The deduced amino acid sequence sellckchem of the open reading frame corresponds to a protein containing 144 amino acids, indicating that PfI2 has the shortest amino acid sequence between I2 homologs. Sequence alignment com bined with visual inspection of PfI2 showed an total identity of 28% and 34% identity amongst amino acids at positions 5 to 105 of PfI2 when in contrast to human I2. The usage of PSORTII application unveiled a putative nuclear localization signal. The PfI2 sequence, located in human I2 and proven for being essential protein contains two peptides KTISW and KHYNE that match flawlessly on the or RV F motif and HYNE motifs responsible for binding to PP1c. However, 2 most important variations had been observed for interaction with PP1c just isn't current from the PfI2 sequence and 2nd, the KSQKW sequence of human I2 includes a Q residue as opposed to V or I on the RV F consensus sequence.
The examination of PfI2 working with protein secondary structure prediction soft ware sellectchem PsiPred predicted the RV F motif is usually a part of an unstructured area, even though the HYNE motif is inside an heli taking place between positions 70 and 120. This structure is in agreement with that identified in mammalian I2. This analysis is in accordance together with the framework prediction presented in PlasmoDB. A ma imum likehood phylogenetic tree was created beneath the JTT I G model together with the help of two outgroups composed of two well described PP1 regula tors Inhibitor 3 and LRR1. In this tree PfI2 segregates with orthologues from other Plasmodium species as well because the apicomple an Theileria parva, but inside of the I2 household on a well supported branch separate from the I3 loved ones.
This examination clearly identi fies PfI2 as being a PP1c inhibitor two relatives member. E pression of PfI2 protein by P. falciparum and localization studies To investigate the e pression of PfI2 by P. falciparum, polyclonal antibodies towards the recombinant PfI2 protein have been raised. As presented in Figure 2A lane 1, the recombinant protein whose amino acid sequence was confirmed by MALDI TOF mass spectrometry, Mammalian target of rapamycin migrated at about twenty kDa, in agree ment with all the anomalous electrophoretic behavior of inhibitors with the PP1 family. the e pected molecular fat of endogenous PfI2 is sixteen. 7 KDa. Whilst these antibodies recognized the recombinant protein, they were unable to react with any bands in total e tracts of asynchronous blood stage parasites. So as to detect endogenous PfI2, we carried out immunoprecipita tion e periments with anti PfI2 sera or pre bleed sera with total parasite e tracts. Immunoblots with anti PfI2 anti bodies showed the presence of the band at twenty kDa inside the immunoprecipitates with anti PfI2, when the pre bleed serum detected no specific band.