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首页> 外文期刊>Protein Expression and Purification >Optimized protocol for soluble prokaryotic expression, purification and structural analysis of human placenta specific-1(PLAC1)
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Optimized protocol for soluble prokaryotic expression, purification and structural analysis of human placenta specific-1(PLAC1)

机译：用于可溶性原核表达，纯化和人胎盘特异性纯化和结构分析的优化方案（PLAG1）

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摘要

Placenta specific-1 (PLACI) has been recently introduced as a small membrane-associated protein mainly involved in placental development. Expression of PLACI transcript has been documented in almost one hundred cancer cell lines standing for fourteen distinct cancer types. The presence of two disulfide bridges makes difficult to produce functional recombinant PLACI in soluble form with high yield. This limitation also complicates the structural studies of PLACI, which is important for prediction of its physiological roles. To address this issue, we employed an expression matrix consisting of two expression vectors, five different E. coli hosts and five solubilization conditions to optimize production of full and truncated forms of human PLAC1. The recombinant proteins were then characterized using an anti-PLAC1-specific antibody in Western blotting (WB) and enzyme linked immunosorbent assay (ELISA). Structure of full length protein was also investigated using circular dichroism (CD). We demonstrated the combination of Origami (TM) and pCold expression vector to yield substantial amount of soluble truncated PLACI without further need for solubilization step. Full length PLACI, however, expressed mostly as inclusion bodies with higher yield in Origami (TM) and Rosetta2. Among solubilization buffers examined, buffer containing Urea 2 M, pH 12 was found to be more effective. Recombinant proteins exhibited excellent reactivity as detected by ELISA and WB. The secondary structure of full length PLAC1 was considered by CD spectroscopy. Taken together, we introduced here a simple, affordable and efficient expression system for soluble PLAC1 production. (C) 2017 Elsevier Inc. All rights reserved.
机译：最近胎盘特定-1（PLAII）最近被引入主要涉及胎盘开发的小膜相关蛋白。 Placi转录物的表达已记录在几乎一百个癌细胞系中，该癌细胞含量在近一百个癌细胞系中被记载。两种二硫键的存在难以以高产率的可溶形式产生功能性重组抛光。这种限制也使PLAIN的结构研究复杂化，这对于预测其生理作用非常重要。为了解决这个问题，我们使用由两种表达载体，五种不同的大肠杆菌宿主和五个溶解条件组成的表达矩阵，以优化人类Plac1的完整和截短形式的生产。然后使用蛋白质印迹（WB）中的抗Plac1特异性抗体和酶联免疫吸附测定（ELISA）进行重组蛋白。还使用圆形二色性（CD）研究全长蛋白质的结构。我们证明了折纸（TM）和PCOLD表达载体的组合，以产生大量的可溶性截短的抛光，而无需进一步需要溶解步骤。然而，全长PLACI主要表达为折纸（TM）和Rosetta2中具有更高产量的包容体。在检查溶解缓冲剂中，发现含有尿素2m，pH 12的缓冲液更有效。重组蛋白表现出优异的ELISA和WB检测的反应性。 CD光谱法考虑全长PLAC1的二次结构。我们一起介绍了一种简单，实惠，高效的可溶性PLAC1生产的表达系统。 （c）2017年Elsevier Inc.保留所有权利。

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