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首页> 外文会议>International symposium on medical and pharmaceutical biotechnology >Increased solubility of recombinant human nerve growth factor expressed in Escherichia coli AD494 (DE3)
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Increased solubility of recombinant human nerve growth factor expressed in Escherichia coli AD494 (DE3)

机译：在大肠杆菌AD494（DE3）中表达重组人神经生长因子的溶解度增加

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The coding sequence of 118-amino acid mature human nerve growth factor (NGF) was amplified from human placenta and cloned into pET28a, when E. coli BL21 (DE3) was used as the host strain, the expression level was ～20%, however, all of the recombinant NGF were insoluble judged by SDS-PAGE. In order to improve the solubility, we first replaced the kanamycin resistance gene in the pET28a-NGF expression vector with ampicillin resistance gene using a special DNA recombination system, then the ampicillin resistant pET28a-NGF was introduced into another host strain, E. coli AD494 (DE3) carrying a kanamycin resistance gene on the bacterial genome. Under the same growth condition, the NGF expression level in E. coli AD494 (DE3) was similar as in E. coli BL21 (DE3), but the soluble recombinant protein represented ～30% of the recombinant protein as shown by SDS-PAGE.
机译：118-氨基酸成熟人神经生长因子（NGF）的编码序列从人胎盘中扩增，并克隆到PET28a中，当使用大肠杆菌BL21（DE3）作为宿主菌株时，表达水平为约20％，但，所有重组NGF都是通过SDS-PAGE判断的不溶性。为了改善溶解度，首先使用特殊的DNA重组系统用氨苄青霉素抗性基因替换PET28A-NGF表达载体中的卡那霉素抗性基因，然后将氨苄青霉素抗性PET28A-NGF引入另一个宿主菌株，E.COLI AD494 （DE3）在细菌基因组上携带卡那霉素抗性基因。在同一生长条件下，大肠杆菌AD494（DE3）中的NGF表达水平与大肠杆菌BL21（DE3）相似，但可溶性重组蛋白代表〜30％的重组蛋白，如SDS-PAGE所示。

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