End Joining-Mediated Gene Expression in Mammalian Cells Using PCR-Amplified DNA Constructs that Contain Terminator in Front of Promoter.
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.
Innovation Center, Yamaguchi University, Tokiwadai, Ube, 755-8611, Japan. nakamura@crc.yamaguchi-u.ac.jp.|Yamaguchi University Biomedical Engineering Center (YUBEC), Tokiwadai, Ube, 755-8611, Japan. nakamura@crc.yamaguchi-u.ac.jp.
Mikiko
M
Nakamura
Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, 755-8611, Japan.
Ayako
A
Suzuki
Innovation Center, Yamaguchi University, Tokiwadai, Ube, 755-8611, Japan.|Department of Environmental and Preventive Medicine, Faculty of Medicine, Oita University, Yufu, 879-5503, Japan.
Junko
J
Akada
Department of Applied Chemistry, Faculty of Engineering, Yamaguchi University, Tokiwadai, Ube, 755-8611, Japan.
Keisuke
K
Tomiyoshi
Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, 755-8611, Japan.|Yamaguchi University Biomedical Engineering Center (YUBEC), Tokiwadai, Ube, 755-8611, Japan.
Hisashi
H
Hoshida
Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, 755-8611, Japan. rinji@yamaguchi-u.ac.jp.|Yamaguchi University Biomedical Engineering Center (YUBEC), Tokiwadai, Ube, 755-8611, Japan. rinji@yamaguchi-u.ac.jp.
Rinji
R
Akada
D053938:DNA Fragmentation; D017931:DNA Primers; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006367:HeLa Cells; D006514:Hepatitis B Surface Antigens / Q000737:chemistry; D006801:Humans; D019913:Nuclear Localization Signals; D021141:Nucleic Acid Amplification Techniques; D009842:Oligopeptides; D020675:Peroxisomes / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D013728:Terminator Regions, Genetic; D014162:Transfection
D017931:DNA Primers; D006514:Hepatitis B Surface Antigens; D019913:Nuclear Localization Signals; D009842:Oligopeptides; C088449:seryl-lysyl-leucine
FLAG-tag; Gene expression; Mutation; Non-homologous end joining (NHEJ); Peroxisome localization signal sequence; Polymerase chain reaction (PCR); Transcriptional terminator; Transfection