{"doi":"10.1186/1475-2859-9-28","title":"Microbial polyhydroxyalkanote synthesis repression protein PhaR as an affinity tag for recombinant protein purification","abstract":"<jats:title>Abstract</jats:title>\n          <jats:sec>\n            <jats:title>Background</jats:title>\n            <jats:p>PhaR which is a repressor protein for microbial polyhydroxyalkanoates (PHA) biosynthesis, is able to attach to bacterial PHA granules <jats:italic>in vivo</jats:italic>, was developed as an affinity tag for <jats:italic>in vitro</jats:italic> protein purification. Fusion of PhaR-tagged self-cleavable Ssp DnaB intein to the N-terminus of a target protein allowed protein purification with a pH and temperature shift. During the process, the target protein was released to the supernatant while PhaR-tagged intein was still immobilized on the PHA nanoparticles which were then separated by centrifugation.</jats:p>\n          </jats:sec>\n          <jats:sec>\n            <jats:title>Results</jats:title>\n            <jats:p>Fusion protein PhaR-intein-target protein was expressed in recombinant <jats:italic>Escherichia coli</jats:italic>. The cell lysates after sonication and centrifugation were collected and then incubated with PHA nanoparticles to allow sufficient absorption onto the PHA nanoparticles. After several washing processes, self-cleavage of intein was triggered by pH and temperature shift. As a result, the target protein was released from the particles and purified after centrifugation. As target proteins, enhanced green fluorescent protein (EGFP), maltose binding protein (MBP) and β-galactosidase (lacZ), were successfully purified using the PhaR based protein purification method.</jats:p>\n          </jats:sec>\n          <jats:sec>\n            <jats:title>Conclusion</jats:title>\n            <jats:p>The successful purification of EGFP, MBP and LacZ indicated the feasibility of this PhaR based <jats:italic>in vitro</jats:italic> purification system. Moreover, the elements used in this system can be easily obtained and prepared by users themselves, so they can set up a simple protein purification strategy by themselves according to the PhaR method, which provides another choice instead of expensive commercial protein purification systems.</jats:p>\n          </jats:sec>","journal":"Microbial Cell Factories","year":2010,"id":615707,"datarank":0.4566783656585135,"base_score":3.044522437723423,"endowment":3.044522437723423,"self_citation_contribution":0.4566783656585135,"citation_network_contribution":0.0,"self_endowment_contribution":0.4566783656585135,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":20,"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":1587140,"name":"Zhi Hui Wang","orcid":null,"position":1,"is_corresponding":false},{"id":1587141,"name":"Guo Qiang Chen","orcid":null,"position":2,"is_corresponding":false},{"id":263324,"name":"Shuang Zhang","orcid":"0000-0003-4927-7230","position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Microbial polyhydroxyalkanote synthesis repression protein PhaR as an affinity tag for recombinant protein purification","abstract":"<jats:title>Abstract</jats:title>\n          <jats:sec>\n            <jats:title>Background</jats:title>\n            <jats:p>PhaR which is a repressor protein for microbial polyhydroxyalkanoates (PHA) biosynthesis, is able to attach to bacterial PHA granules <jats:italic>in vivo</jats:italic>, was developed as an affinity tag for <jats:italic>in vitro</jats:italic> protein purification. Fusion of PhaR-tagged self-cleavable Ssp DnaB intein to the N-terminus of a target protein allowed protein purification with a pH and temperature shift. During the process, the target protein was released to the supernatant while PhaR-tagged intein was still immobilized on the PHA nanoparticles which were then separated by centrifugation.</jats:p>\n          </jats:sec>\n          <jats:sec>\n            <jats:title>Results</jats:title>\n            <jats:p>Fusion protein PhaR-intein-target protein was expressed in recombinant <jats:italic>Escherichia coli</jats:italic>. The cell lysates after sonication and centrifugation were collected and then incubated with PHA nanoparticles to allow sufficient absorption onto the PHA nanoparticles. After several washing processes, self-cleavage of intein was triggered by pH and temperature shift. As a result, the target protein was released from the particles and purified after centrifugation. As target proteins, enhanced green fluorescent protein (EGFP), maltose binding protein (MBP) and β-galactosidase (lacZ), were successfully purified using the PhaR based protein purification method.</jats:p>\n          </jats:sec>\n          <jats:sec>\n            <jats:title>Conclusion</jats:title>\n            <jats:p>The successful purification of EGFP, MBP and LacZ indicated the feasibility of this PhaR based <jats:italic>in vitro</jats:italic> purification system. Moreover, the elements used in this system can be easily obtained and prepared by users themselves, so they can set up a simple protein purification strategy by themselves according to the PhaR method, which provides another choice instead of expensive commercial protein purification systems.</jats:p>\n          </jats:sec>","is_dataset_classified":null,"base_score":3.044522437723423,"endowment":3.044522437723423,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"20459707","pmcid":"PMC2873406","openalex_id":"https://openalex.org/W1992420603","authors":[],"funders":[],"total_grants":0,"fwci":1.3642,"citation_percentile":0.79000889,"influential_citations":0,"citation_trend":[{"year":2012,"count":3},{"year":2013,"count":1},{"year":2015,"count":1},{"year":2016,"count":2},{"year":2017,"count":3},{"year":2020,"count":2},{"year":2024,"count":2},{"year":2025,"count":1}],"oa_status":"gold","license":"cc-by","oa_locations":[{"url":"https://microbialcellfactories.biomedcentral.com/counter/pdf/10.1186/1475-2859-9-28","host_type":"journal"},{"url":"https://microbialcellfactories.biomedcentral.com/counter/pdf/10.1186/1475-2859-9-28","host_type":"publisher"},{"url":"https://link.springer.com/content/pdf/10.1186/1475-2859-9-28.pdf","host_type":"publisher"},{"url":"https://doi.org/10.1186/1475-2859-9-28","host_type":"journal"},{"url":"https://pubmed.ncbi.nlm.nih.gov/20459707","host_type":"repository"},{"url":"https://doaj.org/article/df15ec75d3ce4be98000ed85db2c7322","host_type":"repository"},{"url":"https://www.ncbi.nlm.nih.gov/pmc/articles/2873406","host_type":"repository"},{"url":"http://www.biomedcentral.com/1475-2859/9/28/abstract","host_type":"BioMedCentral"},{"url":"http://www.biomedcentral.com/1475-2859/9/28","host_type":"BioMedCentral"},{"url":"http://www.biomedcentral.com/content/pdf/1475-2859-9-28.pdf","host_type":"BioMedCentral"},{"url":"https://europepmc.org/articles/PMC2873406","host_type":"Europe_PMC"},{"url":"https://europepmc.org/articles/PMC2873406?pdf=render","host_type":"Europe_PMC"}],"fields_of_study":["biodegradable polymer synthesis and properties","Biopolymer Synthesis and Applications","Polymer-Based Agricultural Enhancements"],"mesh_terms":["Affinity Labels","Cloning, Molecular","Hydrogen-Ion Concentration","Recombinant Proteins","Repressor Proteins","Temperature","Escherichia coli Proteins","Inteins","Nanoparticles","Polyhydroxyalkanoates"],"keywords":["Intein","FLAG-tag","Maltose-binding protein","Fusion protein","Tandem affinity purification","Target protein","Biochemistry","Escherichia coli","Protein purification","Protein tag","Myc-tag","Green fluorescent protein","Centrifugation","Recombinant DNA","Affinity chromatography","Biology","Protein biosynthesis","lac operon","Enzyme","RNA","Gene"],"sdg_mappings":[],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-08-02T20:48:06.471063Z","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":[]}