Affinity chromatography medium based on recombinant protein A as well as preparation method and application of affinity chromatography medium

A technology of recombinant protein and chromatographic medium, which is applied in the biological field to achieve the effects of reducing steric hindrance, improving alkali resistance and high dynamic load

Pending Publication Date: 2022-04-29
SEPAX TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, how to obtain recombinant protein A and its affinity medium with high dynamic load and higher and more stable alkali resistance will face greater challenges

Method used

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  • Affinity chromatography medium based on recombinant protein A as well as preparation method and application of affinity chromatography medium
  • Affinity chromatography medium based on recombinant protein A as well as preparation method and application of affinity chromatography medium
  • Affinity chromatography medium based on recombinant protein A as well as preparation method and application of affinity chromatography medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1 Gene synthesis and plasmid preparation

[0068] 1. Gene synthesis

[0069] The coding gene of recombinant protein A (SEQ ID NO.7 and SEQ ID NO.8) was assembled from 50 double-stranded DNAs with 25-30 base pairs, as described in the reference (Krebs, et al. Synthesis of a gene forsensory rhodopsin I and its functional expression in Halobacterium halobium.Proc Natl Acad.Sci US A.1993,90(8):3486-3490), that is, 25 pairs of complementary DNA oligonucleotides were prepared by a DNA synthesizer, and then After phosphorylation, DNA oligonucleotide annealing, and ligation of 25 double-stranded DNAs, the entire synthetic gene is generated. The gene sequence encoding recombinant protein A of SEQ ID NO.7 is shown in SEQ ID NO.9, and the gene sequence encoding recombinant protein A of SEQ ID NO.8 is shown in SEQ ID NO.10.

[0070] 2. Preparation of expression vector

[0071] Insert the coding genes SEQ ID NO.9 and SEQ ID NO.10 of the synthetic recombinant protein A ...

Embodiment 2

[0072] Production and purification of embodiment 2 recombinant protein A (recombinant protein A encoded by amino acid sequence SEQ ID NO.7)

[0073] The production of recombinant protein A was carried out in a 10L fermenter, using LB medium, induced by 200mM IPTG, and confirmed whether the expression was successful by SDS-PAGE. After the end of expression, the cells were collected by centrifugation, and then the cells were resuspended in 1X PBS solution, followed by breaking the cells with a high-pressure homogenizer (18000 psi), and then centrifuging to remove cell debris. After clarification and filtration, the recombinant protein A was captured with a Ni affinity chromatography column. The chromatography medium was Polar MC60-Ni Excel (Sepax PN#: 270660800), and the column specification was 10cm (ID)*37cm (L). Impurities were rinsed with 2 times the column volume of 1X PBS solution after the sample, and the target protein was eluted with 0.1M phosphate buffer + 0.2M imidazo...

Embodiment 3

[0074] Example 3 Coupling of recombinant protein A and polymethacrylate microspheres

[0075] Activate hydrophilic polymethacrylate microspheres: 2L Monomix MC45-SEC (45μm, SepaxPN#: 280145950) ball into a 10L reactor, add 2L 1.5M NaOH and stir evenly, then react at 45°C and 150rpm for 1.5h, then add 2L 1,4-butanediol diglycidyl ether, continue After reacting for 1.5 h, the base balls were washed with ethanol and water respectively to obtain activated base balls.

[0076] Coupling of recombinant protein A produced in Example 2 to activated radical spheres: put 2L of activated radical spheres into a 10L reactor, then add 1L of 0.5M sodium carbonate solution and stir to disperse the activated radical spheres evenly, then add 1L of recombinant protein A solution (1X PBS solution containing 40 g of recombinant protein A in a reduced state), and finally 426 g of Na 2 SO 4And stir evenly, react at 30°C and 150rpm for 16h, add 10g of thioglycerol to continue the reaction for 3h a...

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Abstract

The invention discloses an affinity chromatography medium based on recombinant protein A as well as a preparation method and application of the affinity chromatography medium. A chimera ZA or ZD is obtained by recombining an A or D structural domain and a mutated Z structural domain of the wild protein A, the mutation of the ZA chimera relative to the Z structural domain comprises Asp 53 Glu and Ala 54 Ser, and the mutation of the ZD chimera relative to the Z structural domain comprises Ala 42 Thr, Leu 44 Val, Ala 46 Gly, Asp 53 Glu and Ala 54 Ser. Alkali resistance is further improved through the mutations without affecting the space structure of the whole structural domain, a His-Tag tag and a flexible connecting arm are added on the basis of the mutants, and the activity freedom degree of a chimera is guaranteed, so that the binding capacity of the chimera with a target molecule is improved; the finally obtained affinity medium can obtain higher dynamic loading capacity and stronger alkali resistance.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an affinity chromatography medium based on recombinant protein A and its preparation method and application. Background technique [0002] In recent years, tremendous progress has been made in the treatment of various cancers, rheumatoid arthritis, viral infections and other diseases using antibody therapy. So far, the FDA has approved more than 100 monoclonal antibody drugs, and there is still a very rich R&D pipeline. Therefore, the demand for the production and purification of antibody drugs has also increased. The purification of antibody drugs generally goes through the capture stage and the purification stage. The capture stage uses an affinity chromatography medium, which is formed by coupling a protein ligand that can specifically bind to an immunoglobulin on a solid phase carrier, such as Protein A ligand, protein G ligand, protein L ligand, etc. [0003] Prote...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21C07K1/04C07K1/22C07K1/36B01D15/38C12R1/19
CPCC07K14/31C12N15/70B01D15/3809C07K2319/30C07K2319/21C07K2319/70
Inventor 吕小林杨克毛慧明
Owner SEPAX TECH
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