End Joining-Mediated Gene Expression in Mammalian Cells Using PCR-Amplified DNA Constructs that Contain Terminator in Front of Promoter

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• 作者：Mikiko Nakamura ; Ayako Suzuki ; Junko Akada ; Keisuke Tomiyoshi…
• 关键词：Gene expression ; Polymerase chain reaction (PCR) ; Transfection ; Mutation ; Non ; homologous end joining (NHEJ) ; Transcriptional terminator ; FLAG ; tag ; Peroxisome localization signal sequence
• 刊名：Molecular Biotechnology
• 出版年：2015
• 出版时间：December 2015
• 年：2015
• 卷：57
• 期：11-12
• 页码：1018-1029
• 全文大小：3,533 KB
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• 作者单位：Mikiko Nakamura (1) (4)
  Ayako Suzuki (2)
  Junko Akada (1) (5)
  Keisuke Tomiyoshi (3)
  Hisashi Hoshida (2) (4)
  Rinji Akada (2) (4)
  
  1. Innovation Center, Yamaguchi University, Tokiwadai, Ube, 755-8611, Japan
  4. Yamaguchi University Biomedical Engineering Center (YUBEC), Tokiwadai, Ube, 755-8611, Japan
  2. Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, 755-8611, Japan
  5. Department of Environmental and Preventive Medicine, Faculty of Medicine, Oita University, Yufu, 879-5503, Japan
  3. Department of Applied Chemistry, Faculty of Engineering, Yamaguchi University, Tokiwadai, Ube, 755-8611, Japan
  
• 刊物主题：Biotechnology; Biochemistry, general; Cell Biology; Protein Science; Biological Techniques; Human Genetics;
• 出版者：Springer US
• ISSN：1559-0305

文摘

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. Keywords Gene expression Polymerase chain reaction (PCR) Transfection Mutation Non-homologous end joining (NHEJ) Transcriptional terminator FLAG-tag Peroxisome localization signal sequence