Multi-step purification strategy for RANTES wild-type and mutated analogues expressed in a baculovirus system.
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.
Comuzzi
B
B
Department of Laboratory Medicine, Scientific Institute H.S. Raffaele and School of Medicine, Milan, Italy.
Arcelloni
C
C
Polo
S
S
Nardese
V
V
Lusso
P
P
Paroni
R
R
D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D018946:Chemokine CCL5 / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019075:Electrophoresis, Capillary; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D013058:Mass Spectrometry; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
D018946:Chemokine CCL5; D011994:Recombinant Proteins