State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East China University of Science and Technology, Shanghai, China.
PLoS One. 2012;7(9):e45891. doi: 10.1371/journal.pone.0045891. Epub 2012 Sep 27.
Protein refolding is an important process to recover active recombinant proteins from inclusion bodies. Refolding by simple dilution, dialysis and on-column refolding methods are the most common techniques reported in the literature. However, the refolding process is time-consuming and laborious due to the variability of the behavior of each protein and requires a great deal of trial-and-error to achieve success. Hence, there is a need for automation to make the whole process as convenient as possible. In this study, we invented an automatic apparatus that integrated three refolding techniques: varying dilution, dialysis and on-column refolding. We demonstrated the effectiveness of this technology by varying the flow rates of the dilution buffer into the denatured protein and testing different refolding methods. We carried out different refolding methods on this apparatus: a combination of dilution and dialysis for human stromal cell-derived factor 1 (SDF-1/CXCL12) and thioredoxin fused-human artemin protein (Trx-ARTN); dilution refolding for thioredoxin fused-human insulin-like growth factor I protein (Trx-IGF1) and enhanced fluorescent protein (EGFP); and on-column refolding for bovine serum albumin (BSA). The protein refolding processes of these five proteins were preliminarily optimized using the slowly descending denaturants (or additives) method. Using this strategy of decreasing denaturants concentration, the efficiency of protein refolding was found to produce higher quantities of native protein. The standard refolding apparatus configuration can support different operations for different applications; it is not limited to simple dilution, dialysis and on-column refolding techniques. Refolding by slowly decreasing denaturants concentration, followed by concentration or purification on-column, may be a useful strategy for rapid and efficient recovery of active proteins from inclusion bodies. An automatic refolding apparatus employing this flexible strategy may provide a powerful tool for preparative scale protein production.
蛋白质复性是从包涵体中回收有活性的重组蛋白的重要过程。通过简单稀释、透析和柱上复性方法进行复性是文献中报道的最常见技术。然而，由于每种蛋白质的行为变化多端，复性过程既耗时又费力，需要大量的反复试验才能成功。因此，需要自动化来使整个过程尽可能方便。在这项研究中，我们发明了一种自动装置，集成了三种复性技术：稀释、透析和柱上复性。我们通过改变变性蛋白中稀释缓冲液的流速并测试不同的复性方法来证明该技术的有效性。我们在该装置上进行了不同的复性方法：人基质细胞衍生因子 1（SDF-1/CXCL12）和硫氧还蛋白融合人 artemin 蛋白（Trx-ARTN）的稀释-透析组合；硫氧还蛋白融合人胰岛素样生长因子 I 蛋白（Trx-IGF1）和增强型荧光蛋白（EGFP）的稀释复性；牛血清白蛋白（BSA）的柱上复性。使用逐渐降低变性剂（或添加剂）的方法初步优化了这五种蛋白质的复性过程。使用这种降低变性剂浓度的策略，发现蛋白质复性的效率产生了更高数量的天然蛋白质。标准的复性装置配置可以支持不同应用的不同操作；它不仅限于简单的稀释、透析和柱上复性技术。通过逐渐降低变性剂浓度，然后在柱上进行浓缩或纯化进行复性可能是从包涵体中快速高效回收有活性蛋白质的一种有用策略。采用这种灵活策略的自动复性装置可为制备规模的蛋白质生产提供强大的工具。
