Wood D W, Derbyshire V, Wu W, Chartrain M, Belfort M, Belfort G
Howard P. Isermann Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
Biotechnol Prog. 2000 Nov-Dec;16(6):1055-63. doi: 10.1021/bp0000858.
To reduce the number of recovery steps during downstream processing and to overcome the limitations of present fusion-based affinity separations, a controllable self-splicing protein element in the form of a mini-intein was used to optimize the recovery of proteins for both batch and flow purification strategies. The ability to recover purified proteins was demonstrated using a tripartite fusion consisting of a maltose binding domain, a truncated intein as a controllable linker molecule, and a protein of interest. To characterize expression level, solubility, cleavage rates, pH and temperature controllability, and protein activity, recombinant human acidic fibroblast growth factor (aFGF) was used as a model protein. A simple mass transport model, based on cleavage reaction-limited mass transfer and constant dispersion, was successfully used to predict product concentration and peak shape in relation to critical process parameters (with no fitting parameters). Insight into the nature of the cleavage reaction and its regulation was obtained via temperature- and pH-dependent kinetic data.
为减少下游加工过程中的回收步骤数量，并克服当前基于融合的亲和分离的局限性，一种以小型内含肽形式存在的可控自我剪接蛋白元件被用于优化蛋白质回收，适用于分批和连续纯化策略。通过由麦芽糖结合结构域、作为可控连接分子的截短内含肽以及目标蛋白组成的三方融合体，展示了回收纯化蛋白质的能力。为了表征表达水平、溶解度、切割速率、pH和温度可控性以及蛋白质活性，重组人酸性成纤维细胞生长因子（aFGF）被用作模型蛋白。一个基于切割反应限制传质和恒定扩散的简单传质模型成功用于预测与关键工艺参数相关的产物浓度和峰形（无拟合参数）。通过温度和pH依赖性动力学数据，深入了解了切割反应的性质及其调控。
