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Method for producing peptides containing non-natural amino acids

A technology of unnatural amino acids and amino acids, applied in the field of producing peptides containing unnatural amino acids, can solve problems such as reducing fidelity

Pending Publication Date: 2022-07-22
CHUGAI PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, it has also been reported that L31-deficient strains show reduced fidelity (NPL4)

Method used

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  • Method for producing peptides containing non-natural amino acids
  • Method for producing peptides containing non-natural amino acids
  • Method for producing peptides containing non-natural amino acids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0292] Example 1. Construction of E. coli strains

[0293] A strain lacking ompT (Protease 7) (defined as an L31intact strain) and an L31short strain expressing the 1st to 62nd amino acids of the N-terminal of the L31 protein were constructed. During ribosome purification, protease 7 is known to degrade between amino acids 62 and 63 of the L31 protein.

[0294] E. coli strains were constructed according to the procedure of Quick and Easy Conditional Knockout Kit (loxP / Cre) (Gene Bridges).

[0295] Preparation of functional cassettes with added homology arms

[0296] PCR reactions were performed using PrimeSTAR HS DNA polymerase (Takara Bio Inc., R010A) using the cassette loxP-PGK-gb2-neo-loxP (Gene Bridges, A003) as template. The obtained PCR product was purified using the QIAquick PCRPurification Kit (QIAGEN, 28104). The concentration of the functional cassette is approximately 200 ng / μL. PCR was performed using Oligol (SEQ ID NO: 3) and Oligo2 (SEQ ID NO: 4) as primers...

Embodiment 2

[0299] Example 2. Method for preparing ribosomes

[0300] Culture of Escherichia coli

[0301] The W3110 strain (WT) was grown.

[0302] The bacterial cells of the W3110 strain were inoculated into the pre-medium (glycerol 5g / L, yeast extract 6g / L, KH 2 PO 4 4g / L, K 2 HPO 4 9.3g / L) and pre-incubated. The precultured bacterial cells were added to 30L main medium (glycerol 10g / L, yeast extract 10g / L, polypeptone N15g / L, KH 2 PO 4 4g / L, MgSO 4 ·7H 2 O 2.4g / L, FeSO 4 ·7H 2 O 0.04g / L, CaCl 2 ·2H 2 O 0.04g / L, decyl alcohol LG-1090.24g / L), make OD 600 is 0.1. Cultures were performed in 50-L culture vessels. Cells were incubated at 37°C for 5.4 hours, when the OD 600 Recycled when reaching 33.5. The culture solution was aliquoted into 500 mL and left at room temperature for 1 hour and at 4°C for another hour. Thereafter, the mixture was centrifuged at 6000×g for 10 minutes, the pellet was suspended in D-PBS(-) (Takara Bio Inc., T9181), and then centrifuged aga...

Embodiment 3

[0325] Example 3. Analysis of percent degradation of L31

[0326] The percent degradation of L31 in WT-10MG ribosomes, WT-5MG ribosomes, and WT-0MG ribosomes was analyzed.

[0327] Sample preparation method

[0328] Ribosomes (40 pmol) were diluted with 38 μL of water. 1% trifluoroacetic acid was added to precipitate ribosomal RNA. Centrifugation was performed and the supernatant was mixed 1:1 with matrix (50% acetonitrile, 5 mg / mL sinapic acid) and crystallized for MALDI / MS by spotting 1 [mu]L on the plate.

[0329] Analysis by MALDI / MS

[0330] Measurements were performed on a mass spectrometer (ABS CIEX·TOF / TOF 5800) using linear positive mode. Calibration was performed using ribosomal proteins as internal standards. Use properly calibrated measurements. The percentage of intact L31 was calculated by dividing the MS intensity of the intact L31 by the sum of the MS intensity of the intact L31 and the MS intensity of the short L31 (short L31). Measurements were mad...

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Abstract

The present invention reveals that translation of mRNA encoding a peptide containing a non-natural amino acid in a translation system comprising a ribosome containing a modified L31 protein can increase the amount of translated peptide. In addition, the present invention reveals that the relative amount of by-products can also be reduced by using such ribosomes. The modified L31 protein has an amino acid sequence in which 6 or more amino acid residues are deleted from the C-terminal of the amino acid sequence of the wild-type Escherichia coli L31 protein.

Description

technical field [0001] The present invention relates to methods of producing peptides comprising unnatural amino acids and libraries comprising the same. The present invention also relates to engineered L31 proteins and the like for use in these methods. [0002] Background of the Invention [0003] Drug discovery methods involving the selection of drug candidates from multiple peptide libraries containing multiple unnatural amino acids have recently been devised. In particular, mRNA display libraries containing unnatural amino acid peptides using cell-free translation systems and the like are increasingly regarded as promising due to their diversity and ease of screening. There are many reports on methods of synthesizing peptides containing unnatural amino acids using translation systems (NPL 1 and NPL 2). However, translation synthesis is inefficient in the methods described in these documents. [0004] In vivo, peptides are synthesized through amino acid polymerization ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K7/04C07K7/64C12N1/15C12N1/19C12N1/21C12N5/10C12N15/09C12N15/11C12N15/63C12P21/02C40B30/04C40B40/10G01N33/15G01N33/50
CPCC12P21/02C40B30/04C12N15/70C40B40/08C07K14/245G01N33/68G01N2500/02C12N15/67C12N15/63C12N15/1062C12N15/11
Inventor 吉井早贵子下村康一郎石野聪
Owner CHUGAI PHARMA CO LTD
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