Cardiovascular Research Institute, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 14 Medical Drive, Singapore 117599, Singapore.
Cardiovascular Research Institute, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 14 Medical Drive, Singapore 117599, Singapore.
J Immunol Methods. 2014 Jan 31;403(1-2):37-51. doi: 10.1016/j.jim.2013.11.015. Epub 2013 Nov 26.
This is a first report of recombinant production of human prepro-Urocortin 2 in Escherichia coli by N-terminal fusion with a triple His₆-SUMO-eXact tag and its subsequent use as an antigen for the production and screening of very high affinity monoclonal antibodies. The rationale for this combinatorial construct is that the His tag allows first step protein purification of insoluble and soluble proteins, the SUMO tag enhances protein expression level and solubility, while the eXact tag facilitates anion-triggered on-column cleavage of the triple tag to recover pure native proteins in a simple two-step protein purification procedure. Compared with an eXact fusion alone, the presence of the SUMO moiety enhanced overall expression levels by 4 to 10 fold but not the solubility of the highly basic prepro-Urocortin 2. Insoluble SUMO-eXact-preproUCN2 was purified in milligram quantities by denaturing IMAC and solubilized in native phosphate buffer by on-column refolding or step-wise dialysis. Only a small fraction of this solubilized protein was able to bind onto the eXact™ affinity column and cleaved by NaF treatment. To test whether binding and cleavage failure was due to improperly refolded SUMO-eXact-preproUCN2 or to the presence of N- and C-terminal sequences flanking the eXact moiety, we created a SUMO-eXact-thioredoxin construct which was overexpressed mainly in the soluble form. This protein bound to and was cleaved efficiently on the eXact™ column to yield native thioredoxin. Solubilized SUMO-eXact-preproUCN2 was used successfully to generate two high affinity mouse monoclonal antibodies (KD~10⁻¹⁰ and 10⁻¹¹ M) specific to the pro-region of Urocortin 2.
这是首次在大肠杆菌中通过 N 端融合三聚组氨酸-SUMO-eXact 标签重组生产人前尿皮质素 2 的报告，并将其随后用作产生和筛选高亲和力单克隆抗体的抗原。这种组合构建的原理是，His 标签允许对不溶性和可溶性蛋白进行第一步蛋白纯化，SUMO 标签提高蛋白表达水平和溶解度，而 eXact 标签则有利于阴离子触发的在柱切割三标签，以在简单的两步蛋白纯化过程中回收纯天然蛋白。与单独的 eXact 融合相比，SUMO 部分的存在将整体表达水平提高了 4 到 10 倍，但对高度碱性前尿皮质素 2 的溶解度没有影响。不溶性 SUMO-eXact-preproUCN2 通过变性 IMAC 以毫克级纯化，并通过在柱复性或逐步透析在天然磷酸盐缓冲液中溶解。只有一小部分这种可溶蛋白能够结合到 eXact™亲和柱上，并通过 NaF 处理切割。为了测试结合和切割失败是否是由于 SUMO-eXact-preproUCN2 不正确折叠或由于 eXact 部分侧翼的 N 和 C 末端序列的存在，我们创建了一个主要以可溶性形式表达的 SUMO-eXact-硫氧还蛋白构建体。该蛋白有效地结合并在 eXact™柱上切割，得到天然硫氧还蛋白。可溶的 SUMO-eXact-preproUCN2 成功用于产生两种针对 Urocortin 2 前体的高亲和力小鼠单克隆抗体（KD~10⁻¹⁰ 和 10⁻¹¹ M）。
