Optimisation of production of a domoic acid-binding scFv antibody fragment in Escherichia coli using molecular chaperones and functional immobilisation on a mesoporous silicate support
Optimisation of production of a domoic acid-binding scFv antibody fragment in Escherichia coli using molecular chaperones and functional immobilisation on a mesoporous silicate support
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DOI：
10.1016/j.pep.2006.08.009
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发表时间：
2007-03-01
影响因子：
1.6
通讯作者：
Wall, J. Gerard
中科院分区：
文献类型：
--
作者：
Hu, Xuejun;O'Hara, Liam;Wall, J. Gerard
软骨藻酸是一种强有力的神经毒素，可通过摄入受污染的贝类导致人类的健忘症贝类中毒。我们从大肠杆菌周质中制备并纯化了抗软骨藻酸单链抗体(ScFv)片段。共生产大肠杆菌Dna KJE分子伴侣后，功能蛋白的产量增加了100倍，但共生产GroESL导致了单链抗体的溶解性降低。肽-脯氨酰异构酶触发因子的共同产生导致了未加工的单链抗体在大肠杆菌细胞质中的积累。这是由于重组多肽在细胞质膜转位方面存在明显的瓶颈。共表达大肠杆菌二硫键异构酶dsbC通过延迟宿主细菌细胞的裂解而增加单链抗体的产量，尽管这一效应与分子伴侣的共同产生没有协同作用。同时，使用冷休克启动子来生产蛋白质会导致大量scFv多肽的积累，这些多肽主要以不溶的形式存在，不能被伴侣挽救。利用优化的金属亲和层析程序对ScFv进行纯化，将纯化的蛋白固定在介孔硅酸盐载体上时结合软骨藻酸。这项工作概述了应用分子伴侣/折叠催化剂筛选方法来提高抗体片段生产的潜在好处，用于传感器开发等应用。(C)2006 Elsevier Inc.保留所有权利。
Domoic acid is a potent neurotoxin that can lead to amnesic shellfish poisoning in humans through ingestion of contaminated shellfish. We have produced and purified an anti-domoic acid single-chain Fragment variable (scFv) antibody fragment from the Escherichia coli periplasm. Yields of functional protein were increased by up to 100-fold upon co-production of E. coli DnaKJE molecular chaperones but co-overproduction of GroESL led to a reduction in solubility of the scFv. Co-production of the peptidyl-prolyl isomerase trigger factor resulted in accumulation of unprocessed scFv in the E. coli cytoplasm. This was due to an apparent bottleneck in translocation of the cytoplasmic membrane by the recombinant polypeptide. Co-expression of the E. coli disulfide bond isomerase dsbC increased scFv yields by delaying lysis of the host bacterial cells though this effect was not synergistic with molecular chaperone co-production. Meanwhile, use of a cold-shock promoter for protein production led to accumulation of greater amounts of scFv polypeptide which was predominantly in insoluble form and could not be rescued by chaperones. Purification of the scFv was achieved using an optimised metal affinity chromatography procedure and the purified protein bound domoic acid when immobilised on a mesoporous silicate support. The work outlines the potential benefit of applying a molecular chaperone/folding catalyst screening approach to improve antibody fragment production for applications such as sensor development. (c) 2006 Elsevier Inc. All rights reserved.