title: High yield of recombinant human Apolipoprotein A-I expressed in Pichia pastoris by using mixed-mode chromatography.
abstract: A vast majority of the cardioprotective properties exhibited by High-Density Lipoprotein (HDL) is mediated by its major protein component Apolipoprotein A-I (ApoA1). In order to develop a simplified bioprocess for producing recombinant human Apolipoprotein A-I (rhApoA1) in its near-native form, rhApoA1was expressed without the use of an affinity tag in view of its potential therapeutic applications. Expressed in Pichia pastoris at expression levels of 58.2 mg ApoA1 per litre of culture in a reproducible manner, the target protein was purified by mixed-mode chromatography using Capto™ MMC ligand with a purity and recovery of 84% and 68%, respectively. ApoA1 purification was scaled up to Mixed-mode Expanded Bed Adsorption chromatography to establish an 'on-line' process for the efficient capture of rhApoA1 directly from the P. pastoris expression broth. A polishing step using anion exchange chromatography enabled the recovery of ApoA1 up to 96% purity. Purified ApoA1 was identified and verified by RPLC-ESI-Q-TOF mass spectrometry. This two-step process would reduce processing times and therefore costs in comparison to the twelve-step procedure currently used for recovering rhApoA1 from P. pastoris.
authors.affiliation: IPB, Biotechnologie des Protéines Recombinantes à Visée Santé, Bordeaux, France.|Univ. Bordeaux, Biotechnologie des Protéines Recombinantes à Visée Santé, Bordeaux, France.|Centre for Bio-Separation Technology, VIT University, Vellore, India.
authors.forename: Vignesh
authors.initials: V
authors.lastname: Narasimhan Janakiraman
authors.affiliation: IPB, Biotechnologie des Protéines Recombinantes à Visée Santé, Bordeaux, France.|Univ. Bordeaux, Biotechnologie des Protéines Recombinantes à Visée Santé, Bordeaux, France.
authors.forename: Abdelmajid
authors.initials: A
authors.lastname: Noubhani
authors.affiliation: Centre for Bio-Separation Technology, VIT University, Vellore, India.
authors.forename: Krishnan
authors.initials: K
authors.lastname: Venkataraman
authors.affiliation: Centre for Bio-Separation Technology, VIT University, Vellore, India.
authors.forename: Mookambeswaran
authors.initials: M
authors.lastname: Vijayalakshmi
authors.affiliation: IPB, Biotechnologie des Protéines Recombinantes à Visée Santé, Bordeaux, France. xavier.santarelli@ipb.fr.|Univ. Bordeaux, Biotechnologie des Protéines Recombinantes à Visée Santé, Bordeaux, France. xavier.santarelli@ipb.fr.
authors.forename: Xavier
authors.initials: X
authors.lastname: Santarelli
mesh_terms: D016632:Apolipoprotein A-I / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002852:Chromatography, Ion Exchange; D006801:Humans; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry
chemical_list: C505693:APOA1 protein, human; D016632:Apolipoprotein A-I; D011994:Recombinant Proteins
keywords: Anion exchange chromatography polishing; Apolipoprotein A-I (ApoA1); Expanded bed adsorption; Mixed mode chromatography; Pichia pastoris; Recombinant protein production