Improved Purification of Human Granzyme A/B and Granulysin Using a Mammalian Expression System.
Improved Purification of Human Granzyme A/B and Granulysin Using a Mammalian Expression System.
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使用哺乳动物表达系统改善了人类颗粒A/B和颗粒素的纯化。
DOI：
10.3389/fimmu.2022.830290
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发表时间：
2022
影响因子：
7.3
通讯作者：
Hoft DF
中科院分区：
文献类型：
--
作者：
Rasi V;Hameed OA;Matthey P;Bera S;Grandgenett DP;Salentinig S;Walch M;Hoft DF
细胞毒性淋巴细胞在识别感染或肿瘤靶细胞后，释放胞浆溶细胞颗粒中包含的蛋白质。这些细胞毒性颗粒蛋白(即颗粒酶、颗粒溶素和穿孔素)是人体细胞免疫中的关键免疫介质。高纯度细胞毒蛋白的可获得性是了解其在免疫中的功能以及参与脓毒症和自身免疫的机制的基础。回收天然细胞毒蛋白的方法可能存在问题，导致：1)额外蛋白质的共同纯化，混淆了对功能的解释，以及2)高纯度蛋白质的低产率。单个细胞溶解成分的重组蛋白表达可以克服这些挑战。使用哺乳动物表达系统是最佳的翻译后修饰和避免内毒素污染的首选。其中一些蛋白质已被建议用于宿主导向的人类疗法(例如颗粒酶A)，或用于治疗全身感染或肿瘤，如颗粒溶素。我们报道了一种使用HEK293T细胞的新型表达系统，该系统可以经济高效地纯化高产量的人颗粒酶(颗粒酶A和颗粒酶B)和颗粒溶酶，其生物学活性比以往报道的要高。得到的蛋白质没有天然污染物，折叠正确，并保持酶活性。重要的是，这些改进还导致了第一次从哺乳动物系统中高产率地纯化具有生物活性的重组人颗粒溶素。该方法可以作为一种模板来纯化许多其他分泌的细胞蛋白，并可能导致人类医学的进步。
Cytotoxic lymphocytes release proteins contained within the cytoplasmic cytolytic granules after recognition of infected or tumor target cells. These cytotoxic granular proteins (namely granzymes, granulysin, and perforin) are key immunological mediators within human cellular immunity. The availability of highly purified cytotoxic proteins has been fundamental for understanding their function in immunity and mechanistic involvement in sepsis and autoimmunity. Methods for recovery of native cytotoxic proteins can be problematic leading to: 1) the co-purification of additional proteins, confounding interpretation of function, and 2) low yields of highly purified proteins. Recombinant protein expression of individual cytolytic components can overcome these challenges. The use of mammalian expression systems is preferred for optimal post-translational modifications and avoidance of endotoxin contamination. Some of these proteins have been proposed for host directed human therapies (e.g. - granzyme A), or treatment of systemic infections or tumors as in granulysin. We report here a novel expression system using HEK293T cells for cost-effective purification of high yields of human granzymes (granzyme A and granzyme B) and granulysin with enhanced biological activity than previous reports. The resulting proteins are free of native contaminants, fold correctly, and remain enzymatically active. Importantly, these improvements have also led to the first purification of biologically active recombinant human granulysin in high yields from a mammalian system. This method can be used as a template for purification of many other secreted cellular proteins and may lead to advances for human medicine.
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影响因子：
3.7
作者：
Hauptmann A;Podgoršek K;Kuzman D;Srčič S;Hoelzl G;Loerting T
通讯作者：
Loerting T
影响因子：
4.4
作者：
Ernst, WA;Thoma-Uszynski, S;Modlin, RL
通讯作者：
Modlin, RL
DOI：
10.1007/978-1-4939-6673-8_18
发表时间：
2017-01-01
期刊：
BACTERIAL PATHOGENESIS: METHODS AND PROTOCOLS
影响因子：
--
作者：
Leon, Diego Lopez;Fellay, Isabelle;Walch, Michael
通讯作者：
Walch, Michael
影响因子：
11.4
作者：
Martin, SJ;AmaranteMendes, GP;Green, DR
通讯作者：
Green, DR
影响因子：
82.9
作者：
Dotiwala F;Mulik S;Polidoro RB;Ansara JA;Burleigh BA;Walch M;Gazzinelli RT;Lieberman J
通讯作者：
Lieberman J