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Application of hydrophobic cyclic peptide ligand in purification of human immunoglobulin G

A human immunoglobulin, hydrophobic technology, applied in the field of human immunoglobulin G purification, computer simulation and downstream protein separation and purification, which can solve the problems of difficult commercial utilization and low protein purity.

Active Publication Date: 2021-03-12
上海佰君生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The research group of Professor Sun Yan from Tianjin University used 6 key residues of SpA: F132, Y133, H137, E143, R146 and K154 to construct a simulation system, and used molecular docking and molecular dynamics simulation to screen the peptide library. The purity of human IgG can be obtained at The linear octapeptide molecules FYWHCLDE and FYTHCAKE with a recovery rate of more than 88% and a recovery rate of more than 65% have good purification effects, but there is still a long way to go before commercial application, which is based on the relationship between protein A and IgG. Hydrophobic interaction model is used to simulate, but the obtained polypeptide sequence is based on electrostatic interaction and hIgG binding. It is well known in the art that electrostatic interaction is not specific adsorption, and it is easy to adsorb other biomass molecules. The purity of the obtained protein is not high, and it is difficult to commercialize use

Method used

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  • Application of hydrophobic cyclic peptide ligand in purification of human immunoglobulin G
  • Application of hydrophobic cyclic peptide ligand in purification of human immunoglobulin G
  • Application of hydrophobic cyclic peptide ligand in purification of human immunoglobulin G

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Build a simulation system

[0031] Select the crystal conformation of the SpA-hIgG1 complex whose ID is 1FC2 from the PBD database, and extract the C on the SpA and hIgG-Fc fragments H 3 The binding region, the amino acid sequence of the binding region is Ser383-Asn389 (SNGQPEN), and the three-dimensional coordinates of each amino acid residue in the binding region are selected as the coordinate information of the simulation system.

Embodiment 2

[0032] Example 2 Obtaining a Biomimetic Peptide Library

[0033] Use NAMD software to calculate the distance between each residue in the Ser383-Asn389 sequence, take Cys as the connecting residue, insert 5 to 7 amino acid residues X around Cys, and form a circular polytitanium chain-[XXX-C-XXXX ]-, -[XX-C-XXXX]-, -[XX-C-XXXXX]-, -[XXX-C-XXX]-, -[XX-C-XXX]-, using AutoDock molecular docking program for amino acid Positioning, determine the type of amino acid residue X that matches the Ser383-Asn389 coordinate system and has the best binding position. X represents 19 common amino acid residues except Cys, which can be stably combined with hIgG-Fc by calling the GROMOS program The end-to-end biomimetic peptide library of cyclic 6-peptide, cyclic 7-peptide and cyclic 8-peptide sequences, which contains a total of 4256 sequences.

Embodiment 3

[0034] Example 3 Primary Screening of Polypeptide Library

[0035] The peptide molecules in the peptide library were sequentially docked with the three-dimensional structure of hIgG-Fc using LeDock molecular docking software. The docking results showed that most of the peptides could bind to the Fc fragment, and the binding affinity ranged from -3.5 to -7.6kcal / mol Among them, considering the size of the affinity and the screening efficiency, the polypeptide molecules (a total of 1135) with a binding affinity lower than -6.0kcal / mol (absolute value of affinity greater than 6.0kcal / mol) were finally selected for the next round of screening.

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Abstract

The invention relates to an application of a hydrophobic cyclic peptide ligand in purification of human immunoglobulin G. The sequence of the hydrophobic cyclic peptide ligand is -[HWG-C-AKTE], -[YF-C-WRHE]-, -[WV-C-LHHYF]-, -[PYF-C-TIE]- and -[FY-C-DEHL], and the hydrophobic cyclic peptide ligand is obtained through computer simulation biomimetic design. According to the biomimetic design method,an amino acid sequence Ser383-Asn389 on a CH3 binding region on a SpA and hIgG-Fc fragment is used as a simulation system, a hydrophobic cyclic peptide ligand capable of specifically binding to hIgGis determined through LeDock molecular docking, FlexX molecular docking and Amber molecular dynamics simulation screening, and the cyclic peptide ligand chromatography medium is adopted to purify andresearch hIgG from a human serum sample. The purity of 96% and the recovery rate of 92% can be obtained.

Description

technical field [0001] The present invention relates to the technical field of purification of human immunoglobulin G, which adopts computer simulation technology to bionically design an affinity peptide ligand library of human IgG, and then uses affinity chromatography test to verify the purification effect of peptide molecules on human IgG, which belongs to the field of bionic design Computer simulation and downstream protein separation and purification technology field. Background technique [0002] At present, antibodies are widely used in medical diagnosis and disease treatment. In 2006, the global sales of more than 20 kinds of antibody drugs approved by FDA exceeded 17 billion US dollars. Among them, IgG is the main antibody component in serum, accounting for about 75% of serum Ig. , is also the class of antibodies with the greatest demand. The preparation of antibody drugs is usually completed by precipitation, ion exchange chromatography, hydrophobic interaction ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C07K1/22G16B15/00
CPCC07K16/00C07K1/22G16B15/00Y02A90/10
Inventor 钮雪琴
Owner 上海佰君生物科技有限公司
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