Abstract:
:Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are routinely used in HBV diagnostics worldwide and are of potential interest as carriers of foreign peptides (e.g., immunological epitopes and targeting addresses, and/or as vessels for packaged diagnostic and therapeutic nanomaterials). Despite numerous reports exploiting different expression systems, a rapid and comprehensive large-scale methodology for purification of HBc VLPs from yeast is still lacking. Here, we present a convenient protocol for highly efficient production and rapid purification of endotoxin-free ayw subtype HBc VLPs from the methylotrophic yeast Pichia pastoris. The HBc gene expression cassette along with the geneticin resistance gene was transferred to the P. pastoris genome via homologous recombination. A producer clone was selected among 2000 transformants for the optimal synthesis of the target protein. Fermentation conditions were established ensuring biomass accumulation of 163g/L. A simple combination of pH/heat and salt treatment followed by a single anion-exchange chromatography step resulted in a more than 90% pure preparation of HBc VLPs, with a yield of about 3.0mg per 1g of wet cells. Purification is performed within a day and may be easily scaled up if necessary. The quality of HBc VLPs was verified by electron microscopy. Mass spectrometry analysis and direct polyacrylamide gel staining revealed phosphorylation of HBc at at least two sites. To our knowledge, this is the first report of HBc phosphorylation in yeast.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Freivalds J,Dislers A,Ose V,Pumpens P,Tars K,Kazaks Adoi
10.1016/j.pep.2010.09.010subject
Has Abstractpub_date
2011-02-01 00:00:00pages
218-24issue
2eissn
1046-5928issn
1096-0279pii
S1046-5928(10)00258-5journal_volume
75pub_type
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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doi:10.1016/j.pep.2009.11.003
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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doi:10.1006/prep.2000.1266
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abstract::Pullulanases are well-known starch-debranching enzymes that are widely used for hydrolysis of a-1,6-glycosidic linkages in starch, pullulan, amylopectin, and other oligosaccharides. Escherichia coli is a popular heterologous expression host for generating target enzymes. However, cells have to be disrupted to obtain t...
journal_title:Protein expression and purification
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abstract::Arginine has been effectively used in various column chromatographies for improving recovery and resolution, and suppressing aggregation. Here, we have tested the effectiveness of arginine as an eluent in dye-affinity column chromatography using Blue-Sepharose, which binds enzymes requiring adenyl-containing cofactors...
journal_title:Protein expression and purification
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doi:10.1016/j.pep.2006.10.005
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journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2017.01.005
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journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2006.07.002
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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abstract::Protein aggregation is a main barrier hindering structural and functional studies of a number of interesting biological targets. The E6 oncoprotein of Human Papillomavirus strain 16 (E6(16)) is difficult to express under a native soluble form in bacteria. Produced as an unfused sequence, it forms inclusion bodies. Fus...
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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abstract::Soluble oligomers and fibrillar deposits of amyloid beta (Abeta) are key agents of Alzheimer's disease pathogenesis. However, the mechanism of amyloid aggregation and its interaction with live cells still remain unclear requiring the preparation of large amounts of pure and different Abeta peptides. Here we describe a...
journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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journal_title:Protein expression and purification
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abstract::Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent. The aim of this study is to produce large quantities of highly pure and bioactive recombinant human TRAIL. Here, TRAIL was expressed in soluble form by pH-stat fed-batch cultivation and purified using a rapid and simple tw...
journal_title:Protein expression and purification
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