Wang Zhongjie, Böttcher Dominique, Bornscheuer Uwe T, Müller Christian
Zoological Institute and Museum, Department of Animal Physiology, University of Greifswald, 17489 Greifswald, Germany.
Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, University of Greifswald, 17489 Greifswald, Germany.
Methods Protoc. 2025 Aug 3;8(4):89. doi: 10.3390/mps8040089.
The expression of recombinant proteins in heterologous hosts is a common strategy to obtain larger quantities of the "protein of interest" (POI) for scientific, therapeutic or commercial purposes. However, the experimental success of such an approach critically depends on the choice of an appropriate host system to obtain biologically active forms of the POI. The correct folding of the molecule, mediated by disulfide bond formation, is one of the most critical steps in that process. Here we describe the recombinant expression of hirudin, a leech-derived anticoagulant and thrombin inhibitor, in the yeast (formerly known and mentioned throughout this publication as ) and in two different strains of , one of them being especially designed for improved disulfide bond formation through expression of a protein disulfide isomerase. Cultivation of the heterologous hosts and expression of hirudin were performed at different temperatures, ranging from 22 to 42 °C for the bacterial strains and from 20 to 30 °C for the yeast strain, respectively. The thrombin-inhibitory potencies of all hirudin preparations were determined using the thrombin time coagulation assay. To our surprise, the hirudin preparations of were considerably less potent as thrombin inhibitors than the respective preparations of both strains, indicating that a eukaryotic background is not per se a better choice for the expression of a biologically active eukaryotic protein. The hirudin preparations of both strains exhibited comparable high thrombin-inhibitory potencies when the strains were cultivated at their respective optimal temperatures, whereas lower or higher cultivation temperatures reduced the inhibitory potencies.
在异源宿主中表达重组蛋白是一种常见策略，用于出于科学、治疗或商业目的获取大量“目标蛋白”（POI）。然而，这种方法的实验成功与否关键取决于选择合适的宿主系统以获得具有生物活性形式的POI。由二硫键形成介导的分子正确折叠是该过程中最关键的步骤之一。在此，我们描述了水蛭来源的抗凝剂和凝血酶抑制剂水蛭素在酵母（在本出版物中始终称为）以及两种不同的菌株中的重组表达，其中一种菌株经过特殊设计，通过表达一种蛋白二硫键异构酶来改善二硫键的形成。分别在不同温度下培养异源宿主并表达水蛭素，细菌菌株的培养温度范围为22至42°C，酵母菌株的培养温度范围为20至30°C。使用凝血酶时间凝固试验测定所有水蛭素制剂的凝血酶抑制效力。令我们惊讶的是，菌株的水蛭素制剂作为凝血酶抑制剂的效力明显低于两种菌株各自的制剂，这表明真核背景本身并非表达具有生物活性的真核蛋白的更好选择。当两种菌株在各自的最佳温度下培养时，它们的水蛭素制剂均表现出相当高的凝血酶抑制效力，而较低或较高的培养温度会降低抑制效力。
