Xia Z, Kam C M, Huang C, Powers J C, Mandle R J, Stevens R L, Lieberman J
Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115, USA.
Biochem Biophys Res Commun. 1998 Feb 13;243(2):384-9. doi: 10.1006/bbrc.1998.8102.
Granzyme B (GranB), a serine protease stored in the granules of cytotoxic T lymphocytes and natural killer cells, can initiate target cell apoptosis. To produce large amounts of purified active enzyme, recombinant murine granzyme B (rGranB) was expressed from baculovirus in insect cells. The expressed rGranB is secreted into the culture medium and can be readily purified to homogeneity by one-step affinity chromatography to yield 1.5 mg enzyme per liter insect cell medium. RGranB is recognized by a GranB-specific anti-peptide antibody and is active against synthetic substrate Boc-Ala-Ala-Asp-SBzl with kinetic constant (kcat/Km 45,000 M-1s-1) comparable to purified human GranB, RGranB processes the caspase pro-CPP32 into its enzymatically active form and induces DNA fragmentation in isolated nuclei in the presence of cytosolic factors. The ability to express enzymatically active rGranB using the baculovirus system will help elucidate the role of this granzyme in the immune response.
颗粒酶B（GranB）是一种储存在细胞毒性T淋巴细胞和自然杀伤细胞颗粒中的丝氨酸蛋白酶，可引发靶细胞凋亡。为了大量生产纯化的活性酶，重组鼠颗粒酶B（rGranB）在昆虫细胞中通过杆状病毒表达。表达的rGranB分泌到培养基中，可通过一步亲和层析轻松纯化至同质，每升昆虫细胞培养基可产生1.5毫克酶。rGranB可被GranB特异性抗肽抗体识别，对合成底物Boc-Ala-Ala-Asp-SBzl具有活性，其动力学常数（kcat/Km为45,000 M-1s-1）与纯化的人GranB相当。rGranB将半胱天冬酶原CPP32加工成其酶活性形式，并在存在胞质因子的情况下诱导分离细胞核中的DNA片段化。利用杆状病毒系统表达具有酶活性的rGranB的能力将有助于阐明这种颗粒酶在免疫反应中的作用。
