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Ribosome display complex and production method therefor

A technology of ribosome display and manufacturing method, which is applied in the field of ribosome display complex and its manufacturing, and can solve the problems of difficult technology and the like

Active Publication Date: 2019-02-05
KANEKA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, for polypeptides, ribosomes, or RD complexes containing multiple cysteines, lysines, histidines, and tryptophans, modifications to target cysteines should be designed without compromising their functions. The technique of the amino acid method is not easy

Method used

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  • Ribosome display complex and production method therefor
  • Ribosome display complex and production method therefor
  • Ribosome display complex and production method therefor

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preparation example Construction

[0104] (1) Preparation process of ribosome display complex

[0105] In this step, mRNA molecules are translated using a cell-free peptide synthesis system using ribosomes to obtain ribosome complexes including unmodified polypeptide chains, mRNA molecules, and ribosomes.

[0106] The cell-free peptide synthesis system using ribosomes is a system for synthesizing polypeptides from mRNA in vitro using compounds required for polypeptide synthesis based on RNA information carried out in cells. Specifically, mRNA molecules are added to a reaction system containing proteins, ribosomes, tRNA, amino acids, NTP, buffers, etc. required for mRNA translation, energy regeneration, etc. , to synthesize a polypeptide corresponding to the added mRNA. Since a kit for synthesizing a cell-free peptide is commercially available, it is sufficient to use a commercially available kit other than the mRNA molecule.

[0107] The ribosome display complex prepared in this step includes mRNA, a polypept...

Embodiment 1

[0165] (1) Preparation of RNA library

[0166] In this (1) project, for the use of NNK method to make 10 12 The method of the RNA library of the above RNA is described, the RNA has a (NNK) comprising (NNK) 10 [wherein, N represents A, U, G or C, K represents G or U, and NNK corresponds to all codons] the sequence of the sequence.

[0167] To make this RNA library, use a figure 1 The structure of the template DNA (base sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2). Specifically, using the reaction solution having the composition shown in Table 1, a 5' fragment was prepared using the plasmid as a template DNA in the PCR cycle in Table 2. In Table 1, 5FFnew_130816 is the forward primer, and Ma3frag_R0502 is the reverse primer.

[0168] [Table 1]

[0169]

[0170] [Table 2]

[0171]

[0172] Next, using the reaction solution having the composition shown in Table 3, the 3' fragment of the template DNA was prepared according to the PCR cycle in Table 4. In...

Embodiment 2

[0211] Embodiment 2: the cyclization reaction of peptide

[0212] Using a recombinant cell-free protein synthesis kit ("PURE frex (registered trademark)" manufactured by GeneFrontier Corporation), RNA having a FLAG sequence, a His6 sequence, and a nucleotide sequence encoding a TEV protease site (SEQ ID NO. 8) 2.5×10 13 The molecules were reacted at 37°C for 35 minutes to prepare an RD complex, and anti-FLAG (registered trademark) M2 antibody-bound agarose beads (manufactured by Sigma-Aldrich, 2 μL) were added to the reaction solution to bind to the RD complex. Further, tris(2-carboxyethyl) sodium salt (pH 7, final concentration 0.5 mM) was added as a reducing agent and Figure 4 Each modification reagent at the indicated final concentration of 2 mM was stirred at 4° C. for 3 hours, thereby cyclizing the peptide in the RD complex on the beads. After the reaction, the RD complex was released from the beads by adding FLAG peptide (sequence: DYKDDDDK, 5 mg). Separate and remov...

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Abstract

The present invention addresses the problem of providing: a ribosome complex which can be prepared without involving a complicated reaction procedure, and in which a presented polypeptide is modifiedwithout impairing the function of a ribosome (particularly, the function for producing a polypeptide library); and a method for preparing the ribosome complex. The ribosome display complex productionmethod according to the present invention is characterized by comprising: a step for translating an mRNA molecule using a cell-free peptide synthesis system utilizing a ribosome, to obtain a ribosomecomplex including an unmodified polypeptide chain, the mRNA molecule, and the ribosome; and a step for reacting a side-chain reactive functional group included in the unmodified polypeptide chain witha modification reagent to modify the unmodified polypeptide chain.

Description

technical field [0001] The present invention relates to a novel ribosome display complex and its manufacturing method. Background technique [0002] In recent years, studies have been conducted to select therapeutic drugs for specific diseases, molecules with high affinity to target molecules, and the like from polypeptide libraries of various amino acid sequences. The reason for this is considered to be that the specificity and selectivity of polypeptides are high, and therefore they have fewer side effects than low-molecular-weight compounds. In addition, DNA encoding a polypeptide in which the amino acid sequence of a specific portion is randomized can be easily obtained by PCR technology using random primers. Therefore, compared with a library of low-molecular-weight compounds, it also has the advantage of being able to easily prepare a library containing a large number of polypeptides. Advantages such as libraries (Non-Patent Documents 1 to 3). [0003] On the other h...

Claims

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Application Information

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IPC IPC(8): C12N15/09C07K2/00C12P21/00C40B40/10
CPCC40B40/10C07K1/1077C12N15/1041C12P21/00C12N15/09C07K2/00
Inventor 北宽士鹿仓敏裕前田博文高津庆士
Owner KANEKA CORP
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