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MOLECULE LIBRARY CONSTRUCTED ON THE BASIS OF BACKBONE STRUCTURE OF MICROPROTEIN コモンズ

外国特許コード F180009478
整理番号 2013001940
掲載日 2018年9月18日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2013JP007238
国際公開番号 WO 2014103203
国際出願日 平成25年12月9日(2013.12.9)
国際公開日 平成26年7月3日(2014.7.3)
優先権データ
  • 特願2012-285734 (2012.12.27) JP
発明の名称 (英語) MOLECULE LIBRARY CONSTRUCTED ON THE BASIS OF BACKBONE STRUCTURE OF MICROPROTEIN コモンズ
発明の概要(英語) 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.
従来技術、競合技術の概要(英語) 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).
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND TECHNOLOGY
  • 発明者(英語)
  • HONDA SHINYA
  • WATANABE HIDEKI
  • YAMASAKI KAZUHIKO
国際特許分類(IPC)
指定国 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
参考情報 (研究プロジェクト等) Biomedical Research Institue,Molecular and Cellular Breeding Research Group

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