Figure 6
Scale-up and purification of IFP fusion proteins expressed in L. tarentolae. A) Culture volume of IFP-His expressing Leishmania cells was scaled-up in nine 150 cm2-tissue culture flasks (1-9), followed by incubation under static flat condition for four days (1d-4d). Samples for IR analysis were taken every 24 h. B) Scaled-up culture volumes were pooled and IFP-His protein was purified by affinity chromatography using a HisTrap HP column coupled to the Äkta-Purifier FPLC system. Aliquots of the 1-mL unbound (U), wash (W) and elution (E) fractions were IR-scanned in the wells of a 96-well microtiter plate. C) Expression of ANAC42-IFP-His fusion protein was scaled-up and the protein was enriched manually on a Protino Ni-IDA 150 column by circulating the protein-containing sample three times. Aliquots of the fractions unbound after each circulation (U1, 2, 3), washing (W1, 3) and elution (E1 - E6) were IR-scanned in the wells of a 96-well microtiter plate. Red signal in the image indicates moderate and white signal high IFP concentration.