Computational protocol: CYP5122A1, a Novel Cytochrome P450 Is Essential for Survival of Leishmania donovani

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Protocol publication

[…] Overlapping sets of primers for the CYP450 protein genes were designed from sequence available with the Leishmania major genome database (LmjF27.0090, Gene DB hosted by the Wellcome Trust Sanger Institute, Hinxton, UK). Using L. donovani genomic DNA as template, an insert was amplified with relevant primers and cloned into pGEM-TEasy vector. Sequencing was carried out by the di-deoxy method , at sequencing facility of Delhi University (New Delhi, India). The sequence was submitted to the CYP450 Nomenclature Committee (http://drnelson.uthsc.edu/cytochromep450.html) for assignment. Alignment of the L. donovani sequence with other CYP450s was performed using ClustalW hosted at the European Bioinformatics Institute . The protein sequence derived was subjected to secondary structure prediction program PSIPRED hosted at the Bloomsburry Center for Bioinformatics . The derived protein sequence of CYP5122A1 was also submitted to the SWISS-MODEL homology modelling server hosted at the Swiss Institute of Bioinformatics . The model generated was viewed in Pymol molecular graphic software (educational use). The stereochemical qualities of this 3D model were checked using Ramachandran plot generated by PROCHECK (EMBL-EBI, UK) (http://www.ebi.ac.uk/thornton-srv/software/ PROCHECK/). WHATCHEK (http://www.cmbi.kun.nl/gv/whatcheck/) server was used to assess the packing quality of the protein model .For CYP5122A1 allelic replacement constructs, ORFs encoding neomycin and hygromycin resistance were inserted between 0.4 kb of 5′ and 0.37 kb of 3′ of CYP5122A1 homologous regions in the pBlueScript SK+ vector to generate the constructs pBSK+CYP5122A1Neo and pBSK+CYP5122A1Hyg respectively. Half knockouts (HKOs) were generated with these constructs through transfection of log phase promastigotes as described previously . To generate double knockout parasites, second allelic replacement construct was transfected into the HKO parasites. Proper insertion of cassettes was confirmed by PCR to detect the presence of replacement constructs into the transformant genomic DNA. Primers were also designed using the intergenic sequence upstream of CYP5122A1 gene, the antibiotic resistance marker ORFs and the CYP5122A1 gene, such that amplification using these primers of expected molecular weight would only occur upon insertion at the correct locus. The leishmanial expression vector pXG-GFP +2 (a kind gift from Dr. S. Beverley) containing the CYP5122A1 gene was used for complementation of CYP5122A1 through episomal expression in the HKO parasites. Complementation was confirmed by GFP expression and detection of fusion m-RNA and GFP-fusion protein in the transformants. […]

Pipeline specifications

Software tools Clustal W, PSIPRED, PyMOL, PROCHECK, WHAT_CHECK
Application Protein structure analysis
Organisms Leishmania donovani
Diseases Infection, Leishmaniasis, Visceral, Parasitic Diseases
Chemicals Adenosine Triphosphate, Amphotericin B, Ergosterol, Neomycin, Sterols