The Expression, Purification And Bioactivity Assay Of The Pheromone 3 Of Euplotes Octocarinatus In Escherichia Coli

Conjugation is a sexual model of reproduction, a feature for ciliates. During conjugation, the complementary mating type cells unite pairwise undergo meiosis, exchange gametic nuclei and separate again. For Euplotes octocarinatus, the interaction between cells begins with mutual recognition, which is induced by mating type specific signal substances, the pheromones. However, the molecular mechanism of the process remains unknown.Pheromones are characterized as acidic, cysteinc-rich polypeptides with a molecular mass of about 11 kDa. In the study, several recombinant expression vectors were constructed in vitro by using the technique of gene engineering, making pheromone 3 be expressed under the control of different promoters and signal pcptides.To obtain the fusion pheromone in the E.coli cytoplasm, a series of recombinant vectors are constructed including pGEX-6Pl-G3, pRSetc-G3, pET32M-G3 and pMal-c2x-G3. Among them the engineering strain of E.coli BL21/pGEX-6Pl-G3 was induced with 1.0 mmol/L IPTG at 37 . The results of SDS-PAGE show a prominent band appears at the 37kDa, however, most of the fusion protein exits in the form of the inclusion bodies.To obtain the secret fusion protein in the culture medium, the engineering strain , E.coli BL21/pExSecl-G3, was constructed. The percent of the fusion protein in that of the bacterium bodies is 23.6%, and the produced ZZ:G3 fusion protein present a single band after purified by affinity IgG-sepharose.The systematic comparison and analysis of expression of pheromone 3 of Euplotes in ? coli have been performed in this study. Engineering bacteria, screened for efficient expression of pheromone 3, express the proteins in type of insoluble (inclusion) and soluble forms, respectively. The results of this research provide basal information for studying the structure and function of pheromone, for elucidating the process of recognition between the pheromone and receptor and the mechanism of mating of Euplotes.