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Characterization of guanine nucleotide exchange activity of DH domain of human FGD2.
Protein Expression and Purification ( IF 1.4 ) Pub Date : 2020-07-16 , DOI: 10.1016/j.pep.2020.105693
Junlan Chuan 1 , Shiyu He 2 , Tian Xie 3 , Ganggang Wang 3 , Zhenglin Yang 4
Affiliation  

FGD2, a member of FGD family, contains a Dbl homology domain (DH) and two pleckstrin homology domains segregated by a FYVE domain. The DH domain has been deduced to be responsible for guanine nucleotide exchange of CDC42 to activate downstream factors. Our aim was to build a prokaryotic expression system for the DH domain and to examine its guanine nucleotide exchange activity toward CDC42 in vitro. A recombinant vector, which was successfully constructed based on pGEX-6P-1, was employed to express the DH domain of human FGD2 (FGD2-DH) in E. coli BL21 (DE3). Purified FGD2-DH behaved as a homogeneous monomer with an estimated molecular weight that corresponded to the theoretical molecular weight and was predicted to be an α-helix protein by circular dichroism spectroscopy. FGD2-DH displayed weak guanine nucleotide exchange activity in vitro and very weak interactions with CDC42 following glutaraldehyde cross-linking.



中文翻译:

人FGD2 DH结构域的鸟嘌呤核苷酸交换活性的表征。

FGD2的成员FGD2包含一个Dbl同源域(DH)和两个由FYVE域分隔的pleckstrin同源域。推测DH结构域负责CDC42的鸟嘌呤核苷酸交换以激活下游因子。我们的目的是建立一个DH域的原核表达系统,并在体外研究其对CDC42的鸟嘌呤核苷酸交换活性。基于pGEX-6P-1成功构建的重组载体用于在大肠杆菌中表达人FGD2的DH结构域(FGD2-DH)BL21(DE3)。纯化的FGD2-DH表现为均质单体,其估计分子量与理论分子量相对应,并且通过圆二色谱法预测为α-螺旋蛋白。FGD2-DH体外显示弱的鸟嘌呤核苷酸交换活性戊二醛交联后与CDC42的相互作用非常弱。

更新日期:2020-08-22
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