Environmental and cellular inappropriate conditions can cause damages to cells nucleotide poll. Deamination and oxidation damages interfere with cell�s vital reactions. Inosine triphosphate pyrophosphatase (ITPA), an evolutionary conserved enzyme, plays a critical role in elimination of non-canonical bases. In human genome, the ITPA gene is located on chromosome 20 short arm and transcribed into three different alternative transcripts in various tissues. The main alternative variant is variant I (coding isoform a), which is expressed in all human�s cells. Although, ITPA variant II (coding isoform b) is expressed in large cell carcinoma, astrocytoma grade IV, brain and Embryonic Stem Cells (ESCs). Comparing with isoform a, isoform b is 17 amino acids shorter in length. It�s not yet clear whether the variant II can code any protein or not.
Materials and methods
RNA was extracted from cancerous cell line. cDNA was synthesized by oligo dt and random hexamer primers. Primers specific to the variant II amplified the relevant cDNA. The PCR fragment was cloned into TA vector. By applying restriction enzymes, cloned fragment was subcloned in the pET expression system. For its expres sion, pET32a vector was transformed into BL21 E.coli strain, which can code T7 RNA polymerase. Expression was induced by exertion IPTG into the liquid bacterial culture (LB).
Sequence file was analyzed and the accuracy of cloned fragment was endorsed by sequencing. Expression of isoform b was validated on SDS-PAGE.
The size of the cloned fragment was confirmed by protein ladder. The size difference between two isoforms was observed on SDS-PAGE, approving the precision of cloned fragment. This cloned variant, can be used for further investigations such as, activation analysis, protein-protein interaction and sub-cellular localization.