Thi Kieu Oanh Nguyen, Dayoung Ryu, Minh Quan Nguyen, Huynh Kim Khanh Ta, Thi Luong Vu, Han Choe
{"title":"利用蛋白二硫异构酶 b'a' 结构域从大肠杆菌中高效生产人白细胞介素-3。","authors":"Thi Kieu Oanh Nguyen, Dayoung Ryu, Minh Quan Nguyen, Huynh Kim Khanh Ta, Thi Luong Vu, Han Choe","doi":"10.1002/biot.202300581","DOIUrl":null,"url":null,"abstract":"<p>Human interleukin-3 (IL3) is a multifunctional cytokine essential for both clinical and biomedical research endeavors. However, its production in <i>Escherichia coli</i> has historically been challenging due to its aggregation into inclusion bodies, requiring intricate solubilization and refolding procedures. This study introduces an innovative approach employing two chaperone proteins, maltose binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a'), as N-terminal fusion tags. Histidine tag (H) was added at the beginning of each chaperone protein gene for easy purification. This fusion of chaperone proteins significantly improved IL3 solubility across various <i>E. coli</i> strains and temperature conditions, eliminating the need for laborious refolding procedures. Following expression optimization, H-PDIb'a'-IL3 was purified using two chromatographic methods, and the subsequent removal of the H-PDIb'a' tag yielded high-purity IL3. The identity of the purified protein was confirmed through liquid chromatography coupled with tandem mass spectrometry analysis. Biological activity assays using human erythroleukemia TF-1 cells revealed a unique two-step stimulation pattern for both purified IL3 and the H-PDIb'a'-IL3 fusion protein, underscoring the protein's functional integrity and revealing novel insights into its cellular interactions. This study advances the understanding of IL3 expression and activity while introducing novel considerations for protein fusion strategies.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":null,"pages":null},"PeriodicalIF":3.2000,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202300581","citationCount":"0","resultStr":"{\"title\":\"Efficient production of human interleukin-3 from Escherichia coli using protein disulfide isomerase b'a' domain\",\"authors\":\"Thi Kieu Oanh Nguyen, Dayoung Ryu, Minh Quan Nguyen, Huynh Kim Khanh Ta, Thi Luong Vu, Han Choe\",\"doi\":\"10.1002/biot.202300581\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Human interleukin-3 (IL3) is a multifunctional cytokine essential for both clinical and biomedical research endeavors. 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Efficient production of human interleukin-3 from Escherichia coli using protein disulfide isomerase b'a' domain
Human interleukin-3 (IL3) is a multifunctional cytokine essential for both clinical and biomedical research endeavors. However, its production in Escherichia coli has historically been challenging due to its aggregation into inclusion bodies, requiring intricate solubilization and refolding procedures. This study introduces an innovative approach employing two chaperone proteins, maltose binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a'), as N-terminal fusion tags. Histidine tag (H) was added at the beginning of each chaperone protein gene for easy purification. This fusion of chaperone proteins significantly improved IL3 solubility across various E. coli strains and temperature conditions, eliminating the need for laborious refolding procedures. Following expression optimization, H-PDIb'a'-IL3 was purified using two chromatographic methods, and the subsequent removal of the H-PDIb'a' tag yielded high-purity IL3. The identity of the purified protein was confirmed through liquid chromatography coupled with tandem mass spectrometry analysis. Biological activity assays using human erythroleukemia TF-1 cells revealed a unique two-step stimulation pattern for both purified IL3 and the H-PDIb'a'-IL3 fusion protein, underscoring the protein's functional integrity and revealing novel insights into its cellular interactions. This study advances the understanding of IL3 expression and activity while introducing novel considerations for protein fusion strategies.
Biotechnology JournalBiochemistry, Genetics and Molecular Biology-Molecular Medicine
CiteScore
8.90
自引率
2.10%
发文量
123
审稿时长
1.5 months
期刊介绍:
Biotechnology Journal (2019 Journal Citation Reports: 3.543) is fully comprehensive in its scope and publishes strictly peer-reviewed papers covering novel aspects and methods in all areas of biotechnology. Some issues are devoted to a special topic, providing the latest information on the most crucial areas of research and technological advances.
In addition to these special issues, the journal welcomes unsolicited submissions for primary research articles, such as Research Articles, Rapid Communications and Biotech Methods. BTJ also welcomes proposals of Review Articles - please send in a brief outline of the article and the senior author''s CV to the editorial office.
BTJ promotes a special emphasis on:
Systems Biotechnology
Synthetic Biology and Metabolic Engineering
Nanobiotechnology and Biomaterials
Tissue engineering, Regenerative Medicine and Stem cells
Gene Editing, Gene therapy and Immunotherapy
Omics technologies
Industrial Biotechnology, Biopharmaceuticals and Biocatalysis
Bioprocess engineering and Downstream processing
Plant Biotechnology
Biosafety, Biotech Ethics, Science Communication
Methods and Advances.