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

Protein Expr Purif. 2007 Jan;51(1):49-58. doi: 10.1016/j.pep.2006.07.009. Epub 2006 Jul 21.

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.

MeSH terms

  • Animals
  • C-Reactive Protein / biosynthesis*
  • C-Reactive Protein / isolation & purification*
  • C-Reactive Protein / metabolism
  • CHO Cells
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Circular Dichroism
  • Complement C1q / metabolism
  • Cricetinae
  • Cricetulus
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Protein Structure, Quaternary
  • Recombinant Proteins / isolation & purification*
  • Serum Amyloid P-Component / biosynthesis*
  • Serum Amyloid P-Component / isolation & purification*
  • Serum Amyloid P-Component / metabolism
  • Spectrometry, Fluorescence

Substances

  • Recombinant Proteins
  • Serum Amyloid P-Component
  • PTX3 protein
  • Complement C1q
  • C-Reactive Protein