Expression and Purification of Recombinant Human Apolipoprotein A-I in Chinese Hamster Ovary Cells

Schmidt, Hartmut; Genschel, Janine; Haas, Regina; Büttner, Janine; Manns, Michael P.

doi:10.1006/prep.1997.0753

Cited by 17 publications

(13 citation statements)

References 51 publications

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“…Their results revealed both an increased catabolism and altered association with the HDL fraction of apoA‐I isolated from affected patients [33]. To gain further insight into the metabolism of apoA‐I Iowa , we established recombinant expression of this protein and its wildtype in transfected CHO cells [37]. After purification by reversed phase chromatography we radiolabelled the recombinant form of apoA‐I Iowa , and injected it simultaneously with wild type in New Zealand White (NZW) rabbits using 131 I and 125 I, respectively [38, 39].…”

Section: In Vivo Characterization Of Amyloidogenic Apoa‐imentioning

confidence: 99%

Apolipoprotein A‐I induced amyloidosis

et al. 1998

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Amyloidosis is characterized by extracellular deposits of protein fibrils with a high content of L L-sheets in secondary structure. The protein forms together with proteoglycans amyloid fibrils causing organ damage and serious morbidity. Intact apolipoprotein A-I (apoA-I) is an important protein in lipid metabolism regulating the synthesis and catabolism of high density lipoproteins (HDL). Usually, apoA-I is not associated with amyloidosis. However, four naturally occuring mutant forms of apoA-I are known so far resulting in amyloidosis. The most important feature of all variants is the very similar formation of N-terminal fragments which were found in the amyloid deposits (residues 1^83 to 1^94). The new insights in the understanding of the association of apoA-I with HDL, its metabolism, and its hypothesized structural findings may explain a common mechanism for the genesis of apoA-I induced amyloidosis. Here we summarized the specific features of all known amyloidogenic variants of apoA-I and speculate about its metabolic pathway, which may have general implications for the metabolism of apoA-I.z 1998 Federation of European Biochemical Societies.

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Section: In Vivo Characterization Of Amyloidogenic Apoa‐imentioning

confidence: 99%

Apolipoprotein A‐I induced amyloidosis

et al. 1998

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“…Most biopharmaceutical glycoproteins are currently produced by culture systems using animal cells such as Chinese hamster ovary (CHO) cells or human fibroblast cells in which human-like glycans are attached (Jones et al 2003;Kariya et al 2002;Schmidt et al 1997). However, there are some reports of accidental emergence of organisms harboring infectious prion (PrP Sc ) diseases and of closure of good manufacturing practice (GMP) production facilities due to contamination by animal viral pathogens .…”

Section: Introductionmentioning

confidence: 98%

Glycoengineering in plants for the development of N-glycan structures compatible with biopharmaceuticals

Yoo

Lee

et al. 2014

Plant Biotechnol Rep

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Plants and plant cells are emerging as promising alternatives for biopharmaceutical production with improved safety and efficiency. Plant cells are capable of performing post-translational modifications (PTMs) similar to those of mammalian cells and are safer than mammalian cells with regard to contamination by infectious pathogens, including animal viruses. However, a major obstacle to producing biopharmaceuticals in plants lies in the fact that plant-derived N-glycans include plant-specific sugar residues such as b1,2-xylose and a1,3-fucose attached to a pentasaccharide core (Man 3 GlcNAc 2 ) as well as b1,3-galactose and a1,4-fucose involved in Lewis a (Le a ) epitope formation that can evoke allergic responses in the human body. In addition, sugar residues such as a1,6-fucose, b1,4-galactose and a2,6-sialic acid, which are thought to play important roles in the activity, transport, delivery and halflife of biopharmaceuticals are absent among the N-glycans naturally found in plants. In order to take advantage of plant cells as a system in which to produce biopharmaceuticals development of plants producing N-glycan structures compatible with biopharmaceuticals is necessary. In this article we summarize the current state of biopharmaceutical production using plants as well as what is known about N-glycosylation processes occurring in the endoplasmic reticulum and Golgi apparatus in plants. Finally, we propose and discuss a strategy for and the associated technical barriers of producing customized N-glycans via removal of enzyme genes that add plantspecific sugar residues and introducing enzyme genes that add sugar residues absent in plants.

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“…The production of ApoA1 secreted in CHO cell culture (up to 80 µg/mL) and its subsequent purification have been performed by the following methods: either centrifugation, filtration, delipidation with ethanol:ether at −20°C, solubilization in 6 M urea followed by reverse phase chromatography with further elimination of acetonitrile (yield 50%, purity 90%) ; or precipitation with ammonium sulfate, centrifugation followed by hydrophobic interaction chromatography with elution with propylene glycol and then immunoaffinity chromatography (yield 6%, purity 90%). In 1997, Schmidt et al compared several techniques: PrepSDS‐PAGE (drawbacks: time‐consuming, fragmentation during electroelution, removal of SDS), size exclusion chromatography (drawbacks: further concentration steps owing to sample volume, low flow rates), ion exchange chromatography (drawbacks: removal of salts), immunoaffinity (drawbacks: loss of capacity, removal of acetic acid and NaSCN, concentration step), and reverse phase chromatography (drawbacks: removal of acetonitrile and TFA). They found reverse‐phase chromatography to be most efficient with high recovery (80–90%) and high purity (90%) but low production at the beginning of the process.…”

Section: Introductionmentioning

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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Expression and Purification of Recombinant Human Apolipoprotein A-I in Chinese Hamster Ovary Cells

Cited by 17 publications

References 51 publications

Apolipoprotein A‐I induced amyloidosis

Apolipoprotein A‐I induced amyloidosis

Glycoengineering in plants for the development of N-glycan structures compatible with biopharmaceuticals

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

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