title: An alternative method of enhancing the expression level of heterologous protein in Escherichia coli.
abstract: Though numerous strategy options are available for achieving high expression levels of genes in Escherichia coli, not every gene can be efficiently expressed in this organism. By investigating the relationship between the mRNA secondary structure of translational initiation region (TIR) and gene expression in E.coli, we establish a simple method to design sequences of appropriate TIR (from -35 to +36) that meet a specific expression level as we need. Using this method, overexpression of native human humor necrosis factor α and extracellular domain of Her2/neu protein (aa 23-146) in E. coli were achieved. Differences in expression appeared was mainly related to the efficiency of translation initiation and the stability of mRNA secondary structure, because the intracellular mRNA levels analyzed by real-time RT-PCR were quite similar. Our approach can overcome the steric hindrance of translation startup, and therefore promote translation smoothly to acquire high expression of exogenous protein.
authors.affiliation: State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Yin
authors.affiliation: State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Tian
authors.affiliation: State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Lichen
authors.initials: L
authors.lastname: Bao
authors.affiliation: State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Dai
authors.affiliation: State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China. Electronic address: xdgao@cpu.edu.cn.
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China. Electronic address: wbyao@cpu.edu.cn.
authors.forename: Wenbing
authors.initials: W
authors.lastname: Yao
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D013816:Thermodynamics; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C508053:ERBB2 protein, human
keywords: Expression level; Translation efficiency; Translation initiation region; mRNA secondary structure; ΔG