Comprehensive genome-wide analysis of WDR superfamily of Plasmodium falciparum, cloning and production of PfWDR protein involved in chromatin remodelling

Abstract

Background: WD40 repeats (WDRs) proteins comprise one of the largest and functionally diverse protein families in eukaryotes. WD40 gene family is of special interest because of its diverse functionality and mutidomain context; however this family remains uncharacterized in P. falciparum the most virulent species of human malaria. The present study gives a comprehensive in silico analysis of PfWDR gene family and cloning and production of a PfWD40 gene involved in chromatin assembly. Methods & Materials: PfWDR genes were extracted from PlasmoDB v 9.0 by gene text search, BLASTp and HMM search. Domain architecture of extracted genes was drawn by SMART and Pfam. Subcellular localization prediction was carried by Euk-mPLoc 2.0 server, PATS, Mitoprot and predict NLS. Expression of all PfWDR genes was analysed using MeV software as per transcriptome and proteome data available at PlasmoDB. Human homologs were assigned by BLASTp and literature search. Evolutionary relationships were drawn using phylip by NJ method. Further, one of WDR genes i.e. the chromatin assembly factor 1 (PfCAF-1) was amplified, cloned in pETM 11 vector and expressed in E. Coli B834 cellls. Expressed protein was purified by Ni-NTA chromatography. Results: In our present analysis, we identified 80WDR genes in P. falciparum and classified them in two families and twenty one subfamilies based on domain composition. While based on their functions,we categorized PfWDRproteins in eleven classes. By subcellular localization prediction, PfWDR proteins were found to be targeted to various cellular compartments verifying their diversified functions. Approximately fifty PfWD40 genes were predicted to have human homologs. Importantly, we were able to annotate some of PfWD40 genes based on their orthologs in other species. TheORF (1341bp) of the PfCAF-1was amplified and cloned in pETM 11.When transformed E. coli B834 cells were induced at 16 ◦Cwith 0.25mM IPTG, 50% of expressed protein was found to be in soluble form. The recombinant protein was purified and identity was confirmed by western blotting. Conclusion: This study provides a genomic glimpse of PfWDR gene family. The purified recombinant PfCAF-1 can be further exploited for the functional and structural characterization.