Multi-step purification strategy for RANTES wild-type and mutated analogues expressed in a baculovirus system

J Chromatogr B Biomed Sci Appl. 2000 Jan 14;737(1-2):47-54. doi: 10.1016/s0378-4347(99)00361-8.

Abstract

RANTES (regulated on activation, normal T cell expressed and secreted), a C-C chemokine, is one of the major HIV-suppressive factors produced by CD8+ T cells. Wild-type RANTES and genetically modified analogues were expressed in a baculovirus system and purified from cell culture supernatants employing a multi-step strategy based on affinity and RP-HPLC. Quantification and purity control of the final proteins were carried out by capillary electrophoresis using the synthetic or the recombinant wild-type RANTES as a reference. The procedure here reported requires only three days to obtain 0.016-0.270 mg of the pure and characterised proteins, starting from 370-900 ml of culture media, and is suitable for the analysis of a large number of RANTES analogues.

MeSH terms

  • Baculoviridae / genetics
  • Cell Line
  • Chemokine CCL5 / genetics
  • Chemokine CCL5 / isolation & purification*
  • Chemokine CCL5 / pharmacology
  • Chromatography, Affinity / methods
  • Chromatography, High Pressure Liquid / methods
  • Electrophoresis, Capillary
  • Enzyme-Linked Immunosorbent Assay
  • Humans
  • Mass Spectrometry
  • Mutation*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / pharmacology

Substances

  • Chemokine CCL5
  • Recombinant Proteins