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CRYSTALLIZATION-PROMOTING POLYPEPTIDE meetings

Foreign code F150008266
Posted date Mar 31, 2015
Country WIPO
International application number 2014JP062215
International publication number WO 2014181786
Date of international filing May 7, 2014
Date of international publication Nov 13, 2014
Priority data
  • P2013-097698 (May 7, 2013) JP
Title CRYSTALLIZATION-PROMOTING POLYPEPTIDE meetings
Abstract The purpose of the present invention is to provide a versatile and effective method for promoting the crystallization of a protein of interest in X-ray crystal structure analysis. The abovementioned problem can be solved by (1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO:1, (2) a polypeptide comprising the amino acid sequence represented by SEQ ID NO:2, and (3) a polypeptide comprising the amino acid sequence represented by SEQ ID NO:2, wherein 1 to 18 amino acids have been deleted from the N-terminal side, and/or any of 1 to 3 amino acids have been deleted from the C-terminal side.
Outline of related art and contending technology BACKGROUND ART
The structural analysis of protein X-ray crystal structure analysis method, NMR method, or electron microscope analysis is mainly used. In which, X-ray crystal structure analysis method, an electron microscope image can be obtained with higher accuracy than molecules. In addition, as compared to large molecules NMR in molecular weight can also be applied. Therefore, X-ray crystal structure analysis method, as a method for structural analysis of protein, a general-purpose. However, X-ray crystal structure analysis method for analysis of proteins, protein crystal must be made. In the single molecule protein, due to low weak X-ray scattering, the analysis is not possible. Protein crystals, since the diffraction light is amplified, scattering intensity can be measured, by calculating the electron density it, to create a model structure of the protein molecule becomes possible.
However, the crystallization of the protein X-ray crystal structure analysis method is also the largest barrier. Protein crystallization conditions, for example even between proteins belonging to the same family are greatly different from, and precipitation pH is very sensitive to changes of the concentration. In addition, crystallisation of proteins is not seen in current mechanism. Therefore, the crystallization of the protein, and a large amount of conditions necessary to conduct a study, a large amount of labor, time, and costly. Then, in the X-ray crystal structure analysis method, for good-quality crystal cannot be obtained, often cannot be protein structure analysis. The use of a protein X-ray crystal structure analysis in order to obtain, for examining the condition of the large number of crystal kit developed crystallization, crystallization conditions to be used in the study was conducted to the robot. Further, a zero-gravity field (space) the crystallization, the crystallization by irradiation with laser or magnetic field but have been examined, and both say that sufficient effect was unacceptable.
Scope of claims (In Japanese)請求の範囲 [請求項1]
(1)配列番号1で表されるアミノ酸配列からなるポリペプチド、
(2)配列番号2で表されるアミノ酸配列からなるポリペプチド、
(3)配列番号2で表されるアミノ酸配列において、N末側から1~18個のアミノ酸が欠失、及び/若しくはC末側から1~3個のいずれかのアミノ酸が欠失したアミノ酸配列からなるポリペプチド、又は
(4)前記ポリペプチド(1)、ポリペプチド(2)、又はポリペプチド(3)のアミノ酸配列において、1若しくは複数のアミノ酸が欠失、置換、挿入、及び/若しくは付加されたアミノ酸配列からなり、かつタンパク質のC末端若しくはN末端に直接若しくはペプチドリンカーを介して融合させた融合タンパク質として発現させた場合に当該融合タンパク質の結晶化を促進する活性を示す、改変体ポリペプチド。

[請求項2]
 前記改変体ポリペプチドが、配列番号3、又は4で表されるアミノ酸配列からなる、請求項1に記載のポリペプチド。

[請求項3]
 請求項1又は2に記載のポリペプチドが、タンパク質のC末端又はN末端に、直接又はペプチドリンカーを介して融合した、融合タンパク質。

[請求項4]
 請求項1又は2に記載のポリペプチド又は請求項3に記載の融合タンパク質をコードするポリヌクレオチド。

[請求項5]
 請求項4に記載のポリヌクレオチドを含む組換えベクター。

[請求項6]
 請求項5に記載の組換えベクターを含む形質転換細胞。

[請求項7]
 請求項3に記載の融合タンパク質をコードするポリヌクレオチドを含む組換えベクターを構築する工程、
前記組換えベクターを用いて融合タンパク質を発現させる工程、及び
前記発現した融合タンパク質を精製する工程、
を含む融合タンパク質の製造方法。

[請求項8]
 請求項3に記載の融合タンパク質をコードするポリヌクレオチドを含む組換えベクターを構築する工程、
前記組換えベクターを用いて融合タンパク質を発現させる工程、
前記発現した融合タンパク質を精製する工程、及び
精製された融合タンパク質の多量体形成を確認する工程、
を含む請求項7に記載の融合タンパク質の製造方法。

[請求項9]
 請求項3に記載の融合タンパク質をコードするポリヌクレオチドを含む組換えベクターを構築する工程、
前記組換えベクターを用いて融合タンパク質を発現させる工程、
前記発現した融合タンパク質を精製する工程、
精製された融合タンパク質の多量体形成を確認する工程、
精製された融合タンパク質を結晶化させる工程、及び
X線照射によりタンパク質結晶を確認する工程、
を含むタンパク質結晶の製造方法。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL UNIVERSITY CORPORATION HOKKAIDO UNIVERSITY
  • Inventor
  • YAO Min
  • KOMODA Keisuke
  • YU Jian
  • TANAKA Yoshikazu
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
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