Production of stable anti-digoxin Fv in Escherichia coli.
Production of stable anti-digoxin Fv in Escherichia coli.
复制标题
在大肠杆菌中生产稳定的抗地高辛 Fv。
DOI：
10.1016/0161-5890(92)90060-b
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发表时间：
1992
影响因子：
3.6
通讯作者：
Ng,SC
中科院分区：
文献类型：
--
作者：
Anthony,J;Near,R;Wong,SL;Iida,E;Ernst,E;Wittekind,M;Haber,E;Ng,SC
我们已经建立了一个细菌表达-输出系统，并用它在大肠杆菌中表达了（14 mg l-1）抗地高辛抗体（26-10）的可变区片段（Fv）。表达输出质粒含有T7启动子和E.大肠杆菌信号序列ompA [Movvaet al.，J.Biol.Chem.255，27-29（1980）]和phoA [Inouye等人，149，434-439（1982）]融合到重链（VH）和轻链（VL）可变区序列以产生人工顺反子。使用该系统制备的26-10 Fv蛋白是可溶的，不像许多其他表达系统以包涵体的形式产生不溶性蛋白。将26-10个VHand VL蛋白在其成熟的N-末端切割并输出到细菌周质中，在那里它们可以容易地被提取并在哇巴因-琼脂糖凝胶上亲和纯化。26-10 Fv与地高辛结合的亲和力和特异性与整个26-10抗体相似（Fv的Ka为1.3 × 109 M −1，IgG的Ka为7 × 109 M −1）。26-10 Fv与其它Fv片段相比表现出显著的稳定性。26-10 Fv的链解离半衰期为48小时，而McPC 603 Fv的报告半衰期为1.5小时。我们提出的质子NMR谱的26-10 Fv作为初步证据，这种表达输出系统可以用来促进分析的溶液结构的26-10 Fv的NMR。
We have created a bacterial expression-export system and have used it to express (14 mg l−1) the variable region fragment (Fv) of an anti-digoxin antibody (26-10) inEscherichia coli. The expression-export plasmid contains a T7 promoter and theE. colisignal sequencesompA[Movvaet al., J. biol. Chem.255, 27–29 (1980)] andphoA[Inouyeet al., J. Bacterial.149, 434–439 (1982)] fused to heavy chain (VH) and light chain (VL) variable region sequences to generate an artificial cistron. The 26-10 Fv protein made using this system was soluble, unlike many other expression systems which produce insoluble proteins in the form of inclusion bodies. The 26-10 VHand VLproteins were cleaved at their mature N-termini and exported into the bacterial periplasm where they could be easily extracted and affinity purified on ouabain-Sepharose. 26-10 Fv bound to digoxin with similar affinity and specificity as the whole 26-10 antibody (Ka, for Fv, 1.3 × 109M−1,Kafor IgG, 7 × 109M−1). 26-10 Fv appears to be remarkably stable in comparison with other Fv fragments. The half-life for chain dissociation of 26-10 Fv was 48 hr compared to the reported 1.5 hr half-life of McPC603 Fv. We present the proton NMR spectra of the 26-10 Fv as preliminary evidence that this expression-export system can be used to facilitate the analysis of the solution structure of 26-10 Fv by NMR.