title: A novel strategy for the purification of a recombinant protein using ceramic fluorapatite-binding peptides as affinity tags.
abstract: In 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 (20-50mM sodium phosphate, pH 6-8.4). Sodium phosphate (500mM) or 1M 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.
authors.affiliation: Department of Biochemical Engineering, School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany; Centro de Biotecnología FEMSA, Tecnológico de Monterrey, Campus Monterrey, Ave. Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, Mexico.
authors.forename: Tuhidul
authors.initials: T
authors.lastname: Islam
authors.affiliation: Centro de Biotecnología FEMSA, Tecnológico de Monterrey, Campus Monterrey, Ave. Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, Mexico.
authors.forename: José Manuel
authors.initials: JM
authors.lastname: Aguilar-Yañez
authors.affiliation: Centro de Biotecnología FEMSA, Tecnológico de Monterrey, Campus Monterrey, Ave. Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, Mexico.
authors.forename: Jesús
authors.initials: J
authors.lastname: Simental-Martínez
authors.affiliation: Centro de Biotecnología FEMSA, Tecnológico de Monterrey, Campus Monterrey, Ave. Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, Mexico.
authors.forename: Cesar Ivan
authors.initials: CI
authors.lastname: Ortiz-Alcaraz
authors.affiliation: Centro de Biotecnología FEMSA, Tecnológico de Monterrey, Campus Monterrey, Ave. Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, Mexico.
authors.forename: Marco
authors.initials: M
authors.lastname: Rito-Palomares
authors.affiliation: Department of Biochemical Engineering, School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany; National Council for Research and Technology, Buenos Aires, Argentina. Electronic address: m.fernandez-lahore@jacobs-university.de.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Fernandez-Lahore
mesh_terms: D000345:Affinity Labels / Q000737:chemistry*; D001031:Apatites / Q000737:chemistry*; D002516:Ceramics; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D001031:Apatites; D010455:Peptides; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C025105:fluorapatite
keywords: Affinity chromatography; Ceramic fluorapatite; Peptide affinity tags; Peptide synthesis; Production and purification of recombinant proteins; Retention behavior