End JoiningMediated Gene Expression in Mammalian

Authors: Mikiko Nakamura Ayako Suzuki Junko Akada Keisuke Tomiyoshi Hisashi Hoshida Rinji Akada

Publish Date: 2015/09/08

Volume: 57, Issue: 11-12, Pages: 1018-1029

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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 proteincoding 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 Cterminal SerLysLeu 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 Cterminal proteincoding sequences and suggest that direct gene expression with PCRamplified DNA is useful for highthroughput protein analysis in mammalian cellsWe 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 TargetDriven RD JST Japan and the YU “PumpPriming Program” for fostering research activitiesMN 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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