title: High-level expression of LMW-GS and α-gliadin genes promoted by the expressed tag sequence of 5' end in Escherichia coli.
abstract: Wheat storage protein genes, especially low molecular weight glutenin subunit (LMW-GS) and gliadin genes are difficult to be expressed in Escherichiacoli, mainly due to the presence of highly repetitive sequences. In order to establish a high efficiency expression system for these genes, five different expression plasmids combining with 9 genes, viz. 6 LMW-GS and 3 α-gliadin genes isolated from common wheat and related species, were studied for heterologous expression in E. coli. In this study, when an expressed tag sequence encoding signal peptide, His-S or GST-tag was fused to the 5' end of LMW-GS or gliadin gene as the leading sequence, all recombination genes could be stably expressed at a high level. On the contrast, as expected, the inserted genes encoding mature protein failed without an expressed tag sequence. This result indicated that using expressed tag sequences as leading sequences could promote LMW-GS and gliadin genes to be well expressed in E. coli. Further transcriptional analysis by quantitative real-time PCR (qRT-PCR) showed transcription levels of recombination genes (e.g. GST-Glutenin, His-S-Glutenin and SP(∗)-His-Glutenin) were 4-fold to 33-fold higher than those of the LMW-GS genes, which suggested these expressed tag sequences might play an important role in stimulating transcription. The possible molecular mechanism under this phenomenon was discussed.
authors.affiliation: Key Laboratory of Genetics and Biotechnology, College of Life Science, Capital Normal University, Beijing, China.
authors.forename: Peihong
authors.initials: P
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Genetics and Biotechnology, College of Life Science, Capital Normal University, Beijing, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Wang
authors.affiliation: Key Laboratory of Genetics and Biotechnology, College of Life Science, Capital Normal University, Beijing, China.
authors.forename: Jiansheng
authors.initials: J
authors.lastname: Gao
authors.affiliation: Key Laboratory of Genetics and Biotechnology, College of Life Science, Capital Normal University, Beijing, China.
authors.forename: Xiaoran
authors.initials: X
authors.lastname: Zheng
authors.affiliation: Key Laboratory of Genetics and Biotechnology, College of Life Science, Capital Normal University, Beijing, China.
authors.forename: Jianing
authors.initials: J
authors.lastname: Feng
authors.affiliation: School of Veterinary & Life Sciences, Murdoch University and Australian Export Grains Innovation Centre, Perth, Australia.
authors.forename: Wujun
authors.initials: W
authors.lastname: Ma
authors.affiliation: Key Laboratory of Genetics and Biotechnology, College of Life Science, Capital Normal University, Beijing, China.
authors.forename: Yueming
authors.initials: Y
authors.lastname: Yan
authors.affiliation: Key Laboratory of Genetics and Biotechnology, College of Life Science, Capital Normal University, Beijing, China. Electronic address: lixiaohui@cnu.edu.cn.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Li
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D020224:Expressed Sequence Tags / Q000378:metabolism*; D005903:Gliadin / Q000032:analysis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins
chemical_list: D011994:Recombinant Proteins; D005903:Gliadin
keywords: Escherichia coli; Expressed tag sequence; Heterologous expression; Low molecular weight subunit; Repetitive sequence; α-Gliadin