Master of Science (MS)
Semester of Degree Completion
Michael A. Menze
In nature several organisms exhibit anhydrobiosis, the outstanding feature to survive in extreme desiccation by entering into a state of dormancy known as diapause. The cyst of the brine shrimp Artemia franciscana shows anhydrobiosis by entering into a diapause phase. Previous studies showed a correlation between anhydrobiosis and expression of highly hydrophilic polypeptides termed late embryogenesis abundant (LEA) proteins. However, the precise molecular mechanisms of LEA proteins are still unknown. The presence of multiple LEA proteins in Artemia suggests that some of them might work together. Here, I aimed to express different combination of two LEA proteins from Artemia franciscana in the Drosophila melanogaster cell lines Kc167 and S2R+ by using the multicistronic vector pAc5-STABLE2-Neo. Immunoblot confirmed concurrent expression of both mCherry-LEA3m and GFP-LEA6 proteins in the Kc167 cells transfected with LEA3m+LEA6 construct. However, in three other Kc167 clones, although Western blot verified expression of mCherry tagged LEA proteins transcribed at first position of the vector, GFP tagged LEA protein cloned at second position of the vector was not detected. Another assumption that consensus ribosome recognition sequence of Drosophila would improve expression of LEA proteins in Drosophila cells was supported by the images of fluorescence microscopy. The final goal, simultaneous expression of two LEA proteins without the fluorescent reporters, was partially successful as immunoblot identified only DDK tagged first LEA proteins but not 6X His tagged second LEA proteins. Nonetheless, our results showed that expression of two LEA proteins concurrently in the Drosophila cells is possible by using the multicistronic vector instead of conventional two vectors system.
Islam, Kazi Nazrul, "Expression of Artemia LEA Proteins in Drosophila melanogaster Cells Using Multicistronic Vector Constructs" (2016). Masters Theses. 2454.