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GST and FLAG with PEP-cDNA. PEP-cDNA was sub-cloned in pGEX6p2 prokaryotic expression vector in order to label this gene with GST to purify PEP protein for further biochemical analysis and identifying related proteins thereafter. FLAG-PEP recombinant DNA was produced and sub-cloned in
pUcD3 eukaryotic expression vector to express tagged-PEP protein for transient transfection analysis and identifying intracellular localization of PEP protein in future experiments. PEP-cDNA was amplified
in different PCR reactions using pEGFP-PEP vector and 2 sets of primers introducing specific restriction sites at the ends of PEP. PCR products with BamHI/SalI restriction sites were treated by restriction enzymes and inserted into the pGEX6p2, downstream of GST tag. PEP-cDNA containing
BamHI/ApaI restriction sites and FLAG gene (which amplified using pUcD3-FLAG-PEX3 vector) were used as templates in secondary PCR for amplifying FLAG-PEP recombinant DNA. FLAG-PEP fragment was treated by enzymatic digestion and inserted into the pUcD3 eukaryotic expression vector.
pGEX6p2-PEP and pUcD3-FLAG-PEP constructed vectors were transformed into the one shot TOP10 and JM105 bacterial competent cells, respectively. Positive colonies were selected for plasmid preparation. Results confirmed correct amplification of the expected products. PEP-cDNA in both PCR
reactions encompasses 630 bp. FLAG fragment containing designed sites was 77 bp and FLAG-PEP fragment was 700 bp. Sequencing of constructed vectors confirmed that PEP-cDNA was tagged appropriately and inserted free of mutation and in frame with GST and FLAG.