Phytocystatins are a family of plant cysteine-protease inhibitors of great interest due to their biotechnological application in culture improvement. It was shown that their expression in plants increases resistance to herbivory by insects and improves tolerance to both biotic and abiotic stress factors. In this work, owing to the economical relevance of the source organism, a phytocystatin from hop (Humulus lupulus), Hop1, was produced by heterologous expression in E. coli Lemo21 (DE3) cultivated in auto-inducing ZYM-5052 medium and purified by immobilized metal ion affinity and size exclusion chromatography. Thermal denaturation assays by circular dichroism showed that Hop1 exhibited high melting temperatures ranging from 82 °C to 85 °C and high thermal stability at a wide pH range, with ΔG₂₅'s higher than 12 kcal/mol. At 20 °C and pH 7.6, the dimeric conformation of the protein is favored according to size exclusion chromatography and analytical ultracentrifugation data, although monomers and higher order oligomers could still be detected in a lesser extent. The crystal structure of Hop1 was solved in the space groups P 2 2₁ 2₁ and C 2 2 2₁ at resolutions of 1.80 Å and 1.68 Å, respectively. In both models, Hop1 is folded as a domain-swapped dimer where the first inhibitory loop undergoes a significant structural change and interacts with their equivalent from the other monomer forming a long antiparallel beta strand, leading to loss of inhibitory activity.

植物胱抑素是植物半胱氨酸蛋白酶抑制剂的一个重要家族，因为它们在培养改良中的生物技术应用。结果表明，它们在植物中的表达增加了昆虫对草食动物的抵抗力，并提高了对生物和非生物胁迫因素的耐受性。在这项工作中，由于源生物的经济相关性，通过在大肠杆菌中进行异源表达产生了来自蛇麻草（Hulus lupulus）Hop1的植物半胱氨酸蛋白酶抑制剂。Lemo21（DE3）在自诱导ZYM-5052培养基中培养，并通过固定化的金属离子亲和力和尺寸排阻色谱法纯化。通过圆二色性热变性分析表明，表现出HOP1高的熔化温度范围为82℃至85℃和高的热稳定性，在宽的pH范围内，ΔG ₂₅的高于12千卡/摩尔。在20°C和pH 7.6下，尽管尺寸较小的单体和较高级的低聚物仍可检测到，但根据尺寸排阻色谱和超离心分析数据，该蛋白的二聚体构象更为有利。Hop1的晶体结构在空间群P 2 2 ₁ 2 ₁和C 2 2 2 _1中被解析分辨率分别为1.80Å和1.68Å。在这两种模型中，Hop1都折叠为域交换的二聚体，其中第一个抑制环发生重大结构变化，并与它们的等效物相互作用，形成长的反平行β链的其他单体，导致抑制活性降低。