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Protein Expr Purif. 2007 Jan;51(1):49-58. Epub 2006 Jul 21.

High-level expression and efficient purification of recombinant human long pentraxin PTX3 in Chinese hamster ovary cells.

Author information

1
Tecnogen SCpA, Località La Fagianeria I-81015 Piana di Monteverna (CE), Italy. rivieccio@tecnogen.it

Abstract

PTX3 is a secreted multimeric glycoprotein which plays a key role in innate immunity by activating the classical complement pathway through specific recognition of the C1q subunit. A method is described for the high level expression of the recombinant human PTX3 in Chinese hamster ovary cells (CHO), adapted to a suspension growth in spinner flasks containing a serum-free chemically defined medium and producing about 50 mg of PTX3/L of culture. A purification procedure to produce a homogeneous protein preparation from the supernatant, by means of anion exchange, hydroxyapatite and size exclusion chromatography, is also reported. This three-step protocol allows us to obtain PTX3 with a recovery yield close to 70%, a purity degree exceeding 95%, and a final host cell protein (HCP) content lower than 150 ppm. The recombinant purified PTX3 retains its biological activity, as demonstrated by C1q binding ELISA assay, and displays a complex quaternary structure characterized by a high secondary structure content quite different from human short pentraxin C-reactive protein (CRP) and serum amyloid P component (SAP), as determined by circular dichroism, fluorescence analysis, and native and SDS-PAGE experiments.

PMID:
16931047
DOI:
10.1016/j.pep.2006.07.009
[Indexed for MEDLINE]

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