Expression And Identification Of A Putative Treh Of Chlamydomonas Reinhardtii In E.coli

Trehalose is an inducible component in the organisms. It plays great role in protection and stabilization of biological structures such as cell membrane, protein and nucleic acid under varies stress condition and significantly enhance organism's resistance. The study of trehalose and its biological metabolism always attracts many interests. Recently trehalose has been detected in Chlamydomonas reinhardtii for the first time. In this paper, we cloned a putative trehalase gene (treh) cDNA fragment from C. reinhardtii and express this putative gene in E. coli. We proved that the purified recombinant protein do behaves as trehalose hydrolysis function in vitro. This result filled up the blank of trehalose metabolism study in C. reinhardtii.The main contents and results of this experiment are as follows:1. Extracted total RNA from C. reinhardtii and reverse transcripted it into cDNA. Used specific primers to amplify the putative treh gene from the cDNA with PCR method. Cloned the treh into vector pUC19 with restriction enzyme cutting sites of EcoRâ… and BamHâ… to construct the vector pUC-treh for sequencing of this gene.2. Bioinformatic analysis showed that Treh of C. reinhardtii is highly homologous to that of Oryza sativa, Arabidopsis thaliana and Glycine max.3. Constructed an expressing vector pET32a-treh for expression in E. coli.4. Used IPTG to sucessfully induce treh to express in E. coli. The recombinant Treh protein exists as inclusion bodies in E. coli.5. After denatured and refolded the inclusion bodies,the refolded protein showed low activity of trehalose hydrolysis maybe due to the exist of other proteins in inclusion bodies.6. Further purified the refolded protein through Ni+column according to the his-tag structure in vector pET32a. After secondary round of dialysis, the pure protein showed significant trehalose hydrolysis activity, 80.4U/mg.7. In this experiment, imidazole in the binding/washing and elution buffer of Ni+column system could totally inhibit Treh activity. While 0.05mM EDTA and 1mM Mg2+ did not effect enzyme activity.The results showed that the putative treh cloned from C. reinhardtii do behave as trehalose hydrolysis activity in vitro.