCGTGGTTCGTTCGAGATCGTCGTAAA- (Spacer18)-(C6)-(Azide) -3 '

For (C6), for example, 3′-Amino-Modifier C7 CPG 500 (2-Dimethoxytrityloxymethyl-6-fluorenylmethoxycarbonylamino-hexane-1-succinoyl-long chain alkylamino-CPG) may be used. As (Azide), Azidobutyrate NHS Ester (4-Azido-butan-1-oic acid N-hydroxy succinimide ester) can be used. Any of the above reagents can be purchased and used from Glen Research, etc., and can be reacted according to the phosphoramidite method using an automatic nucleic acid synthesizer to synthesize an azide segment.
Next, for example, an alkyne-peptide-biotin segment having the following structure is synthesized. The following sequence is shown as a structure from N-terminal to C-terminal.

(SEQ ID NO: 3)
Gly (Propargyl) -EDHVAHALAQ- (K-Biotin) -NH ₂

Here, Fmoc-Gly (Propargyl) -OH can be used for Gly (Propargyl), and (K-biotin) -NH ₂ amidates the C-terminal of the lysine residue, Those modified by binding biotin to the side chain can be used.
The azide segment (I) and alkyne segment (II) synthesized as described above,

アジドセグメントとアルキンセグメントとは、１：50〜１：200のモル比で混合し、CuSO₄/Ascorbateを触媒として、20〜30℃にて60〜70時間、反応させることが好ましく、このように反応させることによって下記の反応生成物（III）を得ることができる。

The azide segment and alkyne segment are preferably mixed at a molar ratio of 1:50 to 1: 200 and reacted at 20 to 30 ° C. for 60 to 70 hours using CuSO ₄ / Ascorbate as a catalyst. By reacting, the following reaction product (III) can be obtained.

The reaction product is desalted with a column equilibrated with phosphate buffer (pH 7 to 7.5), the cross-linked reaction product is separated by polyacrylamide gel electrophoresis, and the substrate unit is obtained by staining and analyzing DNA. be able to.
Subsequently, the puromycin segment and the psoralen-amino segment are cross-linked. About 5 μL of the puromycin segment is mixed with about 45 μL of 0.8 to 1.2 M phosphate buffer (pH 8.5 to 9.5), 5 μL of 1 M DTT is added, and the mixture is allowed to stand at room temperature for 0.5 to 2 hours. Reduce to group.
Immediately before the cross-linking reaction, DTT is removed and desalted using a column equilibrated with 10-30 mM phosphate buffer (pH 7.0-7.5).

Subsequently, about 100 μL of phosphate buffer (pH 7.0 to 7.5), about 10 μL of about 1 mM psoralen-amino segment and about 20 μL of about 100 mM cross-linking agent are added and mixed well. Incubate for 40 minutes. As the crosslinking agent, for example, EMCS (6-Maleimidohexanoic acid N-hydroxysuccinide ester) (manufactured by Dojindo Laboratories) can be used.
Thereafter, ethanol precipitation is performed according to a conventional method to precipitate the reaction product, and unreacted EMCS is removed. The resulting precipitate is washed with 70% ethanol and dried under reduced pressure. The reaction product thus obtained is immediately dissolved in the puromycin segment solution reduced as described above (about 20 nmol) and left at 2-6 ° C. overnight. Here, DTT is added so that the final concentration is about 50 mM, and the mixture is left at about 37 ° C. for 20 to 40 minutes to stop the crosslinking reaction of thiol groups. By the above reaction, a reaction solution containing a crosslinked product in which the two segments are crosslinked can be obtained.
The two cross-linked products obtained as described above were mixed in a solution containing 10 to 30 mM Tris-HCl (pH 7.5 to 8.5) and 80 to 120 mM NaCl, and then heated to about 60 ° C., Cool to about 25 ° C over 10 minutes. Next, the sample solution is irradiated with light of 350 to 400 nm (about 1 to 3 W / cm ² ) using, for example, a xenon lamp as a light source for about 20 minutes to expose the sample. A crosslinked product can be obtained by this light irradiation.

Using the linker of the present invention produced as described above, proteins having various enzyme-like activities can be obtained. Hereinafter, the case where a linked enzyme is used and the case where a cleaved enzyme is used will be described separately.
First, the case where a linked enzyme is used will be described.
In the mRNA-linker conjugate production step, the A-type or B-type protein evolution linker and mRNA having a sequence complementary to a part of the main chain of the linker are positioned on the main chain of the linker. At the mRNA binding site, binding is performed by RNA ligase at the mRNA binding site. This produces an mRNA-linker conjugate. Here, the mRNA that can be used for the design of the main chain is as described above.

Here, in the mRNA-linker conjugate production step, annealing is performed prior to ligation using ligase, and then ligation is performed. In addition to RNA ligase for ligation between RNA / DNA, DNA ligase for ligation between RNA and RNA / DNA can also be used. For example, ATP-dependent DNA ligase (EC6.5.1.1), NAD ⁺ -dependent DNA ligase (EC6.5.1.2), RNA ligase (EC6.5.1.3), T4 RNA ligase, TS2126 thermostable phage-derived DNA ligase Etc.
Among these ligases, T4 RNA ligase or TS2126 thermostable phage-derived DNA ligase is preferably used, and T4 RNA ligase is more preferably used from the viewpoint of ligation efficiency.

The volume of the reaction system for performing the ligation reaction is preferably 4 to 100 μL, and more preferably 20 to 40 μL from the viewpoint of reaction efficiency. 20 to 25 μL is more preferable because of the highest reaction efficiency.
The ratio of RNA: linker in this reaction system can range from 3: 1 to 1: 6 in molar ratio, but is approximately 1: (1-6), specifically, 5-100 pmol. It is preferable to use 5 to 100 pmol of mRNA for this linker. In order to increase the reaction efficiency and minimize the residue, it is more preferable to set the ratio to 1: (1-2). In this case, 10 to 20 pmol of linker is reacted with 10 pmol of mRNA.

Annealing prior to the ligation reaction is performed as follows. First, warm the sample solution at about 60-100 ° C for about 2 to about 60 minutes using a heating block, aluminum block, water bath, or other heating device, and then leave it at room temperature for about 2 to about 60 minutes And gently reduce the liquid temperature. Thereafter, it is further cooled to about −5 ° C. to about 10 ° C. Specifically, for example, the sample solution is warmed on an aluminum block at 90 ° C. for 5 minutes, then transferred to an aluminum block at 70 ° C. and placed for 5 minutes, and then a ligation reaction between the mRNA and the linker is performed. .
Ligation is performed, for example, in a Tris-HCl buffer (pH 7.0 to 8.0) containing magnesium chloride, dithiothreitol (hereinafter sometimes abbreviated as “DTT”), ATP, T4 polynucleotide kinase and T4 RNA ligase. And reacting at a desired temperature for a desired time.

For example, about 0.1 to about 2.5 U / μL T4 polynucleotide kinase, about 0.4 to about 5 U / μL T4 RNA ligase, about 2 to about 50 mM magnesium chloride, about 2 to about 50 mM DTT, about 0.2 to about 5 mM The reaction can be carried out in a 10 to 250 mM Tris-HCl buffer (pH 7.0 to 8.0) containing ATP, and is preferably reacted at about 10 ° C. to about 40 ° C. for about 1 minute to about 60 minutes. From the viewpoint of work efficiency and reaction efficiency, it is preferably performed at 20 ° C. to 30 ° C. for 5 minutes to 30 minutes, more preferably at 25 ° C. for 15 minutes.
If necessary, an RNA-degrading enzyme inhibitor such as SUPERase RNase inhibitor (Ambion) can be added to this reaction solution.

  Next, the mRNA-linker-protein conjugate production step is a cell-free translation system, a protein having enzyme-like activity is synthesized using the mRNA as a template, and the protein is located at the protein binding site on the side chain of the linker. Combined. At this time, the aforementioned puromycin or an analog thereof is bound to the protein binding site. Thereby, an mRNA-linker-protein conjugate is obtained.

As the cell-free translation system, those selected from the group consisting of animal-derived cells, plant-derived cells, fungi, and bacteria can be used, but partially or entirely artificially synthesized ones are used. You can also More specifically, Escherichia coli, rabbit reticulocyte, wheat germ extract and the like can be used as a cell-free system (Lamfrom H., Grunberg-Manago M., Biochem. Biophys. Res. Commun., 27, 1 (1967)).
In the above cell-free translation systems, the types of codons used for translation differ depending on the species from which they are derived. For this reason, it is preferable to select a cell-free translation system according to the target gene and the origin of the gene. In addition, it is preferable to design the mRNA sequence and structure so as to suit the translation system to be used.

In this step, it is preferable to use a lysate of mammalian reticulocyte cells as the cell-free translation system, and it is more preferable to use a lysate of reticulocyte cells obtained from rabbit blood. In order to increase the ratio of reticulocytes in the blood, it is preferable to administer acetylphenylhydrazine to the mammal in advance to induce hemolytic anemia and the like, and then collect blood after several days.
The lysate is preferably obtained, for example, by degrading cell-derived mRNA with a nuclease and removing calcium with a chelating agent and then inactivating the nuclease. Cell-derived mRNA is decomposed with micro-coccal nuclease, glycol ether diamine tetraacetic acid (EGTA) is added to chelate calcium, and the nuclease is inactivated (hereinafter referred to as “micro-coccal nuclease-treated”). ) Is more preferable.

For example, a reaction solution containing about 16 to about 400 mM potassium acetate, about 0.1 to about 2.5 mM magnesium acetate, about 0.2 to about 50 mM creatine phosphate, 0 to about 0.25 mM amino acid (the concentrations are all final). (Concentration) In 100 to 100 μL, a translational reaction can be performed by adding a rabbit reticulocyte lysate treated with micrococcal nuclease and the above-mentioned ligated body.
From the viewpoint of reaction efficiency, the amount of rabbit reticulocyte lysate is about 8.5 to about 17 μL, the amount of the above conjugate is about 1.2 to about 2 pmol, the size of the reaction system is about 12.5 to about 25 μL, and about 20 to about 40 ° C. For about 10 to about 30 minutes. The reaction solution used in this case is preferably one containing 80 mM potassium acetate, 0.5 mM magnesium acetate, 10 mM creatine phosphate, 0.025 mM methionine and leucine, and 0.05 mM methionine and leucine, respectively. In terms of production efficiency and work efficiency, it is more preferable to perform translation at about 30 ° C. for about 20 minutes.

If necessary, an RNase inhibitor such as SUPERase RNase inhibitor (manufactured by Ambion) may be added to this reaction solution.
After the translation reaction, it is preferable to bind the translation product protein and the mRNA-linker conjugate under high salt concentration conditions. For example, in the presence of 0.3 to 1.6 M potassium chloride and 40 to 170 mM magnesium chloride (both concentrations are final concentrations) at about 27 to about 47 ° C. for about 30 minutes to about 1.5 hours, the protein is It can be efficiently combined with the connector.
Next, in the cDNA-linker-protein conjugate production step, a cDNA chain is synthesized by reverse transcription using the mRNA as a template. In this step, a cDNA strand is synthesized by performing a reverse transcription reaction under a predetermined condition using the 3 ′ end of the main chain as a reaction start point and the mRNA as a template. As a result, an mRNA / cDNA-linker-protein conjugate is obtained.
The reverse transcription reaction system can be arbitrarily selected, but the mRNA-linker protein conjugate, dNTP Mix, DTT, reverse transcriptase, standard solution, and water from which RNase has been removed (hereinafter referred to as “RNase-free water”). It is preferable to carry out reverse transcription under the conditions of 30 to 50 ° C. for 5 to 20 minutes in this system.

Subsequently, in the immobilization step, the protein having the enzyme-like activity is allowed to act on the substrate linked to the substrate linking site and the desired substrate bound on the solid phase to link them. Thereby, the cDNA-linker-protein conjugate is immobilized on the solid phase.
In this step, the solid phase to be used is not particularly limited, and can be appropriately selected depending on the use for which the conjugate is used. As such a solid phase, those having various shapes serving as a carrier for immobilizing a biomolecule can be used. For example, beads such as styrene beads, glass beads, agarose beads, sepharose beads, magnetic beads; substrates such as glass substrates, silicon (quartz) substrates, plastic substrates, metal substrates (for example, gold foil substrates); glass containers, plastic containers Examples of such containers include membranes made of materials such as nitrocellulose and polyvinylidene fluoride (PVDF).

There is no particular limitation on the method for binding a substrate on which a protein having enzyme-like activity can act on the solid phase as described above, and various methods known to those skilled in the art can be used. For example, it can be carried out by introducing a predetermined molecule in advance into a solid phase binding part provided in the above-mentioned linker and preliminarily binding a receptor molecule that binds to the molecule to the solid phase. Combinations that can be used for such bonding are as described above.
If the solid phase is composed of a plastic material such as a styrene bead or a styrene substrate, a part of the linker can be directly covalently bonded to the solid phase using a known method, if necessary ( (See Qiagen, LiquidChip Applications Handbook, etc.) Therefore, when biotin or an analog thereof is bound to the conjugate, the conjugate can be easily bound to the solid phase by binding avidin to the solid phase.

  Next, in the first washing step, the linked body that has not bound to the solid phase is washed using a buffer solution. The washing solution used here is about 1 to 100 mM Tris-HCl buffer (pH 7.0 to about 10 to about 10 M sodium chloride, about 0.1 to about 10 mM EDTA, about 0.01 to about 1% surfactant. 9.0) or phosphate buffer (pH 7.0 to 9.0) and the like, and 10 to 30 mM Tris- containing 1 to 3 M sodium chloride, 1 to 3 mM EDTA, 0.05 to 0.02% Triton X-100. It is preferable to use a hydrochloric acid buffer (pH 8.0) because of good washing efficiency.

Next, in the recovery step, the immobilized cDNA-linker-protein conjugate is recovered. Thereafter, in the amplification step, the cDNA of the cDNA-linker-protein conjugate recovered as described above is amplified.
By repeating the steps as described above, proteins having enzyme-like activity can be efficiently screened.

When using a cleaved enzyme, it is the same as the above except that the immobilization step to the solid phase is different. That is, it can be carried out by introducing a predetermined molecule in advance into the solid phase binding part provided in the above-mentioned linker and preliminarily binding a receptor molecule that binds to the molecule to the solid phase. Combinations that can be used for such bonding are as described above.
Thereafter, the protein having enzyme-like activity acts on the substrate in the substrate unit bound to the solid phase and cleaves at a predetermined sequence portion. This cleavage reaction is preferably performed in a reaction solution containing a Tris-HCl buffer solution at a predetermined temperature for a predetermined time.
For example, when subtilisin is used, in a reaction having a composition of about 5-20 mM Tris-HCl, about 2.5 to about 10 mM EDTA, and about 0.1 to about 0.4 M sodium acetate, about 5 to about The reaction can be carried out in the temperature range of about 30 to about 45 ° C. for 20 minutes. Moreover, it can also be made to react on various conditions by using the reaction liquid and organic solvent of very high ionic strength as a reaction solution.

As described above, using the linker of the present invention, proteins having various enzyme-like activities can be obtained, and cDNAs corresponding to the proteins can also be obtained.
Alternatively, mRNA / cDNA-linker-protein conjugates can be selected by column chromatography using the affinity of the protein obtained as described above.
Further, by using the linker obtained in the present invention, screening can be performed with high accuracy while meeting the demand for high throughput.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

(Example 1) Synthesis of A-type linker for protein evolution The A-type linker for protein evolution used in this example comprises (a) puromycin segment, (b) psoralen-amino segment, (c) azide segment, and (D) The alkyne-peptide-biotin segment was divided into four segments and synthesized. Among these, (a) and (b) are used for the synthesis of the main chain, and (c) and (d) are used for the synthesis of the substrate unit.
The synthesis of the special DNA used for the synthesis of the above segments (a) to (c) was commissioned to Gene World Co., Ltd. and Japan Bioservice Co., Ltd. The peptide synthesis used in (d) was commissioned to Scrum Co., Ltd.

(1) Synthesis of puromycin segment A puromycin segment having the following structure was synthesized.

    5 '-(S) -TC (F)-(Spacer18)-(Spacer18)-(Spacer18)-(Spacer18) -CC- (Puro) -3'

Here, Thiol-Modifier C6 SS (1-O-Dimethoxytrityl-hexyl-disulfide, 1 ′-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite) was used for (S).
(F) includes Fluorescein-dT (5'-Dimethoxytrityloxy-5- [N-((3 ', 6'-dipivaloyl fluoresceinyl) -aminohexyl) -3-acryimido] -2'-deoxyUridine-3'-succinoyl -long chain alkylamino)) was used.
Puromycin CPG (5′-Dimethoxytrityl-N-trifluoroacetyl-puromycin, 2′-succinoyl-long chain alkylamino-CPG) was used for (Puro).
Spacer Phosphoramidite 18 ((18-0-Dimethoxytritylhexaethylene glycol, 1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite)) was used as (Spacer18).
All of the above reagents were purchased from Glen Research. Using these reagents, reaction was performed according to the phosphoramidite method using an automatic nucleic acid synthesizer to synthesize a puromycin segment schematically shown in FIG.

(2) Synthesis of psoralen-amino segment A psoralen-amino segment having the following structure was synthesized.

(SEQ ID NO: 4)
5 '-(psoralen) -TACGACGATCTCGAACGAACCACCCCCGCCGCCCCCCG- (T-NH2) -CCT-3'

Here, (psoralen) includes Psoralen C6 Phosphoramidite ((6- [4 '-(Hydroxymethyl) -4,5', 8-trimethylpsoralen] -hexyl-1-O- (2-cyanoethyl)-(N, N -diisopropyl) -phosphoramidite)) was used.
(T-NH2) includes Amino-Modifier C6 dT (5'-Dimethoxytrityl-5- [N- (trifluoroacetylaminohexyl) -3-acrylimido] -2'-deoxyUridine, 3 '-[(2-cyanoethyl) -(N, N-diisopropyl)]-phosphoramidite).
All the above reagents were purchased from Glen Research. Using these reagents, reaction was performed according to the phosphoramidite method using an automatic nucleic acid synthesizer, and the psoralen-amino segment schematically shown in FIG. 3 was synthesized.

(3) Synthesis of azide segment An azide segment having the following structure was synthesized.

(SEQ ID NO: 5)
5'-CCCGTGGTTCGTTCGAGATCGTCGTAAA- (Spacer18)-(C6)-(Azide) -3 '

Here, Spacer Phosphoramidite 18 ((18-0-Dimethoxytrityl hexaethyleneglycol, 1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite)) was used as (Spacer18).
For (C6), 3′-Amino-Modifier C7 CPG 500 (2-Dimethoxytrityloxymethyl-6-fluorenylmethoxycarbonylamino-hexane-1-succinoyl-long chain alkylamino-CPG) was used.
As (Azide), Azidobutyrate NHS Ester (4-Azido-butan-1-oic acid N-hydroxy succinimide ester) was used.
All the above reagents were purchased from Glen Research. Using these reagents, reaction was carried out according to the phosphoramidite method using an automatic nucleic acid synthesizer to synthesize azide segments.

(4) Synthesis of alkyne-peptide-biotin segment An alkyne-peptide-biotin segment having the following structure was synthesized. The following sequences are shown as structures from N-terminal to C-terminal.

(SEQ ID NO: 6)
Gly (Propargyl) -EDHVAHALAQ- (K-Biotin) -NH ₂

Here, Fmoc-Gly (Propargyl) -OH was used for Gly (Propargyl). (K-biotin) -NH ₂ is modified by amidating the C-terminus of the lysine residue and binding biotin to the side chain of the lysine residue.
Azide represented by the following formula (I)

And an alkyne represented by the following formula (II):

Were mixed at a molar ratio of 1: 100 and reacted at room temperature for 64 hours using CuSO ₄ / Ascorbate as a catalyst to obtain a reaction product represented by the following formula (III).

(5) Cross-linking of azide segment and alkyne-peptide-biotin segment
10 μL of 25 μM azide segment and 25 μL of 1 mM alkyne-peptide-biotin segment are mixed with a solution consisting of 60 μL of t-butanol, 5 μL of 0.5 mM CuSO _{4 and} 5 μL of 2.5 mM ascorbate for 64 hours with stirring at room temperature. Reacted.
This reaction product was desalted with a microbiospin column 6 (manufactured by Bio-rad) equilibrated with a phosphate buffer (pH 7.2). The desalted cross-linked reaction product was separated by polyacrylamide gel electrophoresis, and the DNA was stained with SybrGold (manufactured by Invitrogen) and analyzed.
The results are shown in FIG. Lane 1 shows a 10 bp DNA step ladder (Promega), Lane 2 shows an azide segment, and Lane 3 shows a cross-linked reaction product. The resulting crosslinked product (F: substrate unit) is schematically shown in FIG.
From this result, it was confirmed that the target crosslinked product (F) was obtained with a yield of about 70%.

(3) Cross-linking of puromycin segment and psoralen-amino segment
5 μL of 4 mM puromycin segment was mixed with 45 μL of 1M phosphate buffer (pH 9.0), 5 μL of 1M DTT was added, and the mixture was allowed to stand at room temperature for 1 hour to reduce the disulfide group of the puromycin segment to a thiol group.
Immediately before the crosslinking reaction, DTT was removed and desalted using NAP-5 Columns (manufactured by GE Healthcare Japan) equilibrated with 20 mM phosphate buffer (pH 7.2).
To 100 μL of 0.2 M phosphate buffer (pH 7.2), add 10 μL of 1 mM psoralen-amino segment and 20 μL of 100 mM cross-linking agent EMCS (6-Maleimidohexanoic acid N-hydroxysuccinide ester) (Dojindo Laboratories) The mixture was thoroughly stirred and reacted at 37 ° C. for 30 minutes.

Thereafter, ethanol precipitation was performed according to a conventional method to precipitate the reaction product, and unreacted EMCS was removed. The precipitate was washed with 200 μL of 70% ethanol and dried under reduced pressure.
This reaction product was quickly dissolved in a puromycin segment solution (about 20 nmol) reduced as described above and left at 4 ° C. overnight.
DTT was added to this sample solution so that the final concentration was 50 mM, and the mixture was allowed to stand at 37 ° C. for 30 minutes to stop the crosslinking reaction of thiol groups, thereby obtaining a reaction solution containing a crosslinked product in which the two segments were crosslinked. .
The obtained cross-linked products were separated by urea-denaturing polyacrylamide gel electrophoresis (12% acrylamide gel, 8M urea, 60 ° C.), and the DNA was stained and analyzed by SybrGold (manufactured by Invitrogen). Lane 1 is a 10 bp DNA step ladder (manufactured by Promega), and lane 2 is a migration result of the cross-linked reaction product.
In addition to the puromycin segment and psoralen-amino segment bands, bands comparable to the sum of these molecular weights were detected, confirming that the desired cross-linked product (E) was obtained. The results are shown in FIG.

  Then, this crosslinked product (E) was precipitated with ethanol in the same manner as described above to remove the unreacted puromycin segment, and to remove the unreacted psoralen-amino segment and its EMCS crosslinked product as follows. Purification was performed by HPLC under conditions.

(HPLC conditions)
Column: Symmetry300 C18, 5μm, 4.6 x 250 mm (Waters)
Buffer A: 0.1M TEAA (triethylammonium acetate, manufactured by Wako Pure Chemical Industries, Ltd.)
Buffer B: 80% acetonitrile (manufactured by Wako Pure Chemical Industries, Ltd., diluted with ultrapure water)
Flow rate: 0.5 ml / min Concentration gradient: The mixing buffer concentration of A and B is changed from 85:15 to 50:50 (A: B) (50 minutes).

  The product fractionated by HPLC was detected again by electrophoresis on a 12% acrylamide gel (8 M urea, 60 ° C.), and the target fraction was concentrated under reduced pressure, and then the same as above was performed with ethanol. Precipitated. Thereafter, the precipitate was dissolved in water to 50 μM.

(6) Photocrosslinking of cross-linked product (E) and cross-linked product (F) 1 pmol cross-linked product (E) and 1.2 pmol cross-linked product (F), 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl. Mix in the solution containing, then heat to 60 ° C. and cool to 25 ° C. over 10 minutes.
Next, the sample was irradiated with 365 nm (2 W / cm ² ) light using a xenon lamp as a light source for 20 minutes, and the sample was exposed to obtain an exposed sample.
The exposed sample was separated by urea-denaturing polyacrylamide gel electrophoresis under the same conditions as described above, and the DNA was stained and analyzed by SybrGold (manufactured by Invitrogen). The results are shown in FIG.
As shown in FIG. 7, in the exposed sample, an electrophoresis band moved to the long chain length side was detected, and it was confirmed that a crosslinked product (G) was generated. The result of the HPLC analysis shown in FIG. 8 also confirmed that the cross-linked product was eluted at a position where the retention time was 31.819 minutes.

(7) Confirmation of cleavage of peptide substrate in cross-linked product (F) by subtilisin
In a buffer containing 0.1 M Tris-HCl (pH 8.0), 10 mM CaCl ₂ , 10 mM NaCl ₂ , 5 μL of the cross-linked product (F) was mixed with 0.1 pmol subtilisin Carlsberg (manufactured by Sigma), 50 The reaction was allowed to proceed for 30 minutes at ° C.
The reaction products were separated by 12% polyacrylamide gel electrophoresis (8 M urea, 60 ° C.), and the DNA was stained and analyzed by SybrGold (Invitrogen). The results are shown in FIG. Lane 1 is a 10 bp DNA step ladder (Promega), Lane 2 is a cross-linked product (F), Lane 3 is a reaction solution treated with subtilisin (treatment product), and Lane 4 is a reaction solution not treated with subtilisin. (Negative control) is shown.
The band of the cross-linked product detected in lane 2 and lane 4 in FIG. 9 was not detected in the treated product of lane 3, but a band was detected on the shorter chain length side. As a result, it was confirmed that the peptide substrate was cleaved by subtilisin.

(Example 2) Synthesis of B-type linker for protein evolution The B-type linker for protein evolution used in this example is composed of (a) puromycin segment, (b ') amino segment, (c') psoralen-azide segment. , And (d) The alkyne-substrate-biotin segment was divided into four segments and synthesized. The synthesis of special DNA used for the synthesis of the segments (a) to (d) was outsourced to Gene World Co., Ltd. and Japan Bioservice Co., Ltd.
The reagents and the like used in the above (a) to (d) are the same as those used in (a) to (d) of Example 1.
(1) Cross-linking of puromycin segment and amino segment
5 μL of 4 mM puromycin segment was mixed with 45 μL of 1M phosphate buffer (pH 9.0), 5 μL of 1M DTT was added, and the mixture was allowed to stand at room temperature for 1 hour to reduce the disulfide group of the puromycin segment to a thiol group.
Immediately before the crosslinking reaction, DTT was removed and desalted using NAP-5Columns (manufactured by GE Healthcare Japan) equilibrated with 20 mM phosphate buffer (pH 7.2).
10 μL of 1 mM amino segment and 20 μL of 100 mM cross-linking agent EMCS (6-Maleimidohexanoic acid N-hydroxysuccinide ester) (Dojindo Laboratories) may be added to 100 μL of 0.2 M phosphate buffer (pH 7.2). The mixture was stirred and reacted at 37 ° C. for 30 minutes.

Thereafter, ethanol precipitation was performed according to a conventional method to precipitate the reaction product, and unreacted EMCS was removed. The precipitate was washed with 200 μL of 70% ethanol and dried under reduced pressure.
This reaction product was quickly dissolved in a puromycin segment solution (about 20 nmol) reduced as described above and left at 4 ° C. overnight.
DTT was added to this sample solution so that the final concentration was 50 mM, and the mixture was allowed to stand at 37 ° C. for 30 minutes to stop the crosslinking reaction of thiol groups, thereby obtaining a reaction solution containing a crosslinked product in which the two segments were crosslinked. .
The obtained cross-linked products were separated by urea-denaturing polyacrylamide gel electrophoresis, and DNA was stained with SybrGold (manufactured by Invitrogen) and analyzed. Urea-denaturing polyacrylamide gel electrophoresis was performed under the same conditions as in Example 1.

As a result, it was confirmed that the desired crosslinked product (E) was obtained.
Next, this crosslinked product (E) was precipitated with ethanol in the same manner as described above. Thereafter, unreacted puromycin-segment was removed, and purification was performed by HPLC under the same conditions as in Example 1. Next, the product fractionated by HPLC was analyzed by urea-denaturing polyacrylamide gel electrophoresis under the same conditions as in Example 1. After concentrating the target fraction under reduced pressure, ethanol was obtained in the same manner as described above. It was precipitated with. Thereafter, the precipitate was dissolved in water to 50 μM.

(Example 3) Cleavage of substrate by B-type linker for protein evolution (1) Preparation of mRNA
Has a T7 promoter region, and a template DNA encoding the B domain of protein A and 1μg prepared, transcription kit ^{(RiboMAX TM Large Scale RNA Production Systems} , Promega) After 1 hour the reaction was purified using the .
(2) Measurement of RNase T1 inhibitory activity
Five RNaseT1 (1 U / μL; Ambion) 1 μL (1 U) was added to 17 μL RNaseT1 buffer to make 18 μL. In addition, one without RNase T1 was prepared for reference.

One of these was used as a negative control, GMP was not added, and GMP was added to the remaining four to a final concentration of 0.2 μM, 2 μM, 20 μM, and 200 μM, respectively, and incubated at 37 ° C. for about 30 minutes.
To each tube, 10 pmol of the already transcribed mRNA was added and incubated for another 30 minutes at 37 ° C. Thereafter, 1 μL of each sample was collected and analyzed by 8M urea 5% denaturing polyacrylamide gel electrophoresis. The result of electrophoresis is shown in FIG.

As shown in FIG. 10, it was found that in the samples to which GMP was added, degradation of mRNA was suppressed in a concentration-dependent manner, and degradation was almost suppressed at 200 μM. On the other hand, in the negative control to which GMP was not added, a band was observed on the low molecular weight side, confirming that most of the original mRNA was degraded.
(3) Degradation of substrate by RNase T1
After completion of protein synthesis from mRNA, a high salt solution was added, and ligation reaction between puromycin and the growing peptide was performed for about 60 minutes. Thereafter, buffer exchange was performed, and a reverse transcription reaction was performed to form an mRNA / cDNA-protein conjugate. Subsequently, the buffer was exchanged with the above RNase T1 buffer, and then incubated at 37 ° C. for about 30 minutes for the purpose of spontaneously dissociating GMP. By this reaction, a band appeared on the lower molecule side than the substrate, indicating that the substrate linked to the substrate unit by RNase T1 was degraded.

  The present invention contributes to the efficiency of evolutionary engineering techniques for creating new proteins or polypeptides, and is useful for research and development for creating peptide / protein drugs or industrial enzymes.

Psoralen-amino segment
Azide segment
Alkyne-peptide-biotin segment

Claims (9)

A main chain having a side chain linking site in the vicinity of the 3 ′ end; a side chain having one end linked to the side chain linking site; and a substrate unit bonded to the main chain via a bridge. A linker;
The main chain is
An mRNA binding site located on the 3 ′ end side of the linker and functioning as a primer for reverse transcription;
A crosslink formation site located on the 5 ′ end side of the linker and forming a crosslink with the substrate unit;
The substrate unit is
A substrate unit side cross-linking site for forming a cross-link with the main chain;
A solid phase binding site having a predetermined molecule that forms a bond with the solid phase;
A substrate linking site to which a substrate capable of acting on a protein having enzyme-like activity bound to the linker is linked;
An RNA ligase recognition sequence in which one end forms an RNA ligase recognition site and the other end is linked to the substrate ligation site after a bridge is formed between the main chains;
The substrate linking site is located between the substrate side cross-linking site and the solid phase binding site and at a position where a protein having an enzyme-like activity bound to the side chain can bind.
The side chain has a protein binding site for binding a protein having enzyme-like activity.
Protein evolution linker with enzyme-like activity. A linker comprising a main chain having a side chain linking site in the vicinity of the 3 ′ end, a side chain having one end linked to the side chain linking site, and a substrate unit;
The main chain is
An RNA ligase recognition site, an mRNA binding site located on the 3 ′ end side of the RNA ligase recognition site and functioning as a primer for reverse transcription,
A main-chain-side cross-linking site located 5 ′ from the RNA ligase recognition site and forming a cross-link with the substrate unit;
,
A double-stranded forming region for forming the RNA ligase recognition site;
Comprising:
The other end of the side chain comprises a protein binding site for binding a protein having enzyme-like activity;
The substrate unit is
A solid phase binding site having a predetermined molecule that forms a bond with the solid phase;
A substrate linking site to which a substrate capable of acting on the protein having the enzyme-like activity is linked;
One end portion forms a bridge with the main chain, and the other end portion is connected to the substrate connecting portion, a cross-linking formation site,
Comprising:
The substrate linking site is located between the cross-linking site and the solid phase binding site and at a position where a protein having enzyme-like activity bound to the side chain can bind.
Protein evolution linker with enzyme-like activity.   The said substrate unit is couple | bonded with the said principal chain by forming either the bridge | crosslinking chosen from the group which consists of a photocrosslinking, a chemical bridge | crosslinking, and an enzymatic bridge | crosslinking. Protein evolution linker with enzyme-like activity.   The protein binding site is composed of puromycin selected from the group consisting of puromycin, 3′-N-aminoacylpuromycin aminonucleoside, and 3′-N-aminoacyladenosine aminonucleoside or a derivative thereof. A linker for protein evolution having the enzyme-like activity according to claim 1 or 2.   5. The protein having enzyme-like activity according to claim 4, wherein the substrate linking site is formed by binding a plurality of substrates with a protein having enzyme-like activity bound to the side chain. Evolution linker.   The combination of the protein having the enzyme-like activity and the substrate is: a polypeptide having an activity equivalent to that of a peptide-degrading enzyme and an arbitrary peptide sequence; a polypeptide having an activity equivalent to that of a glycolytic enzyme and an arbitrary sugar chain; A polypeptide having an activity equivalent to a nucleolytic enzyme and an arbitrary nucleic acid sequence; a polypeptide having an activity equivalent to a lipolytic enzyme and an arbitrary lipid; and a polypeptide having an activity to degrade a synthetic polymer and an arbitrary synthetic polymer; The protein evolution linker according to any one of claims 1 to 5, which is any one selected from the group consisting of:   The combination of the protein having the enzyme-like activity and the substrate comprises a polypeptide having an activity equivalent to that of a nucleic acid ligase and an arbitrary nucleic acid sequence; a polypeptide having an activity equivalent to that of a peptide-linked enzyme and an arbitrary peptide sequence; A polypeptide having an activity equivalent to that of an enzyme and an arbitrary peptide sequence; a polypeptide having an activity equivalent to that of a molecule linking enzyme for linking a molecule to a polypeptide or peptide, an arbitrary peptide sequence, an arbitrary transfer molecule substrate, and a nucleic acid A polypeptide having an activity equivalent to that of a molecule linking enzyme for linking molecules, an arbitrary nucleic acid sequence and an arbitrary transfer molecule substrate; a polypeptide having an activity equivalent to that of a glycosyltransferase, an arbitrary sugar chain and the aforementioned arbitrary sugar A group to be modified with a chain; and a polypeptide having an activity to catalyze a novel ligation and an arbitrary substrate. Et selected, and characterized in that either, protein evolution for linker according to any one of claims 1 to 5. An mRNA-linker, wherein the linker for protein evolution according to any one of claims 1 to 7 and mRNA having a sequence complementary to a part of the main chain of the linker are bound by RNA ligase at the mRNA binding site. A linked body production step;
Synthesizing a protein having enzyme-like activity using the mRNA as a template, and a conjugate generating step for obtaining an mRNA-linker-protein conjugate in which the protein is bound to a protein binding site on the side chain of the linker;
A cDNA-linker-protein conjugate production step of synthesizing a cDNA chain by reverse transcription using the mRNA as a template;
The protein having the enzyme-like activity is allowed to act on the substrate linked to the substrate linking site and the desired substrate bound on the solid phase to link them, and the cDNA-linker-protein conjugate is linked to the solid phase. An immobilization step of fixing to the surface;
A first washing step of washing the solid phase to which the cDNA-linker-protein conjugate is bound with a first buffer;
A recovery step of recovering the immobilized cDNA-linker-protein conjugate;
An amplification step of amplifying the cDNA of the recovered cDNA-linker-protein conjugate,
A screening method for proteins having enzyme-like activity. A linker for protein evolution according to any one of claims 1 to 7, wherein an mRNA having a sequence complementary to a part of the main chain of the linker is bound by an RNA ligase at the mRNA binding site. A body production process;
Synthesizing a protein having enzyme-like activity using the mRNA as a template, and a conjugate generating step for obtaining an mRNA-linker-protein conjugate in which the protein is bound to a protein binding site on the side chain of the linker;
A cDNA-linker-protein conjugate production step of synthesizing a cDNA chain by reverse transcription using the mRNA as a template;
A solid phase binding step of binding the cDNA-linker-protein conjugate to a solid phase via the solid phase binding site;
A first washing step of washing the solid phase to which the cDNA-linker-protein conjugate is bound with a first buffer;
A cleavage step of cleaving the cDNA-linker-protein conjugate from the solid phase by allowing a protein having an enzyme-like activity bound to a protein binding site on the side chain of the linker to act on the substrate linked to the substrate linkage site; ;
A recovery step of recovering the cleaved cDNA-linker-protein conjugate;
An amplification step of amplifying the cDNA of the recovered cDNA-linker-protein conjugate,
A screening method for proteins having enzyme-like activity.

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