Novel affinity peptide ligand of murine polyoma capsomere as well as designing and screening method thereof

A technology of murine polyoma virus and screening method, which is applied in the field of computer simulation and protein separation and purification, and can solve the problems of limiting the application of murine polyoma virus-like particles, complicated operation, and increased cost.

Active Publication Date: 2014-09-10
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, during post-purification treatment, expensive thrombin cleavage is required to remove the GST tag, and the shed GST tag requires additional separation and purification steps to remove it from

Method used

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  • Novel affinity peptide ligand of murine polyoma capsomere as well as designing and screening method thereof
  • Novel affinity peptide ligand of murine polyoma capsomere as well as designing and screening method thereof
  • Novel affinity peptide ligand of murine polyoma capsomere as well as designing and screening method thereof

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Experimental program
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Embodiment 1

[0031] Example 1 Analysis of the mechanism of affinity between VP2-C and capsid particles and the construction of a simplified affinity binding model for VP2-C

[0032] The crystal structure of the complex of VP2-C and capsid particles used in this study was obtained from the PDB database ( http: / / www.rcsb.org / pdb / , No.: 1CN3), wherein the capsid particle contains 5 VP1 (residues 34-316), and VP2-C contains 19 residues (residues 279-297). The molecular dynamics simulation adopts GROMACS4.5.3 and CHARMM27 all-atom standpoint. First, the TIP3P water molecule model was used to dissolve the complex of VP2-C and capsid particles in a cube box (12.163×12.163×12.163nm). Add 225 Na + and 190 Cl - . After energy minimization of the system, the restricted kinetic equilibrium under the normalized (NVT) ensemble of 200 ps and the isothermal and isobaric (NPT) ensemble of 200 ps is sequentially carried out. All simulations use periodic boundary conditions. A distance cutoff of 1.2n...

Embodiment 2

[0035] The construction of embodiment 2 polypeptide library

[0036] The five key residues D286, W287, L289, L293, and Y296 closest to the bottom of the capsid particle are selected as the starting point for polypeptide construction, which can avoid the steric hindrance effect in actual operation to the greatest extent. The fact that W287, L289, L293 and Y296 are almost on the same straight line is also conducive to the design of short peptide ligands without considering the spatial conformation of VP2. Calculated using VMD Therefore, one amino acid can be inserted between every two adjacent hotspot residues. The finally obtained peptide construction mode is an octapeptide library: DWXLXLXY (X is a residue that does not contain Cys). A perl script was used to call CHARMM software to construct a polypeptide library, which contained a total of 6859 sequences, and each sequence contained the above-mentioned 5 hotspot residues.

Embodiment 3

[0037] The docking of embodiment 3 polypeptide and capsid particle

[0038] 1. VINA docking

[0039] Using VINA software, 6859 polypeptides were respectively docked to the binding domains on the surface of the capsid particle lumen, and the scores of all polypeptides were between -4 and -8kcal / mol, which was in line with the moderate affinity requirement of the affinity ligand ( Binding constant at 10 4 ~10 8 m -1 between). In order to avoid missed selection during screening, according to empirical values ​​and distribution results, a total of 1158 polypeptides with binding free energy lower than -6.5kcal / mol were selected.

[0040] 2. RMSD calculation

[0041] In this study, the g_rms program included in the GROMACS4.5.3 software package was used to calculate the RMSD between the hotspot residues in 1158 polypeptide sequences obtained by VINA docking and the corresponding hotspot residues in capsid particles. The smaller the RMSD value, the closer the docking conformati...

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Abstract

The invention discloses a novel affinity peptide ligand of murine polyoma capsomere as well as a designing and screening method thereof. Based on a compound of natural existing capsomere and a minor capsid structure protein VP2-C, a bionic designing process of the affinity peptide ligand of the capsomere is constructed. The method comprises the following steps: firstly, resolving intermolecular interactions in the compound by utilizing a molecular dynamics simulation combined Poisson-Boltzmann solvent accessible surface area (MM/PBSA) method; determining a hot spot residue ligand of the VP2-C and a simplified affinity model of the hot spot residue ligand so as to construct an affinity peptide ligand bank of the capsomere; screening the high-affinity peptide ligands in manners of molecular docking and molecular dynamics simulation by virtue of the MM/PBSA method. The affinity chromatography experiment proves that the obtained affinity peptide ligand DWDLRLLY which ranks the first during the screening process can be used for effectively separating and purifying the capsomere by one step from an escherichia coli cracking supernatant, thereby having wide application prospect in preparation of virus-like particles.

Description

technical field [0001] The invention relates to the technology of using computer simulation to design the affinity peptide ligand of target protein and the use of affinity chromatography technology to purify the target protein, and belongs to the technical field of computer simulation and protein separation and purification in biotechnology. Background technique [0002] Murine polyomavirus-like particles (Virus-Like Particles, VLP) are hollow nanoparticles formed by self-assembly of the main capsid structural protein VP1 (42kDa), and have broad applications in the fields of vaccines, gene therapy, drug carriers, and material science. Application prospect. Murine polyomavirus VLP contains 72 right-slanted capsid particles arranged in T=7d, and each capsid particle is composed of 5 VP1. [0003] After expressed and purified in prokaryotic cells, VP1 exists in the form of capsid particles, and can be assembled into VLPs with uniform shape and stable structure under suitable c...

Claims

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Application Information

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IPC IPC(8): C07K7/06G06F19/16G16B15/30
CPCC07K14/005C12N2710/22022G16B15/00C07K7/06G16B15/30
Inventor 张麟董晓燕李艳英刘晓丹孙彦
Owner TIANJIN UNIV
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