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Affiliative small molecular peptide of IgG (Immunoglobulin G) antibody and application thereof

A small molecule peptide and affinity technology, applied in protein separation and purification, biological field, can solve the problems of inability to provide affinity binding constant information, huge gap between calculation and experiment, high immunogenicity of peptide chain, simple structure and wide application range. , the effect of high antibody selectivity and affinity

Active Publication Date: 2019-04-26
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The molecular simulation platform provides a good example for the design and development of new ligands at the atomic level, but the molecular simulation based on molecular structure thermodynamic data is limited by the huge gap between calculation and experiment, and cannot provide real affinity binding constant information. The target that actually has affinity with the target protein must be confirmed by the final affinity experiment
The currently discovered peptide ligands have shortcomings such as high immunogenicity, poor selectivity, and low loading capacity due to too long peptide chains, so that no small peptide ligands have been put into the market so far, so it is still necessary to design a cheaper, Small molecule small peptide ligand with simpler structure

Method used

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  • Affiliative small molecular peptide of IgG (Immunoglobulin G) antibody and application thereof
  • Affiliative small molecular peptide of IgG (Immunoglobulin G) antibody and application thereof
  • Affiliative small molecular peptide of IgG (Immunoglobulin G) antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1 Design, synthesis and screening of small molecule peptides of the present invention

[0044] According to the directional design of the two α-helices of the domain B domain on the protein A binding site, the binding region is cut into several peptides to establish a small molecule peptide library (the synthesis of the peptide library is entrusted to Shanghai Gil Biochemical Co., Ltd., and then using STD -NMR method is used to screen the interaction ability between the small molecule peptide library and the antibody, and the small molecule peptide with the strongest affinity is screened out, and the binding site between the small molecule peptide and the antibody is further detected by STD-NMR technology at the atomic level , the specific operation steps are as follows:

[0045] It is carried out on 600M NMR (5mm Triple inverse ultra-low temperature probe), the solution environment is 20mM phosphate buffer, and the pulse program is STDDIFFGP. According to th...

experiment example 2

[0051] Experimental example 2 preparation of affinity medium

[0052] The small molecular peptide obtained in Example 1 was prepared into an affinity medium, and the specific steps were as follows:

[0053] (1) Medium activation: The epoxy activation method can be selected to activate the polysaccharide medium containing hydroxyl groups, that is, take 36 g of drained agarose gel 4FF microspheres, fully mix it with 100 ml of acetone in a three-necked flask, and then Add 100ml of epichlorohydrin, react at 30°C and 120rpm for 20 minutes, then add 100ml of 4M sodium hydroxide containing 0.3% sodium borohydride into a constant pressure burette, insert the burette into a three-necked flask, and dissolve the hydrogen within 1 hour After the sodium oxide was added dropwise, the reaction was continued for 2 hours at 30°C and 120rpm. After the reaction was completed, the epoxy-activated agarose 4FF microspheres were drained using a G3 sand core funnel, and then washed with 20% acetone f...

experiment example 5

[0063] Experimental example 5 Stability experiment of ligand

[0064] After the separation and purification of the affinity medium, wash the chromatography column with 1 column volume of pure water, then rinse the column with 5 column volumes of 1M NaOH buffer, and then use 2 column volumes of 20mmol / L PB buffer The solution was finally balanced with 5 column volumes of equilibrium solution, and the measurement and medium cleaning process was repeated 5 times. The chromatographic spectra of the 5 chromatographic operations had good reproducibility, and there was no significant change in the medium loading.

[0065] In summary, the small molecule peptide ligands of this application have the advantages of salt resistance, acid and alkali resistance, and easy regeneration of ligands after they are prepared into affinity media. The loading and elution conditions are mild, which can avoid overacid and The elution conditions such as overalkali will destroy the protein structure, and...

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Abstract

The invention relates to an affiliative small molecular peptide of an IgG (Immunoglobulin G) antibody. A general formula of the affiliative small molecular peptide is FYX1X2X3X4, wherein F representsphenylalanine, Y represents tyrosine, X1 represents histidine, glutamic acid or the phenylalanine, X2 represents isoleucine, leucine or methionine, X3 represents the glutamic acid, the leucine or glycine, and X4 represents proline, the phenylalanine or the histidine. An affiliative medium derived from the small molecular peptide not only can be used for separation and purification, but also can beused for drug coupling and hemodialysis.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the field of protein separation and purification, in particular to a small molecule peptide, in particular to a small molecule peptide with IgG antibody affinity and application thereof. Background technique [0002] Monoclonal antibody drugs (referred to as monoclonal antibodies) are widely used in disease diagnosis, prevention and treatment, and are the fastest growing market in the global pharmaceutical industry. At present, the upstream technology platform for monoclonal antibody preparation is becoming more and more mature, the expression level has reached 5g / L or higher, and the culture scale has reached the scale of 10,000-20,000 liters. In contrast, the downstream separation and purification technology of monoclonal antibody has gradually become a bottleneck. Currently, the downstream purification cost of monoclonal antibody has accounted for 45% to 70% of the total production c...

Claims

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Application Information

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IPC IPC(8): C07K7/06C12N15/11C07K1/22
CPCC07K1/22C07K7/06
Inventor 马光辉郝冬霞黄永东赵岚王伟颖葛佳苏志国周炜清
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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