Abstract
Mammalian gene expression constructs are generally prepared in a plasmid vector, in which a promoter and terminator are located upstream and downstream of a protein-coding sequence, respectively. In this study, we found that front terminator constructs—DNA constructs containing a terminator upstream of a promoter rather than downstream of a coding region—could sufficiently express proteins as a result of end joining of the introduced DNA fragment. By taking advantage of front terminator constructs, FLAG substitutions, and deletions were generated using mutagenesis primers to identify amino acids specifically recognized by commercial FLAG antibodies. A minimal epitope sequence for polyclonal FLAG antibody recognition was also identified. In addition, we analyzed the sequence of a C-terminal Ser-Lys-Leu peroxisome localization signal, and identified the key residues necessary for peroxisome targeting. Moreover, front terminator constructs of hepatitis B surface antigen were used for deletion analysis, leading to the identification of regions required for the particle formation. Collectively, these results indicate that front terminator constructs allow for easy manipulations of C-terminal protein-coding sequences, and suggest that direct gene expression with PCR-amplified DNA is useful for high-throughput protein analysis in mammalian cells.
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Abbreviations
- PCR:
-
Polymerase chain reaction
- SKL:
-
Serine-lysine-leucine
- HBsL:
-
Hepatitis B virus surface antigen large protein
- NHEJ:
-
Non-homologous end joining
- CMV:
-
Cytomegalovirus
- SV40 polyA:
-
Simian virus polyA terminator
- β-globin polyA:
-
Rabbit β-globin polyA terminator
- tRNA:
-
Transfer RNA
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Acknowledgments
We would like to thank Mariko Fujinaga, Sawako Kondo, and Yukie Misumi for their technical assistance. We are also grateful to Fujirebio Inc. for the kind gift of HBsL cDNA. This study was supported in part by JSPS KAKENHI (Grant No. 25660080), the Adaptable and Seamless Technology Transfer Program through Target-Driven R&D (JST, Japan), and the YU “Pump-Priming Program” for fostering research activities.
Author contributions
MN and RA designed the study and wrote the manuscript. MN and AS performed luciferase assays and microscopy. MN, JA, and KT performed Western blotting. MN, RA, and HH analyzed and interpreted the data. All authors approved the final version of the manuscript.
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Nakamura, M., Suzuki, A., Akada, J. et al. End Joining-Mediated Gene Expression in Mammalian Cells Using PCR-Amplified DNA Constructs that Contain Terminator in Front of Promoter. Mol Biotechnol 57, 1018–1029 (2015). https://doi.org/10.1007/s12033-015-9890-1
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DOI: https://doi.org/10.1007/s12033-015-9890-1