title: Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.
abstract: Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13.
authors.affiliation: Laboratorio de Materiales Biotecnológicos (LaMaBio), Universidad Nacional de Quilmes-IMBICE (CONICET), Roque Sáenz Peña 352, B1876BXD, Bernal, Argentina.
authors.forename: Pamela
authors.initials: P
authors.lastname: Kikot
authors.forename: Aise
authors.initials: A
authors.lastname: Polat
authors.forename: Estefania
authors.initials: E
authors.lastname: Achilli
authors.forename: Marcelo
authors.initials: M
authors.lastname: Fernandez Lahore
authors.forename: Mariano
authors.initials: M
authors.lastname: Grasselli
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D010165:Palladium / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D006639:Histidine; D010165:Palladium; D003545:Cysteine
keywords: metal ion affinity chromatography; palladium(II); recombinant protein purification