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High yield of recombinant human Apolipoprotein A‐I expressed in Pichia pastoris by using mixed‐mode chromatography
Author(s) -
Narasimhan Janakiraman Vignesh,
Noubhani Abdelmajid,
Venkataraman Krishnan,
Vijayalakshmi Mookambeswaran,
Santarelli Xavier
Publication year - 2016
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201500245
Subject(s) - pichia pastoris , chromatography , bioprocess , recombinant dna , chemistry , expanded bed adsorption , affinity chromatography , pichia , apolipoprotein b , biochemistry , biology , enzyme , elution , cholesterol , gene , paleontology
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