Iron content of native Lf was approximately 8%15%% [ 12].
Gene density differentiation is clearly observed when counting the number of genes within the reconstructed tPCK subgenomes: the subgenome LF has approximately 1.6 times more genes than the other two subgenomes MF1 and MF2 (the more fractionated subgenomes one and two).
The EC50 values (concentration required to kill 50% of the cells) of LF-A and LF-A/X were approximately 3-fold lower than those of LF-HMA (Fig. 2A, Table 1), and were comparable to the values found in other experiments for LF-A/St (data not shown).
The least fractionated (LF) genome retains approximately 70% of the genes, whereas the medium fractionated (MF1) and most fractionated (MF2) genomes retain ∼46%and∼36%6%, respectively (Wang et al. 2011; Tang et al. 2012).
Its expression at the protein level was assessed using the monoclonal anti-human PRLR antibody, which specifically recognizes the extracellular domain common to all known isoforms (R&D Systems, Inc .. Differences in mRNA levels were reflected at the protein level, with the LF migrating at approximately 85 90 kDa.
In our studies we initially noted that the presence of two additional N-terminal residues in recombinant LF decreased its potency approximately 3-fold.
Transient transfection of CHO-K1s with a mammalian expression vector encoding the full-length coding sequence of the human PRLR LF resulted in an approximately 2-fold increase in receptor levels compared to cells transfected with either empty vector (pcDNA3.1) or PRLR-SF1b encoding a short isoform.
Regarding our results, we found that both variants of amoebae recognize B-holo-Lf with high specificity, and H-holo-Lf and H-holo-Tf did not compete with B-holo-Lf for the EhBholoLf-binding sites despite the high homology in the amino acid sequence, which are approximately 77% between B-Lf and H-Lf and 60% between B-Lf and H-Tf [ 38, 39].
B-holo-Lf (100 135 μM Fe) supported the growth of both nonvirulent and virulent variants over a period of at least 96 h; however, the virulent amoebae were more efficient in using the iron from B-holo-Lf because an increase of approximately 70% in the number of amoeba was observed in this culture.
An approximately 10,000-fold dilution of the LF added to the reaction mixture resulted in a 50% decrease in the hydrolysis rate of MUL.
