title: Expression and refolding of mite allergen pro-Der f1 from inclusion bodies in Escherichia coli.
abstract: 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.
authors.affiliation: Guangdong Provincial Key Laboratory of Allergy & Clinical Immunology, The State Key Laboratory of Respiratory Disease, The Second Affiliated Hospital of Guangzhou Medical University, 250# Changgang Road East, Guangzhou 510260, Guangdong Province, PR China; School of Life Science, South China Normal University, 55# Zhongshan Road West, Tianhe District, Guangzhou 510631, PR China.
authors.forename: Chunfang
authors.initials: C
authors.lastname: Ling
authors.affiliation: Guangdong Provincial Key Laboratory of Allergy & Clinical Immunology, The State Key Laboratory of Respiratory Disease, The Second Affiliated Hospital of Guangzhou Medical University, 250# Changgang Road East, Guangzhou 510260, Guangdong Province, PR China.
authors.forename: Junyan
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Guangdong Provincial Key Laboratory of Allergy & Clinical Immunology, The State Key Laboratory of Respiratory Disease, The Second Affiliated Hospital of Guangzhou Medical University, 250# Changgang Road East, Guangzhou 510260, Guangdong Province, PR China.
authors.forename: Huifang
authors.initials: H
authors.lastname: Chen
authors.affiliation: Guangdong Provincial Key Laboratory of Allergy & Clinical Immunology, The State Key Laboratory of Respiratory Disease, The Second Affiliated Hospital of Guangzhou Medical University, 250# Changgang Road East, Guangzhou 510260, Guangdong Province, PR China.
authors.forename: Zehong
authors.initials: Z
authors.lastname: Zou
authors.affiliation: Guangdong Provincial Key Laboratory of Allergy & Clinical Immunology, The State Key Laboratory of Respiratory Disease, The Second Affiliated Hospital of Guangzhou Medical University, 250# Changgang Road East, Guangzhou 510260, Guangdong Province, PR China.
authors.forename: He
authors.initials: H
authors.lastname: Lai
authors.affiliation: Guangdong Provincial Key Laboratory of Allergy & Clinical Immunology, The State Key Laboratory of Respiratory Disease, The Second Affiliated Hospital of Guangzhou Medical University, 250# Changgang Road East, Guangzhou 510260, Guangdong Province, PR China.
authors.forename: Jianguo
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Guangdong Provincial Key Laboratory of Allergy & Clinical Immunology, The State Key Laboratory of Respiratory Disease, The Second Affiliated Hospital of Guangzhou Medical University, 250# Changgang Road East, Guangzhou 510260, Guangdong Province, PR China; School of Life Science, South China Normal University, 55# Zhongshan Road West, Tianhe District, Guangzhou 510631, PR China. Electronic address: lindq168@scnu.edu.cn.
authors.forename: Deqiu
authors.initials: D
authors.lastname: Lin
authors.affiliation: Guangdong Provincial Key Laboratory of Allergy & Clinical Immunology, The State Key Laboratory of Respiratory Disease, The Second Affiliated Hospital of Guangzhou Medical University, 250# Changgang Road East, Guangzhou 510260, Guangdong Province, PR China. Electronic address: taoailin@gzhmu.edu.cn.
authors.forename: Ailin
authors.initials: A
authors.lastname: Tao
mesh_terms: D000485:Allergens / Q000378:metabolism*; D000818:Animals; D039741:Antigens, Dermatophagoides / Q000378:metabolism*; D060829:Arthropod Proteins / Q000378:metabolism*; D015153:Blotting, Western; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D019803:Glutathione Disulfide / Q000378:metabolism; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood; D002479:Inclusion Bodies / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D058849:Protein Refolding; D039981:Pyroglyphidae / Q000378:metabolism*
chemical_list: D000485:Allergens; D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D007073:Immunoglobulin E; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; C451159:Dermatophagoides farinae antigen f 1; D019803:Glutathione Disulfide
keywords: Allergen; Escherichia coli; IgE reactivities; Pro-rDer f1; Refolded; SEC