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A kind of glutamine synthetase mutant and its application

A technology of glutamine and synthetase, which is applied in the field of biomedicine, can solve the problems of weakened enzyme activity, affecting the growth state of CHO stable cell lines, and CHO cell toxicity, and achieves the goals of reducing usage, good application value, and enhancing affinity Effect

Active Publication Date: 2022-04-08
SUMGEN MAB BEIJING BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the GS screening system for CHO cells mainly has the following problems: high concentration of GS inhibitor MSX will have a toxic effect on CHO cells, which will seriously affect the growth status of the screened CHO stably transformed cell lines, and the MSX pressurized screening process is relatively slow. long
In view of this, the present invention provides a novel GS mutant, the affinity of the GS mutant to the GS inhibitor MSX is enhanced, and its own enzyme activity is relatively weakened, the GS mutant not only effectively reduces The amount of the GS inhibitor MSX used in the cell line screening process is conducive to the rapid acquisition of cell lines with multiple copies of stable integration and high expression of the target protein during the MSX pressurized screening process. to report

Method used

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  • A kind of glutamine synthetase mutant and its application
  • A kind of glutamine synthetase mutant and its application
  • A kind of glutamine synthetase mutant and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1 Structural prediction and mutant prediction of CHO glutamine synthetase

[0085] 1. Structural prediction of CHO glutamine synthetase

[0086] Although there is no information on the crystal structure of CHO glutamine synthetase, Krajewski et al. published the crystal structure of human and dog glutamine synthetase 2QC8 (Crystal structure of human glutamine synthetase in complex with ADP and methionine sulfoximine phosphate in 2008) )、2QJW(Crystal structure of human glutamine synthetase in complex with ADP andphosphate)和2UU7(Crystal structure of apo glutamine synthetase from dog)(参考文献为Crystal structures of mammalian glutamine synthetases illustratesubstrate-induced conformational changes and provide opportunities for drugand herbicide design . Krajewski, W.W., Collins, R., Holmberg-Schiavone, L., Jones, T.A., Karlberg, T., Mowbray, S.L. (2008) J Mol Biol 375: 217-228). Amino acid sequence alignment results show that Chinese hamster (Cricetulus griseus; CHO) g...

Embodiment 2

[0095] Example 2 Determination of Glutamine Synthetase Activity

[0096] 1. Construction of firefly luciferase-GS co-expression vector

[0097] The GS mutant genes (GSm1, GSm9-12), wild-type GS gene (GSwt) and R324C mutant corresponding to CHO predicted above were cloned into the eukaryotic expression vector pcDNA3.1 ( +) In the pcDNA3.1-LUCI-Furin T2A vector with modified backbone (see Figure 4 ). The optimized self-cleaving peptide structure of Furin T2A has an amino acid sequence of RRKRGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 15), and its cleavage efficiency is close to 100%, which can basically guarantee the equal expression of firefly luciferase and GS to be tested, so it can The expression of GS in each experimental group was corrected by quantifying firefly luciferase.

[0098] 2. GS activity assay method

[0099] Use a six-well plate to adhere to culture CHOK1SV GS-KO cells (glutamine synthetase deficiency) (LONZA cells), add 2 mL to each well at a density of 10 5 cel...

Embodiment 3

[0104] Example 3 Construction of antibody eukaryotic expression vector

[0105] Backbone vector pDCH2.1 for testing antibody expression (see Figure 6) is spliced ​​by whole gene synthesis after design, and its components include: EF-1α promoter, CMV enhancer, CMV promoter, WPRE element, HSV TK terminator, f1 ori replication initiation site, SV40 promoter, wild Type CHO GS selection marker, SV40 terminator, ori replication origin site, prokaryotic ampicillin resistance selection marker and AmpR promoter, etc. The vector skeleton is suitable for monoclonal antibody double-gene expression, in which the light chain cloning site is HindIII-BsiWI-BamHI, and the heavy chain cloning site is ClaI-NheI-XhoI. The wild-type CHO GS screening marker can be replaced by the low-activity GS mutant screened above by the NotI-EcoRI double enzyme digestion method. The constructed and prepared plasmid can be used for stable transfection screening of CHO K1 after PvuI linearization and recovery....

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Abstract

The invention discloses a mutant of glutamine synthetase and its application. The mutant of glutamine synthetase contains mutation sites V197W, A250F, and R324K, and the mutant of glutamine synthetase can synthesize glutamine The affinity of the enzyme inhibitor MSX is enhanced, and its own enzyme activity is relatively weakened. The glutamine synthetase mutant is not only conducive to maintaining a better growth state of the cells, but also conducive to the rapid acquisition of multiple copies of MSX during the pressurized screening process. The cell line that integrates and highly expresses the target protein has good application value.

Description

technical field [0001] The present invention belongs to the technical field of biomedicine. Specifically, the present invention relates to a glutamine synthetase mutant and its application. More specifically, the present invention relates to a glutamine synthesizing enzyme containing V197W, A250F, and R324K mutation sites. Enzyme mutants. Background technique [0002] Glutamine synthetase (GS) is the oldest and most widespread enzyme in organisms (Kumada Y, Benson D R, Hillemann D, et al. Evolution of the glutamine synthetase gene, one of the oldest existing and functioning genes [J]. Proceedings of the National Academy of Sciences, 1993, 90(7): 3009-3013.), which is involved in nitrogen metabolism and carbon metabolism in many organisms. GS is one of the key enzymes of nitrogen metabolism in organisms, and nitrogen assimilation is necessary for organisms to maintain life. Therefore, GS and its genes have been widely studied in bacteria, fungi, and plants. Due to its biolo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/85C12N5/10
CPCC12N9/93C12N15/85C12N5/0682C12Y603/01002C12N2800/107C12N2510/02
Inventor 丁晓然缪仕伟谈彬吕明
Owner SUMGEN MAB BEIJING BIOTECH CO LTD
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