Abstract
Background and Objectives: Endoproteases play a key role in cell physiology and also in incidence of diseases. Considering their role in the cell in one hand, and the number of the known ndoproteases in the other hand, they clearly imply the importance of the development of a new repeatable and easy strategy to identify cytoplasmic site specific endoproteases. The aim of this study was to develop a new strategy for identification of novel site-specific endoproteases that are localized to the cytoplasm.
To develop this system, two GAL4-VP16 and HSV.tk-Zeo recombinant fusion proteins have been exploited.
Materials and Methods: Molecular biology methods were used to construct plasmids. Constructed plasmids were amplified using competent bacterial cells and their functional activity was studied using HeLa cells. Different recombinant HeLa cells were developed using constructed vectors and their sensitivity to drugs was studied using MTT assay.
Results: HeLa cells were transfected with the pcDBHZm plasmid containing inducible HSV.tk-Zeo cassette and then selected with G418. The transfected cells were retransfected with pcG4VP16 plasmid, which was being constructed during this study. Sensitivity of the transfected cells to GCV was assayed in comparison to the untransfected (served as negative control) and pCC1 transfected HeLa cells (used as positive control). Data showed that, pcDBHZm transfected cells were 30-fold more resistant to GCV compared to pcDBHZM+pcG4VP16 cells. These results confirmed that the expression of HSV.tk-Zeo was under the control of GAL4-VP16 and in the presence of this protein, HSV.tk-Zeo was expressed and the cells became sensitive to GCV and could be killed in the presence of this drug (negative selection). Having confirmed the feasibility of the selection of the cells with GCV, another plasmid called pcG4VP16 was constructed. In this plasmid some restriction sites were created between GAL4 and VP16 coding sequences in order to incorporate oligonucleotide library or known sequences between GAL4 and VP16 fragments. Inclusion of oligonucleotide library or known endoprotease coding sequences allows us to use this system to identify novel site specific endoprotease or to investigate the distribution of putative endoprotease in different cell types.
Conclusion: Results confirmed the feasibility of the developed system for selection of the cells in which the expression of the HSV.tk-Zeo is placed under the control of GAL4-VP16 and in the presence of this protein, HSV.tk-Zeo cassette is expressed. Expression of HSV.tk-Zeo induces 30-fold higher GCV sensitivity cells resulting to cell death in the in the cells containing active GAL4-VP16 (negative selection). This provides possibility to select the cells containing inactive GAL4-VP16 (due to its proteolytic cleavage) from the cells containing active GAL4-VP16 (due to absence of proteolytic cleavage) in the presence of GCV.