Cloning and expression of the gene encoding putative DEAD box helicase from Streptomyces coelicolor in homologous and heterologous host

Helikaze su esencijalni proteini koji sudjeluju u metabolizmu nukleinskih kiselina. U ovom radu istraživan je slijed nukleotida koji kodira za potencijalnu RNA (DEAD) helikazu (GI u NCBI: 6689196; oznaka gena u StrepDB: SCO2863) iz bakterije Streptomyces coelicolor A3(2). Iako je u genomu ove bakterije pronađeno pet potencijalnih gena koji kodiraju za RNA helikaze, oni do sada nisu izučavani. Odabrani gen kloniran je u dva dijela, nakon čega su DNA fragmenti spojeni u jedinstvenu sekvencu provjerenu sekvenciranjem. Cjeloviti slijed nukleotida ugrađen je u ekspresijske vektore za biosintezu helikaze u homolognom (S. coelicolor) i heterolognom sustavu (Escherichia coli). Razina ekpresije gena ispitana je pomoću SDS–PAGE elektroforeze. metodom Western blotting pokazano je kako se u različitim sojevima bakterije E. coli protein nalazi u netopivoj frakciji. Posebnim genetičkim konstruktom transformirani su protoplasti bakterije S. coelicolor, nakon čega je prisustvo proteina utvrđeno u staničnom lizatu metodom Western blotting.Helicases are essential enzymes that participate in nucleic acid metabolism. In this thesis we explored nucleotide sequence encoding a putative RNA (DEAD box) helicase (GI in NCBI: 6689196; gene annotation in StrepDB: SCO2863) from Streptomyces coelicolor A3(2). In the genome of this bacterium five potential genes encoding for RNA helicases are found, but none of these had been studied. In separate PCRs two fragments of the selected gene were cloned following the ligation of fragments into complete nucleotide sequence which was verified by sequencing. A construct containing the complete nucleotide sequence was cloned into expression vectors for helicase biosynthesis in homologous (S. coelicolor) and heterologous (Escherichia coli) hosts. The level of gene expression was studied by SDS–PAGE electrophoresis. Analysis revealed protein presence in the insoluble fraction of three different E. coli strains. Special genetic construct was used to transform protoplasts of S. coelicolor and DEAD box helicase was confirmed in the cell lysate by Western blotting method