Hohenblum Hubertus, Vorauer-Uhl Karola, Katinger Hermann, Mattanovich Diethard
Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
J Biotechnol. 2004 Apr 8;109(1-2):3-11. doi: 10.1016/j.jbiotec.2003.10.022.
The expression of recombinant trypsinogens from different mammalian origins in Escherichia coli typically leads to the formation of insoluble aggregates. This work describes the high level expression of human trypsinogen 1 in E. coli using the T7 expression system. Direct expression of trypsinogen was not possible, but the N-terminal fusion of the first 11 amino acids of the T7 protein 10 resulted in an expression level of 200 mg g(-1) bacterial dry mass. A refolding procedure was optimized, and a method using continuous feed of denatured product was developed. Thus the working concentration of trypsinogen could be raised four-fold, while the yield of active protein could be maintained at 20-35%. The refolded trypsinogen was converted to trypsin by autocatalytic activation, and the utility for the detachment of mammalian cells in culture was proven.
在大肠杆菌中表达来自不同哺乳动物来源的重组胰蛋白酶原通常会导致不溶性聚集体的形成。这项工作描述了使用T7表达系统在大肠杆菌中高效表达人胰蛋白酶原1。直接表达胰蛋白酶原是不可能的，但T7蛋白10的前11个氨基酸的N端融合导致表达水平达到200 mg g(-1)细菌干重。优化了重折叠程序，并开发了一种使用变性产物连续进料的方法。因此，胰蛋白酶原的工作浓度可以提高四倍，而活性蛋白的产量可以保持在20-35%。重折叠的胰蛋白酶原通过自催化激活转化为胰蛋白酶，并证明了其在培养中用于分离哺乳动物细胞的效用。
