Role of urea on recombinant Apo A-I stability and its utilization in anion exchange chromatography

Angarita, Monica; Arosio, Paolo; Müller‐Späth, Thomas; Baur, Daniel; Falkenstein, Roberto; Kuhne, Wolfgang; Morbidelli, Massimo

doi:10.1016/j.chroma.2014.05.067

Cited by 16 publications

(1 citation statement)

References 27 publications

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“…Despite the use of these tags, the purification steps require many treatments as well as cell lysis, protein precipitation with ammonium sulfate, delipidation, removal of the tags and endotoxin clearance at the end of the purification, in addition to an enzymatic cleavage of the affinity tag. In some cases the protein is mainly found in inclusion bodies, which further complicates the procedure [39]. Another reported approach is the production of apolipoprotein A-I using a baculovirus-insect cell expression system [15][16][17].…”

Section: Discussionmentioning

confidence: 99%

High yield of recombinant human Apolipoprotein A‐I expressed in Pichia pastoris by using mixed‐mode chromatography

Janakiraman

Noubhani

Venkataraman

et al. 2015

Biotechnology Journal

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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.

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Section: Discussionmentioning

confidence: 99%