Single-chain variable fragments (scFvs) have gained increased attention among researchers in both academic and industrial fields owing to simple production in E. coli. The E. coli periplasm has been the site of choice for the expression of scFv molecules due to its oxidizing milieu facilitating correctly formation of disulfide bonds. Hence, the recovery of high-yield and biologically active species from the periplasmic space is a critical step at beginning of downstream processing. TES (Tris/EDTA/Sucrose) as a simple and efficient extraction method has been frequently used but under varied extraction conditions, over literature. This study, for the first time, aimed to interrogate the effects of four independent variables (i.e., Tris–HCl concentration, buffer’s pH, EDTA concentration, and incubation time) and their potential interactions on the functional extraction yield of an scFv antibody from the periplasmic space of E. coli. The results indicated that the Tris–HCl concentration and pH are the most significant variables in the TES method and displayed a positive effect at their lower values on the functional extraction yield. Besides, the statistical analysis revealed 4 significant interactions between different variables. Here is the first report on the successful application of a design of experiment based on a central composite design to establish a generic and optimal TES extraction condition. Accordingly, an optimal condition for TES extraction of scFv molecules from the periplasm of HB2151 at the exponential phase was developed as follows: 50 mM Tris–HCl at pH 7.2, 0.53 mM EDTA, and an incubation time of 60 min.

中文翻译：

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优化Tris / EDTA /蔗糖（TES）周质提取以回收功能性scFv抗体。

由于在大肠杆菌中的简单生产，单链可变片段（scFv）在学术和工业领域的研究人员中越来越受到关注。该大肠杆菌周质已成为scFv分子表达的首选位点，因为它的氧化环境有助于正确形成二硫键。因此，从周质空间中回收高产和具有生物活性的物种是开始下游加工的关键步骤。TES（Tris / EDTA /蔗糖）作为一种简单有效的提取方法，在文献中经常被使用，但是在不同的提取条件下。这项研究首次旨在探讨四个独立变量（即Tris–HCl浓度，缓冲液的pH，EDTA浓度和孵育时间）的影响以及它们对scFv抗体功能提取率的潜在相互作用。大肠杆菌周质空间。结果表明，在TES方法中，Tris-HCl的浓度和pH值是最重要的变量，并且在较低的值下对功能提取率产生积极影响。此外，统计分析还显示了不同变量之间的4个显着相互作用。这是关于成功应用基于中央复合设计的实验设计建立通用和最佳TES提取条件的第一份报告。因此，在指数期从HB2151的周质中用TES提取scFv分子的最佳条件如下：pH 7.2的50 mM Tris-HCl，EDTA 0.53 mM的孵育时间为60分钟。