Antibody-bound protein L and preparation method thereof

A technology for binding proteins and antibodies, applied in the field of protein-purified antibody-binding proteins and their preparation, can solve the problems of low affinity, high cost and long purification time of ProteinL, and achieve cost reduction, easy application and wide tolerance. Effect

Inactive Publication Date: 2017-01-04
NOVOPROTEIN SCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, Protein L on the market includes: GenScript’s Protein L Resin, Biovision’s Recombinant Protein L, etc., but these reagents have low affinity for Protein L, longer purification time, and higher cost

Method used

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  • Antibody-bound protein L and preparation method thereof
  • Antibody-bound protein L and preparation method thereof
  • Antibody-bound protein L and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] (1) Construction of antibody binding protein Protein L expression plasmid

[0031] (1.1) According to the known sequence of the target gene, use the designed and synthesized primers Pr-1 and Pr-2 to carry out PCR amplification, and the two ends of the primers are equipped with restriction enzyme cutting sites of the same tail enzymes BamHI and BgIII, and purify PCR product, PCR reaction conditions are: Step 1, 94°C, 5 minutes; Step 2, 94°C, 20 seconds, 55°C, 20 seconds, 72°C, 30 seconds, 30 cycles; Step 3, 72°C, 5 minutes ; Recover the PCR product, use the same tail enzymes BamHI and BgIII to cut the domain, and use T4 ligase to connect to the carrier; the primer sequence is as follows:

[0032] Pr-1: 5'-AAAAGCTTGAAGGAGATATACATATG-3';

[0033] Pr-2: 5'-TGCTCGAGACAGCAACAACTGCCGCCACCACC-3';

[0034] (1.2) Repeat the above PCR amplification experiment 3 times according to the above steps to obtain an expression plasmid, which contains 8 gene fragments of the B2 domain, t...

Embodiment 2

[0047] This example is the same as Example 1, the difference is: in step (2.2), culture at 37°C until the OD is 0.5-0.6, add 0.1mM IPTG and induce culture at 16°C for 24 hours, at this time the bacteria The liquid OD is 2.1, and 10 g of bacterial cells are collected by centrifugation; the SDS polyacrylamide gel electrophoresis detection picture during the screening of the expression strain is as follows image 3 shown, from image 3 It can be seen that the target protein is well expressed and the bands are clear. ; SDS polyacrylamide gel electrophoresis detection figure as shown in the figure, from Figure 4 It can be seen that the target protein is clearly expressed, and the band size is consistent.

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PUM

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Abstract

The invention provides antibody-bound protein L and a preparation method thereof. The antibody-bound protein L comprises B2 protein domains which are repeated for 8 times; the preparation method of the antibody-bound protein L, provided by the invention, comprises the step of repeatedly copying the protein domains by utilizing an isocaudarner technology, and the obtained antibody-bound protein L can be used for efficiently and specifically capturing large-range antibodies and antibody segments, non-specific binding is reduced and the purity of the antibody segments is improved; the purification time is shortened by utilizing a high-dynamic binding capability of the antibody-bound protein L and the cost is reduced; the antibody-bound protein L provided by the invention has a wide pH (Potential of Hydrogen) tolerance range, is simple and convenient to apply and has stable and reliable quality; the antibody-bound protein L provided by the invention has high affinity and can be specifically bound with the antibodies and the antibody segments.

Description

technical field [0001] The invention relates to the field of protein purification fillers, in particular to an antibody-binding protein, in particular to an antibody-binding protein for protein purification and a preparation method thereof. Background technique [0002] Antibody is a biological macromolecule with complex structure, composed of multiple amino acids. The IgG (immunoglobulin G) used in antibody drugs is prone to heterogeneity changes in the process of cell culture, separation and purification, and these heterogeneities affect the efficacy and safety of antibodies. In the existing system, the antibody fragments are expressed and the samples to be purified often have many impurities, which brings challenges to the purification work. [0003] Antibody-binding protein Protein L, which is highly efficient in protein purification, was first isolated from the cell wall of the anaerobic bacterium Peptostreptococcus major. It is a protein that can specifically bind to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/42C12N15/70
CPCC07K16/42C07K2317/31C07K2317/92C12N15/70C12N2800/101
Inventor 王笃强王梦阳周晓慧李德彬
Owner NOVOPROTEIN SCI INC
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