Expression and refolding of mite allergen pro-Der f1 from inclusion bodies in Escherichia coli
| Autor: | He Lai, Jianguo Zhang, Zehong Zou, Junyan Zhang, Deqiu Lin, Ailin Tao, Chunfang Ling, Huifang Chen |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Proteases
medicine.medical_treatment Blotting Western Gene Expression medicine.disease_cause Protein Refolding Microbiology law.invention Arthropod Proteins Allergen law medicine Escherichia coli Animals Humans Antigens Dermatophagoides House dust mite Inclusion Bodies Expression vector Protease biology Glutathione Disulfide Chemistry Pyroglyphidae Allergens Immunoglobulin E biology.organism_classification Cysteine protease Cysteine Endopeptidases Recombinant DNA Chromatography Gel Electrophoresis Polyacrylamide Gel Biotechnology Peptide Hydrolases |
| Zdroj: | Protein expression and purification. 109 |
| ISSN: | 1096-0279 |
| Popis: | House dust mite (Dermatophagoides farinae) allergen Der f1 is one of the most important indoor allergens associated with asthma, eczema and allergic rhinitis in humans. Therefore, sufficient quantities of Der f1 cysteine protease to be used for both experimental and therapeutic purposes are very much needed. Using recombinant DNA technology, high expression rates of cysteine proteases were obtained. The cDNA sequence encoding pro-Der f1 was cloned and expressed in Escherichia coli using the T7 based expression vector pET-44a and induced by isopropyl-β-d-thiogalactoside at a final concentration of 0.2mM. Recombinant pro-Der f1 (pro-rDer f1) was expressed as an inclusion body and the isolated protease was solubilized, refolded and purified. The protease activities and IgE reactivities of pro-rDer f1 that were refolded by size-exclusion chromatography (SEC) were higher than those obtained by dilution. The pair of pro-rDer f1 polypeptides produced by this method could be used for more effective and safer allergen-specific immunotherapy or to produce enzymatically and immunologically active Der f1 for diagnostic testing and deciphering of immunotherapy mechanisms. |
| Databáze: | OpenAIRE |
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