Möller Christina, Rimkus Niklas, Skala Ferdinand F O, Merouze Maëlle, Böttcher Dominique, Dörr Mark, Bornscheuer Uwe T
Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, University of Greifswald, Greifswald, Germany.
Appl Microbiol Biotechnol. 2024 Dec 16;108(1):536. doi: 10.1007/s00253-024-13374-1.
Cysteine cathepsins such as cathepsin B and L play an important role in numerous diseases like acute pancreatitis or SARS-CoV-2 and therefore have high potential for the development of new therapeutics. To be able to screen for potent and selective inhibitors sufficient amounts of protein are required. Here, we present an easy and efficient protocol for the recombinant expression of soluble and active murine cathepsin B and L. For this, we used the strain E. coli SHuffle® T7 Express which is capable of forming disulfide bridges in the cytoplasm. The enzymes were purified by immobilized nickel ion-affinity chromatography. Using different constructs and media, expression levels were significantly improved and expression yields of 80 ± 2 mg L for procathepsin B, which is 16-fold better than previously reported expression yields for procathepsin B, and 37 ± 2 mg L for procathepsin L, were achieved. After activation with dithiothreitol at slightly acidic pH, in vitro kinetic parameters of both cathepsins were determined using the commonly used synthetic substrates Arg-Arg-AMC or Phe-Arg-AMC. Moreover, to investigate the impact of the short C-terminal propeptide of procathepsin B, it was deleted by site-directed mutagenesis, the shortened target protein was expressed and purified, activated in vitro, and its activity was similar to the variant bearing this C-terminal propeptide. KEY POINTS: • Recombinant gene expression of cathepsin B and L in E. coli SHuffle® T7 Express • Soluble cathepsin expression with high expression yields • Investigation of the short C-terminal propeptide of cathepsin B.
半胱氨酸组织蛋白酶，如组织蛋白酶B和L，在许多疾病（如急性胰腺炎或新型冠状病毒肺炎）中发挥着重要作用，因此在开发新疗法方面具有很高的潜力。为了能够筛选出有效的选择性抑制剂，需要足够量的蛋白质。在此，我们提出了一种简便高效的方案，用于重组表达可溶性且有活性的小鼠组织蛋白酶B和L。为此，我们使用了大肠杆菌SHuffle® T7 Express菌株，该菌株能够在细胞质中形成二硫键。通过固定化镍离子亲和层析法对酶进行纯化。使用不同的构建体和培养基，表达水平得到了显著提高，组织蛋白酶B原的表达产量达到了80±2 mg/L，比先前报道的组织蛋白酶B原的表达产量高16倍，组织蛋白酶L原的表达产量达到了37±2 mg/L。在微酸性pH条件下用二硫苏糖醇激活后，使用常用的合成底物Arg-Arg-AMC或Phe-Arg-AMC测定了两种组织蛋白酶的体外动力学参数。此外，为了研究组织蛋白酶B原短C末端前肽的影响，通过定点诱变将其删除，表达并纯化了缩短的目标蛋白，在体外进行激活，其活性与带有该C末端前肽的变体相似。要点：• 在大肠杆菌SHuffle® T7 Express中重组表达组织蛋白酶B和L • 以高表达产量实现可溶性组织蛋白酶表达 • 研究组织蛋白酶B的短C末端前肽
