Cloning, Expression And Analysis Of Spectrum Of GARP Gene From Euplotes Octocarinatus

Proteins containing single amino acid repeats are related to transcription and signal transduction. The expansion of trinucleotide repeats in genome is related to the phthogenesis of several neurode generative deseases. A GARP (glutamic acid-rich protein) gene was isolated from the macronuclear plasmid mini library of Euplotes octocarinatus. The GARP gene from macronuclear DNA molecule is 460 bp and shows (C₄A₄)₄ telomere structure. One of the three cysteines is encoded by the opal codon TGA(88-90). The deduced protein is a 112-amino acid(aa) with a molecular mass of 12.96 kDa, isoelectric point(pI) 3.82. Micronuclear version of the GARP gene contains two internal eliminated sequences(IES), IES1 and IES2. IES1 is 41 bp long and is flanked by 5'-GA-3 'direct repeats. IES2 is 41 bp and is flanked by 5'-TA--3' direct repeats. RT-PCR indicate that the GARP gene is transcriptional activity.To express the GARP gene in E.coli, the TGA were successfully mutated to TGT by site direct PCR mugagenesis. Recombinant expression plasmid pRSETc-His₆-GARP and pRSETc-GARP was constructed,respectively. His₆-GARP fusion protein and GARP protein were expressed in E. coli B121(DE3) by IPTG induction. Analysis of Western blotting with anti-His antibodies showed fusion protein is 26 KD. His6-GARP protein was purified by chromatography. The purity of His6-GARP protein reached 98 percent. Mouses were immunolized by purified His6-GARP protein. The potency of anti- His6-GARP polyclonal antibody was detected by indirect ELISA assay. The potency of the antibody was as high as 1 : 10,0000. The total protein from Euplotes octocarinatus was analyzed by Western blotting, the protein existed as dimeric and tetra-polymer in Euplotes octocarinatus .His₆-GARP protein can bind Cu²⁺, but GARP can not bind with Cu²⁺. It was also made sure by the ultraviolet spectra of Cu²⁺ titrating GARP. The results from the fluorescence spectra and ultraviolet spectra showed that His₆ tag affect interaction assay between GARP protein and Cu²⁺.