title: Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.
abstract: Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals.
authors.affiliation: INSERM UMR 892-CRCNA, Nantes, France; CNRS UMR 6299, Nantes, France; University of Nantes, Nantes, France.
authors.forename: Ghislaine
authors.initials: G
authors.lastname: Béhar
authors.affiliation: INSERM UMR 892-CRCNA, Nantes, France; CNRS UMR 6299, Nantes, France; University of Nantes, Nantes, France.
authors.forename: Axelle
authors.initials: A
authors.lastname: Renodon-Cornière
authors.affiliation: INSERM UMR 892-CRCNA, Nantes, France; CNRS UMR 6299, Nantes, France; University of Nantes, Nantes, France.
authors.forename: Barbara
authors.initials: B
authors.lastname: Mouratou
authors.affiliation: INSERM UMR 892-CRCNA, Nantes, France; CNRS UMR 6299, Nantes, France; University of Nantes, Nantes, France. Electronic address: frederic.pecorari@univ-nantes.fr.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Pecorari
mesh_terms: D000818:Animals; D019843:Archaeal Proteins / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000378:metabolism; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods; D029968:Escherichia coli Proteins / Q000302:isolation & purification*; D006801:Humans; D055767:Immobilized Proteins; D007074:Immunoglobulin G / Q000302:isolation & purification*; D007202:Indicators and Reagents; D008024:Ligands; D009113:Muramidase / Q000302:isolation & purification*; D013205:Staphylococcal Protein A
chemical_list: D019843:Archaeal Proteins; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D055767:Immobilized Proteins; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D008024:Ligands; C560058:PulD protein, E coli; D013205:Staphylococcal Protein A; C421864:hen egg lysozyme; D009113:Muramidase
keywords: Affinity chromatography; Affitin,; Antibody; Immunoglobulin; Sac7d; Sso7d