A novel strategy for the purification of a recombinant protein using ceramic fluorapatite-binding peptides as affinity tags

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dc.creatorJosé Manuel Aguilar Yáñez
dc.creatorMarco Antonio Rito Palomares
dc.date2014
dc.date.accessioned2018-10-18T21:50:59Z
dc.date.available2018-10-18T21:50:59Z
dc.descriptionIn recent years, affinity fusion-tag systems have become a popular technique for the purification of recombinant proteins from crude extracts. However, several drawbacks including the high expense and low stability of ligands, their leakage during operation, and difficulties in immobilization, make it important to further develop the method. The present work is concerned with the utilization of a ceramic fluorapatite (CFT)-based chromatographic matrix to overcome these drawbacks. A heptapeptide library exhibiting a range of properties have been synthesized and subjected to ceramic fluorapatite (CFT) chromatography to characterize their retention behavior as a function of pH and composition of the binding buffer. The specific binding and elution behavior demonstrates the possible application of CFT-binding peptides as tags for enhancing the selective recovery of proteins by CFT chromatography. To materialize this strategy, a phage-derived CFT-specific sequence KPRSVSG (Tag1) with/without a consecutive hexalysine sequence, KKKKKKKPRSVSG (Tag2), were fused at the C-terminus of an enhanced green fluorescent protein (eGFP). The resulting gene constructs H-eGFP, H-eGFP-Tag1 and H-eGFP-Tag2 were expressed in Escherichia coli strain BL-21, and the clarified cell lysate was applied to the CFT column equilibrated with binding buffer (20-50. mM sodium phosphate, pH 6-8.4). Sodium phosphate (500. mM) or 1. M NaCl in the respective binding buffer was used to elute the fused proteins, and the chromatographic fractions were analyzed by gel electrophoresis. Both the yield and purity were over 90%, demonstrating the potential application of the present strategy. © 2014 The Authors.
dc.identifier.doi10.1016/j.chroma.2014.02.079
dc.identifier.endpage33
dc.identifier.issn219673
dc.identifier.startpage26
dc.identifier.urihttp://hdl.handle.net/11285/630478
dc.identifier.volume1339
dc.languageeng
dc.publisherElsevier
dc.relationhttps://www.scopus.com/inward/record.uri?eid=2-s2.0-84897914844&doi=10.1016%2fj.chroma.2014.02.079&partnerID=40&md5=dff76696611d56a0f0c1ec0a0272d576
dc.relationInvestigadores
dc.relationEstudiantes
dc.rightsinfo:eu-repo/semantics/openAccess
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0
dc.sourceJournal of Chromatography A
dc.subjectAffinity chromatography
dc.subjectElectrophoresis
dc.subjectEscherichia coli
dc.subjectPeptides
dc.subjectPhosphates
dc.subjectPurification
dc.subjectRecombinant proteins
dc.subjectSodium compounds
dc.subjectAffinity tags
dc.subjectChromatographic matrix
dc.subjectEnhanced green fluorescent protein
dc.subjectFluorapatites
dc.subjectGel electrophoresis
dc.subjectPeptide synthesis
dc.subjectRetention behavior
dc.subjectSelective recovery
dc.subjectCeramic materials
dc.subjectceramic fluorapatite
dc.subjectfluorapatite
dc.subjecthybrid protein
dc.subjectrecombinant protein
dc.subjectunclassified drug
dc.subjectarticle
dc.subjectbinding affinity
dc.subjectcell lysate
dc.subjectcolumn chromatography
dc.subjectcontrolled study
dc.subjectgel electrophoresis
dc.subjectnonhuman
dc.subjectpeptide analysis
dc.subjectpeptide library
dc.subjectpeptide synthesis
dc.subjectpriority journal
dc.subjectprotein purification
dc.subjectseparation technique
dc.subjectEscherichia coli
dc.subjectAffinity chromatography
dc.subjectCeramic fluorapatite
dc.subjectPeptide affinity tags
dc.subjectPeptide synthesis
dc.subjectProduction and purification of recombinant proteins
dc.subjectRetention behavior
dc.subjectAffinity Labels
dc.subjectApatites
dc.subjectCeramics
dc.subjectChromatography, Affinity
dc.subjectEscherichia coli
dc.subjectGreen Fluorescent Proteins
dc.subjectPeptides
dc.subjectRecombinant Fusion Proteins
dc.subject.classification7 INGENIERÍA Y TECNOLOGÍA
dc.titleA novel strategy for the purification of a recombinant protein using ceramic fluorapatite-binding peptides as affinity tags
dc.typeArtículo
refterms.dateFOA2018-10-18T21:50:59Z

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