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Soluble fragments of influenza virus pb2 protein capable of binding rna-cap

An influenza virus, soluble technology, applied in the field of soluble fragments of influenza virus PB2 protein that can bind RNA caps, can solve problems such as unknown structure and identity of the binding pocket, controversial results, interference with RNA cap binding, etc.

Inactive Publication Date: 2011-02-09
EURO LAB FUER MOLEKULARBIOLOGIE EMBL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the major obstacle to identifying compounds that interact with the RNA cap-binding pocket of PB2 and that might interfere with RNA cap binding and thus RNA polymerase activity has so far been that the structure and identity of the binding pocket is unknown
[0006] There have been some efforts to elucidate the RNA cap binding site, but the results are controversial

Method used

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  • Soluble fragments of influenza virus pb2 protein capable of binding rna-cap
  • Soluble fragments of influenza virus pb2 protein capable of binding rna-cap
  • Soluble fragments of influenza virus pb2 protein capable of binding rna-cap

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0164] Example 1: Generation of PB2 expression constructs

[0165] The E. coli codon-optimized PB2 gene (Geneart) SEQ ID NO: 25 based on the influenza A / Victoria / 3 / 1975 (H3N2) PB2 amino acid sequence (dela Luna et al., 1989) was cloned into the pET9a (Novagen) vector , which was modified to introduce a 5'Aatll / AscI and 3'Nsil / NotI restriction site pair for targeted truncating exonuclease III and to provide the 3' sequence encoding the C-terminal biotin acceptor peptide GLNDIFEAQKIEWHE . The first non-directional truncation plasmid library (Tarendeau et al., 2007) was generated from the 3' end, pooled and used as a substrate for the 5' deletion reaction. Linearized plasmids encoding inserts of 150 to 250 amino acids were isolated from agarose gels, religated, and then used to transform E. coli BL21A1 containing RIL plasmids (Stratagene) for use with Alexa488 streptavidin (Invitrogen) Expression screening was performed by hybridization to colony blots (Tarendeau et al., 2007...

Embodiment 2

[0174] Example 2 : Identification of minimal cap-binding fragments

[0175] PB2 polypeptide fragments 235-496, 241-483, 268-483 and 290-483 were expressed in E. coli using the plasmid constructs described in Example 1 and the general expression and purification protocol described in Example 3. Purified PB2 fragment 235-496 was found to be soluble only at low concentrations, while fragments 241-483, 268-483 and 290-483 were more soluble and could specifically bind m 7 GTPsepharose column, indicating cap binding activity. For these assays, 0.2 mg of protein was loaded onto 50 μl of 7-methyl-GTP Sepharose 4B resin (GE Healthcare) and incubated for 3 hours at 4°C for binding. After washing with a buffer containing 50mM Tris.HCl (pH 8.0), 200mM NaCl, 2mM DTT, use a solution containing 1mM m 7 Proteins were eluted by centrifugation in the same buffer as GTP and analyzed on SDS-PAGE. During the course of these experiments, it was noted that smaller degradation fragments can also...

Embodiment 3

[0176] Example 3 : Expression and purification of PB2 fragments

[0177] The plasmids were transformed into E. coli using chemical transformation. The protein was expressed in E. coli strain BL21-CodonPlus-RIL (Stratagene) in LB medium. After induction with 0.2 mM isopropyl-β-thiogalactopyranoside (IPTG) for 16 to 20 hours at 25°C, cells were harvested and resuspended in lysis buffer (50 mM Tris HCl (pH 8.0), 300 mM NaCl , 5mM 2-mercaptoethanol) and sonicated. After centrifugation, the clarified lysate was directly loaded onto a nickel affinity column (chelated sepharose from GE Healthcare loaded with Ni 2+ ion). Wash the Ni-sepharose resin thoroughly with 50mM Tris·HCl (pH 8.0), 1M NaCl, 15mM imidazole, mM 2-mercaptoethanol buffer, and then rinse with 50mM Tris·HCl(pH 8.0), 200mM NaCl, 50mM imidazole, 5mM 2-mercapto Wash with ethanol buffer. Then, the protein was eluted with 50 mM Tris·HCl (pH 8.0), 200 mM NaCl, 0.5 M imidazole, 5 mM 2-mercaptoethanol buffer. Purified...

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Abstract

The present invention relates to soluble fragments of the Influenza virus RNA dependent RNA polymerase subunit PB2 and variants thereof, and crystallized complexes thereof comprising an RNA cap analog. This invention also relates to computational methods using the structural coordinates of said complex to screen for and design compounds that interact with the RNA cap binding pocket. In addition, this invention relates to methods identifying compounds that bind to PB2 polypeptide fragments comprising the RNA cap binding pocket, preferably inhibit the interaction with RNA caps or analogs thereof, by using said PB2 polypeptide fragments, preferably in a high-throughput setting. This invention also relates to compounds and pharmaceutical compositions comprising the identified compounds for the treatment of disease conditions due to viral infections caused by negative-sense single stranded RNA viruses.

Description

field of invention [0001] The present invention relates to soluble fragments of influenza virus RNA-dependent RNA polymerase subunit PB2 and variants thereof, and crystallized complexes thereof comprising RNA cap (RNA cap) analogues. The present invention also relates to computational methods for screening and designing compounds that interact with the RNA cap binding pocket using the structural coordinates of said complex. In addition, the present invention relates to the use of PB2 polypeptide fragments to identify, preferably in a high-throughput format, compounds that bind (preferably inhibit interaction with RNA caps or analogs thereof) to PB2 polypeptide fragments comprising an RNA cap binding pocket. method. The present invention also relates to compounds and pharmaceutical compositions comprising the identified compounds for use in the treatment of viral infection conditions caused by negative-sense single-stranded RNA viruses. Background technique [0002] Influen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/00C07K14/11C12Q1/18C07K14/16
CPCC07K2299/00C12N2760/16122G01N2500/04C07K14/005G01N2333/11A61P31/14A61P31/16B65D2501/0036B65D1/0223
Inventor 斯蒂芬·库萨克德尔菲娜·吉利盖达伦·哈特弗兰克·塔伦多
Owner EURO LAB FUER MOLEKULARBIOLOGIE EMBL
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