{"doi":"10.1002/iub.1255","title":"Heterologous expression and characterization of human cellular glutathione peroxidase mutants","abstract":"<jats:title>Abstract</jats:title><jats:p>Cellular glutathione peroxidase (GPx1; EC1.11.1.9) is a major intracellular antioxidant selenoenzyme in mammals. However, the complicated expression mechanism of selenocysteine (Sec)‐containing protein increases the difficulty of expressing human GPx1 (hGPx1) in <jats:italic>Escherichia coli</jats:italic> (<jats:italic>E. coli</jats:italic>). In this study, <jats:italic>hGPx1</jats:italic> gene was cloned from a cDNA library of the human hepatoma cell line HepG2. The codon UGA encoding Sec49 of hGPx1 was first mutated to UGC encoding cysteine (Cys) and then biosynthetically converted to Sec during expression in an <jats:italic>E. coli</jats:italic> BL21(DE3)<jats:italic>cys</jats:italic> auxotrophic system. Seleno‐GPx1<jats:sup>Sec</jats:sup> displayed a low GPx activity of 522 U/μmol. To improve the activity, the other five Cys residues (C2, C78, C115, C156, C202) were mutated to serine (Ser) in one hGPx1 molecule. The mutant seleno‐hGPx1<jats:sup>Ser</jats:sup> showed a high activity of 5278 U/μmol, which was more than 10‐fold enhanced as compared with seleno‐GPx1<jats:sup>Sec</jats:sup>. The activity was the highest among all of those seleno‐proteins obtained by this method so far. Kinetic analysis of seleno‐hGPx1<jats:sup>Ser</jats:sup> showed a typical ping‐pong mechanism, which was similar to those of natural GPxs. This research will be of value in overcoming the problem of limited sources of natural GPx and substantially promotes the research of the characterization of GPx. © 2014 IUBMB Life, 66(3):212–219, 2014</jats:p>","journal":"IUBMB Life","year":2014,"id":658835,"datarank":0.3596842909197557,"base_score":2.3978952727983707,"endowment":2.3978952727983707,"self_citation_contribution":0.3596842909197557,"citation_network_contribution":0.0,"self_endowment_contribution":0.3596842909197557,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":10,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1457440,"name":"Yang Yu","orcid":"0000-0002-3687-9868","position":1,"is_corresponding":false},{"id":514683,"name":"Xixi Liu","orcid":"0000-0003-1992-5970","position":2,"is_corresponding":false},{"id":1719897,"name":"Yinlong Zhang","orcid":null,"position":3,"is_corresponding":false},{"id":1061580,"name":"Tuchen Guan","orcid":"0000-0001-5929-5489","position":4,"is_corresponding":false},{"id":1719898,"name":"Guiqiu Xie","orcid":null,"position":5,"is_corresponding":false},{"id":1719899,"name":"Jingyan Wei","orcid":null,"position":6,"is_corresponding":false},{"id":371302,"name":"Xiao Guo","orcid":"0000-0002-1744-4340","position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Heterologous expression and characterization of human cellular glutathione peroxidase mutants","abstract":"<jats:title>Abstract</jats:title><jats:p>Cellular glutathione peroxidase (GPx1; EC1.11.1.9) is a major intracellular antioxidant selenoenzyme in mammals. However, the complicated expression mechanism of selenocysteine (Sec)‐containing protein increases the difficulty of expressing human GPx1 (hGPx1) in <jats:italic>Escherichia coli</jats:italic> (<jats:italic>E. coli</jats:italic>). In this study, <jats:italic>hGPx1</jats:italic> gene was cloned from a cDNA library of the human hepatoma cell line HepG2. The codon UGA encoding Sec49 of hGPx1 was first mutated to UGC encoding cysteine (Cys) and then biosynthetically converted to Sec during expression in an <jats:italic>E. coli</jats:italic> BL21(DE3)<jats:italic>cys</jats:italic> auxotrophic system. Seleno‐GPx1<jats:sup>Sec</jats:sup> displayed a low GPx activity of 522 U/μmol. To improve the activity, the other five Cys residues (C2, C78, C115, C156, C202) were mutated to serine (Ser) in one hGPx1 molecule. The mutant seleno‐hGPx1<jats:sup>Ser</jats:sup> showed a high activity of 5278 U/μmol, which was more than 10‐fold enhanced as compared with seleno‐GPx1<jats:sup>Sec</jats:sup>. The activity was the highest among all of those seleno‐proteins obtained by this method so far. Kinetic analysis of seleno‐hGPx1<jats:sup>Ser</jats:sup> showed a typical ping‐pong mechanism, which was similar to those of natural GPxs. This research will be of value in overcoming the problem of limited sources of natural GPx and substantially promotes the research of the characterization of GPx. © 2014 IUBMB Life, 66(3):212–219, 2014</jats:p>","is_dataset_classified":null,"base_score":2.3978952727983707,"endowment":2.3978952727983707,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"24659529","pmcid":null,"openalex_id":"https://openalex.org/W1692509206","authors":[],"funders":[],"total_grants":0,"fwci":0.6631,"citation_percentile":0.67885545,"influential_citations":0,"citation_trend":[{"year":2014,"count":1},{"year":2016,"count":1},{"year":2017,"count":1},{"year":2018,"count":2},{"year":2019,"count":1},{"year":2020,"count":1},{"year":2021,"count":3}],"oa_status":"bronze","license":"http://onlinelibrary.wiley.com/termsAndConditions#vor","oa_locations":[{"url":"https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/iub.1255","host_type":"journal"},{"url":"https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/iub.1255","host_type":"publisher"},{"url":"https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1002%2Fiub.1255","host_type":"publisher"},{"url":"https://iubmb.onlinelibrary.wiley.com/doi/pdf/10.1002/iub.1255","host_type":"publisher"},{"url":"https://doi.org/10.1002/iub.1255","host_type":"journal"},{"url":"https://pubmed.ncbi.nlm.nih.gov/24659529","host_type":"repository"}],"fields_of_study":["Selenium in Biological Systems","Organoselenium and organotellurium chemistry","Redox biology and oxidative stress"],"mesh_terms":[],"keywords":["GPX1","Selenocysteine","GPX3","Biochemistry","Heterologous expression","Molecular biology","Glutathione peroxidase","Escherichia coli","Mutant","Amino acid","Complementary DNA","Cysteine","Serine","Chemistry","GPX6","Biology","Glutathione","Gene","Enzyme","Recombinant DNA","Site-directed mutagenesis","Cysteine Auxotrophic Strain","Selenocystein"],"sdg_mappings":[],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-08-12T05:30:47.438852Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}