High-level expression and efficient purification of recombinant human long pentraxin PTX3 in Chinese hamster ovary cells.
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.
Rivieccio
Vincenzo
V
Tecnogen SCpA, Località La Fagianeria I-81015 Piana di Monteverna (CE), Italy. rivieccio@tecnogen.it
Esposito
Adelaide
A
Bellofiore
Piero
P
Palladino
Pasquale
P
Sassano
Marica
M
Colombo
Maurizio
M
Verdoliva
Antonio
A
D000818:Animals; D002097:C-Reactive Protein / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D016466:CHO Cells; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D015922:Complement C1q / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000302:isolation & purification*; D000683:Serum Amyloid P-Component / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D000099153:Pentraxins
D002097:C-Reactive Protein; D015922:Complement C1q; D011994:Recombinant Proteins; D000683:Serum Amyloid P-Component; D000099153:Pentraxins