Top > Search of International Patents > MOLECULE LIBRARY CONSTRUCTED ON THE BASIS OF BACKBONE STRUCTURE OF MICROPROTEIN

MOLECULE LIBRARY CONSTRUCTED ON THE BASIS OF BACKBONE STRUCTURE OF MICROPROTEIN

Foreign code F180009478
File No. 2013001940
Posted date Sep 18, 2018
Country WIPO
International application number 2013JP007238
International publication number WO 2014103203
Date of international filing Dec 9, 2013
Date of international publication Jul 3, 2014
Priority data
  • P2012-285734 (Dec 27, 2012) JP
Title MOLECULE LIBRARY CONSTRUCTED ON THE BASIS OF BACKBONE STRUCTURE OF MICROPROTEIN
Abstract Disclosed is a molecule library composed of a group of multiple molecules, wherein each member of the library is a polypeptide having a randomized sequence domain and a microprotein domain. The microprotein is a protein that is capable of being folded autonomously in a solution to form a specific three-dimensional conformation and comprises an amino acid sequence composed of 30 or less amino acid residues, such as chignolin that comprises the amino acid sequence represented by SEQ ID NO: 1. Also disclosed is a method for identifying a novel functional molecule using the library of the present invention.
Outline of related art and contending technology BACKGROUND ART
Naturally occurring protein or nucleic acid biological macromolecules with a high function method, or to create novel molecule or a directed evolution method is commonly referred to as a technique referred to as (or in vitro evolution) (non-patent document 1) region is present. In recent years, in the development of bio-pharmaceuticals and diagnostic agents as the basic technique, the use thereof has been increased.
Proteins and polypeptides with a target of a directed evolution in, the configuration of the initial library, success or failure of the key factors to create a novel molecular technique (non-patent document 2, 3). This initial forms of the molecular libraries, there are mainly divided into two types. One is 10 residues in length as short chain peptide, almost all molecules in the randomized sequence. The other is, the skeleton structure of the particular protein (fold) using as a substrate to, part of the sequence of the molecule with a comparatively long randomized in a single chain polypeptide. Short-chain peptide libraries each library framework-type protein the following the advantages and disadvantages.
Short-chain peptide library could include, library construction and screening work is relatively easy. 7-10 residues of the peptide in the case of the molecular heterogeneity of is random, the theoretical square of 20 7-10, i.e. 109 -1013 scale on the order of. If the library size in this range, using an existing technique can be used in the preparation of screening was performed with the library. In addition, various kinds of screening are widely used in display even in the technique, the smaller the molecular weight may be easily applied. On the other hand, a short-chain peptides generally have greater flexibility in the molecule, a specific three-dimensional structure in solution does not stably formed, a target receptor or peptide - peptide target - specific binding such as an enzyme in the thermodynamically less stable, high affinity molecules, it is difficult to obtain high specificity molecules have a drawback of a being.
Protein library framework-type, naturally occurring protein (or an artificial protein) specific skeletal structure as a substrate to be used. In many cases, known three-dimensional structure of the protein selected. Protein framework type in the library, rather than the entire molecule, only a part of the region is randomized. And the other portion of a particular sequence, in many cases remains in the native sequence. The reason for this is, unique randomization all areas of the creation of three dimensional structures cannot be expected in some cases. Therefore, with reference to the three-dimensional structure data and the like, which contributes to the stabilization in the original protein structure and conserve amino acid residues, positioned on the surface side of the molecule such as a randomized loop region in many cases. A plurality of loop regions may be randomized. In addition, in recent years, natural proteins but artificially designed artificial protein sequence is sometimes used as a base for the framework structure.
The concept of protein library framework type, the molecular structure of the antibody (immunoglobulin) in a manner simulates. That is, the randomized portion, corresponds to a region of an antibody variable region, and the other portion of the native sequence of the remains, corresponding to the constant region. Antigens in the antibody variable region such that, in the protein framework type library, randomized portion and points to the acquisition of a new function.
Randomized sequences introduced into the protein backbone is, unlike the case of short-chain peptide libraries, and the both ends thereof fastened to a structure a rigid scaffold that can be taken from the fact that the conformation is limited, as a result, due to the flexibility of the molecule can be expected that avoid the disadvantages. On the other hand, the molecule size is relatively big is required and thus, research and development on the difficulty associated with this, when the manufacturing cost of the practical use, a disadvantage of a decrease in storage stability and the like pointed out. In addition, in conformation is not limited to, reverse-active structures cannot be achieved in a potential risk also.
On the other hand, low-molecular-weight, the polypeptide having a stable structure as a library, based on the cyclic oligopeptide backbone also known molecular libraries. However, in order to perform the oligopeptide MACROLACTAM, complicated with the introduction of the functional group chemical reaction operations are needed, the synthesis step is complicated. In addition, the oxidation reaction of the cysteine MACROLACTAM is oligopeptide, such as the use of a reducing environment within the cell is generally difficult to have the drawback of.
As described above protein framework type library, for use as a base for the native protein (or an artificial protein) needs to be selected. 40 Proteins to more than one up to now used (non-patent document 2, 3). The main libraries shown in Table 1, exemplary of some of such.
TABLE 1
(SPA) staphylococcal protein A modified protein Z antibody binding domain of the protein skeleton affibody (non-patent document 4, 5, 6, Patent Document 1, 2) is, (6. 5 kDa) protein of 58 residues, without depending on the intramolecular disulfide bridge and a high stability, maintaining the solubility, the mass production in a bacterial expression system besides allowing an, also a chemical production by solid phase synthesis (non-patent document 7) are. Helix 13 residues on the molecule and generating a library can be variable, up to now the binding molecule is several ten types of for the target protein has been obtained. As diagnostic reagents is the best studied has been progressed affibody, a cell surface receptor and a high affinity for affibody HER-2, applied as a diagnostic imaging molecule (non-patent document 8) are.
Fibronectin 3 domain and a small protein domain β sheet, 2 to 3 amino acid residues in the loop region of randomized ubiquitin from the library against multiple targets such as the binding molecule is acquired (non-patent document 9, patent document 3).
Mini-body, the heavy chain variable domain of monoclonal antibodies from 3 by removal of one β chain designed artificial protein (non-patent document 10). This protein is 61 residues and the full-length, with two loops 2. One of the 2 loop portion is randomized. (10 μm) is a practical use of the low solubility problem but, by mutagenesis (350 μm) and that has a high degree of solubility in body mini-mutations have been reported (non-patent document 11).
74 Residues is, sandwich-like sheet 2 of the β 6 chain linked together by one disulfide bond (non-patent document 12). And the skeleton is included in one loop of the 3. To date, only one of 2 of these loops, have been tested and are randomized.
Cytochrome b562 4 is 106 residues structure protein domain, 2 amino acid residues present a loop-shaped portion 9 to randomize 290 nm a low molecular weight hapten molecules that bind the equilibrium dissociation constant (non-patent document 13) has been obtained.
Oligonucleotide oligo/polysaccharide binding fold (OB - fold) is, the amphipathic nature of the α - helix five-stranded β - barrel capped the scaffold structure (Patent Document 4). 20 In the analysis of the genomic sequence of one or more typically the second most common 28 (non-patent document 14) is mDRiPs.
Β backbone cyclization, disulfide - constrained type affords the formation of the secondary structure, conformation in solution and stabilize the low-molecular-weight protein skeleton (Patent Document 5).
Of the short-chain peptides for stabilizing the α helices, disulfide bridges can be introduced into a coiled-coil structures designed based on protein scaffolds. Arginine - glycine - aspartic acid (RGD) quality of the transgene nucleotide sequence competing for the fibrinogen backbone (non-patent document 15) inhibitory activity.
An artificial protein based on quality (Designed AR protein, DARPin) is, in a repeated structure (non-patent document 16) large proteins. The size of the repeating unit residues in domain 33, does not include the disulfide linkage in the β-turn and anti-parallel helix constituted from the loop.
A - Domain (non-patent document 17) is, in the repeating unit being observed as a skeletal structure, cell surface receptors of various species observed, amino acid residues 35-40 configured as a linking domain.
Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a cell surface receptor belonging to the helper T and, by introducing the recognition sequence of the hypervariable loops to the affinity of the obtained (non-patent document 18).
Antibody (immunoglobulin) is, because of their high specificity binding molecule used most widely as a protein. 12 Immunoglobulin g is made up of subunits the molecular weight of about 15 million of macromolecules and, by enzymatic treatment in the area containing the antigen binding (Fab) having antigen-binding fragment thereof, produced by genetic engineering techniques or a heavy chain variable region (VH) and light chain variable regions (VL) variable region fragment (Fv), linked by a peptide linker VL VH and further a single-chain antibody (scFv) and the like, often as a unit of the binding molecule (non-patent document 19) are in general use. Does not depend on the natural immune repertoire as an artificial antibody, an antibody variable region framework used as protein scaffolds, the complementarity determining regions is randomized HuCAL molecular libraries have been reported (non-patent document 20).
Scope of claims (In Japanese)請求の範囲
[請求項1]
複数の分子の群から構成される分子ライブラリであって、ライブラリの各メンバーは、ランダム化配列部分と微小タンパク質部分とを有するポリペプチドであることを特徴とする分子ライブラリ。
[請求項2]
微小タンパク質が、溶液中で自発的にフォールディングし特定の立体構造を形成する能力を有する30アミノ酸残基以下の直鎖状ポリペプチドからなるタンパク質である、請求項1に記載の分子ライブラリ。
[請求項3]
微小タンパク質が、下記のアミノ酸配列:
Gly Tyr Asp Pro Glu Thr Gly Thr Trp Gly (配列番号1)
からなるシニョリンであるか、あるいは該アミノ酸配列において1個若しくは数個のアミノ酸残基が欠失、置換、挿入または付加されたアミノ酸配列からなるシニョリン変異体である、請求項1または2に記載の分子ライブラリ。
[請求項4]
微小タンパク質が、下記のいずれかのアミノ酸配列:
Xaa Tyr Asp Pro Xaa Thr Gly Thr Trp Xaa (配列番号2)
Tyr Tyr Asp Pro Glu Thr Gly Thr Trp (配列番号3)
Tyr Asp Pro Glu Thr Gly Thr Trp Tyr (配列番号4)
Tyr Asp Pro Xaa Thr Gly Thr Trp (配列番号5)
(式中、Xaaは任意のアミノ酸残基を示す)
からなるシニョリン変異体である、請求項3に記載の分子ライブラリ。
[請求項5]
ライブラリの各メンバーが、下記のアミノ酸配列:
 (Xaa) n-Tyr-Asp-Pro-Xaa-Thr-Gly-Thr-Trp-(Xaa) m
(式中、Xaaは任意のアミノ酸残基を示し、nは0以上の整数であり、m は0以上の整数であり、ただしnとmは同時に0ではない)
からなるポリペプチド分子であることを特徴とする請求項1-4のいずれかに記載の分子ライブラリ。
[請求項6]
ライブラリの各メンバーが、下記のアミノ酸配列:
 -[(Xaa) n-Tyr-Asp-Pro-Xaa-Thr-Gly-Thr-Trp-(Xaa) m] k-
(式中、Xaaは任意のアミノ酸残基を示し、kは2以上の整数であり、各nは独立して0以上の整数であり、各mは独立して0以上の整数である)
からなるポリペプチド分子であることを特徴とする請求項1-4のいずれかに記載の分子ライブラリ。
[請求項7]
ライブラリの各メンバーは、さらに固定配列部分を含む、請求項1-6のいずれかに記載の分子ライブラリ。
[請求項8]
前記固定配列部分が、既知のポリペプチドの全部あるいは一部のアミノ酸配列、または請求項1-6のいずれかに記載の分子ライブラリから選択されたポリペプチドの全部あるいは一部のアミノ酸配列からなる請求項7に記載のライブラリ。
[請求項9]
ライブラリの各メンバーのポリペプチドは、それぞれのポリペプチドをコードするポリヌクレオチドと対応付けられた形態で存在していることを特徴とする請求項1-8のいずれかに記載の分子ライブラリ。
[請求項10]
ライブラリの各メンバーのポリペプチドは、それぞれのポリペプチドをコードするポリヌクレオチドと連結されていることを特徴とする請求項9に記載の分子ライブラリ。
[請求項11]
ライブラリの各メンバーのポリペプチドはバクテリオファージの表層に提示されており、それぞれのポリペプチドをコードするポリヌクレオチドは前記バクテリオファージに内包されている、請求項9に記載の分子ライブラリ。
[請求項12]
請求項1-11のいずれかに記載の分子ライブラリの各メンバーをコードするポリヌクレオチドの群から構成されるポリヌクレオチドライブラリ。
[請求項13]
標的物質に結合しうるポリペプチド分子を同定する方法であって、下記(a)~(c)の工程:
(a) 請求項7-11のいずれかのライブラリを前記標的物質に接触させる工程
(b) 前記ライブラリから前記標的物質と結合するメンバーを選択する工程
(c) 前記選択されたメンバーのアミノ酸配列を決定する工程
を含むことを特徴とする方法。
[請求項14]
アミノ酸配列の決定が、ポリペプチドに対応付けられたポリヌクレオチドの塩基配列を決定することにより行われる、請求項13に記載の方法。
[請求項15]
標的物質がヒト免疫グロブリンである、請求項13または14に記載の方法。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND TECHNOLOGY
  • Inventor
  • HONDA, Shinya
  • WATANABE, Hideki
  • YAMASAKI, Kazuhiko
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN TD TG
Reference ( R and D project ) Biomedical Research Institue,Molecular and Cellular Breeding Research Group

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