On-off switching of gene expression regulated with carbohydrate-lectin interaction.
On-off switching of gene expression regulated with carbohydrate-lectin interaction.
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DOI：
10.1021/bm050255a
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发表时间：
2005-09
影响因子：
6.2
通讯作者：
K. Matsuura;K. Hayashi;Kazukiyo Kobayashi
中科院分区：
文献类型：
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作者：
K. Matsuura;K. Hayashi;Kazukiyo Kobayashi
提出了一种利用糖类和凝集素之间的特异性分子识别来构建基因表达人工调控系统的新策略。重氮偶联法制备的质粒-乳糖偶联物(pActin-乳糖和pGFP-乳糖)保持了与T7RNA聚合酶的转录活性。凝胶位移分析表明，pActin-乳糖偶联物与半乳糖特异性凝集素Rca(120)具有很强的结合亲和力(K(A)=7.6×10(5)M(-1)/Lac-单位)。通过透射电子显微镜(TEM)和原子力显微镜(AFM)观察到这些络合物形成了亚微米级的聚集体。根据pActin的转录量和表达的GFP的荧光强度分别评价结合物的转录和表达活性。这些活性在RCA120浓度增加时被抑制，然后通过向RCA120复合物中加入乳糖、乳糖基神经酰胺脂质体和乳糖载体聚合物而恢复。凝胶位移分析和电子显微镜观察表明，在乳糖衍生物的存在下，复合物的聚集形式部分松弛，从而增加了T7RNA聚合酶的可及性，导致转录活性的恢复。
A novel strategy for artificial regulation system of gene expression applying the specific molecular recognition between carbohydrate and lectin is proposed. Plasmid-lactose conjugates (pActin-lactose and pGFP-lactose) prepared via diazocoupling maintained the transcription activity with T7 RNA polymerase. Gel-shift assay showed that the pActin-lactose conjugates were specifically complexed with galactose-specific lectin RCA(120) with a strong binding affinity (K(a) = 7.6 x 10(5) M(-1) per Lac-unit). The complexes were observed to form aggregates of sub-several micrometer size by means of transmission electron microscopy (TEM) and atomic force microscopy (AFM). The activities of transcription and expression of the conjugates were evaluated, respectively, on the basis of the amount of transcript of pActin and the fluorescent intensity of the expressed GFP. These activities were repressed in the presence of an increasing concentration of RCA120, and then recovered by adding lactose, lactosylceramide-containing liposomes, and lactose-carrying polymers to the conjugate-RCA120 complex. Gel-shift assay and TEM observation revealed that the aggregation form of the complex was relaxed partially in the presence of the lactose derivatives, which increased the accessibility of T7 RNA polymerase to result in the recovery of transcription activity.