PAPER TITLE :EUKARYOTIC INITIATION FACTOR 2B COMPLEX’S PURIFICATION FROM A PREDOMINANTLY DIPLOID ORGANISM, C. ALBICANS BY FLAG-EPITOPE TAGGING OF THE TWO GCDI ALLELES

FUTA JOURNAL OF RESEARCH IN SCIENCE | VOLUME 15 NUMBER 2 2019

Paper Details

  • Author(s) : Egbe NE1,2, Onuh KC1
  • Abstract:

To study protein interaction and expression in cells, the cells are usually tagged to enable location of proteins within cells and for isolation and purification of the protein complexes of interest. This study was carried out to tag the two copies of GCDI gene in Candida albicans with FLAG epitopes to enable maximal protein expression of the tagged allele as well as purification of the tagged gene product by affinity chromatography. To achieve this, we designed a tagging strategy with four FLAG epitopes making use of a mini URA3 blaster (URA3-dpl200) cassette. This tagging cassette was commercially synthesized and a standard PCR based epitope tagging strategy was taken to tag the GCD1 genes with the 4X-FLAG-URA3dpl cassette. Agarose gel electrophoresis and Western blot analysis were used to confirm the success of the epitope tagging. Purification of the protein product complex of the tagged GCDI genes was done by affinity chromatography using ANTI-FLAG M2 Magnetic beads. This strategy enabled the epitope tagging of the two alleles of GCD1, which code for a subunit of the eukaryotic initiation factor 2B (eIF2B), a heteropentameric guanine nucleotide exchange factor that functions during translation initiation. The purified tagged protein complex yielded the expected sized bands on SDS PAGE gel. The sub-complex of the protein subunits was enriched in the purified sample detected by mass spectrometry. Therefore, these data show that the tagging of the two GCD1 alleles with 4xFLAG epitope has successfully allowed the eIF2B to be purified.

KEY WORDS: FLAG epitope tag, GCD1, Candida albicans, Affinity chromatography, Mass Spectrometry