Determination of the reactive site (s) of kininogens for cysteine proteases using technique of site-directed mutagenesis

高分子量和低分子量激肽原以非共价和非竞争性方式强烈地结合半胱氨酸蛋白酶，例如组织蛋白酶B、H和L，钙蛋白酶I和II，木瓜蛋白酶和无花果蛋白酶。据认为，这两种激肽原在半胱氨酸蛋白酶的调节中发挥重要的生理作用。在两种激肽原的共同重链上有两个抑制结构域。推测结构域2和3含有两个区域作为半胱氨酸蛋白酶反应位点的潜在候选物：Gln-Val-Val-Ala-Gly（QVVAG）位于结构域的N-末端的大约三分之二处，Gly位于QVVAG区上游的44个氨基酸残基处。我们已经尝试在真核细胞如BHK中表达野生型蛋白质和具有结构域2和3的缺失以及具有QVVAG和Gly区的缺失或取代的蛋白质，尽管野生型蛋白质和两种结构域2或3缺失的蛋白质的量BHK细胞经地塞米松诱导表达的蛋白质（<50 ng/ml）很低，我们从细胞中收集这些培养基（共约5L），通过Red-Sepharose和Mono Q柱层析部分纯化这三种蛋白质，最终获得约3 μ g每种蛋白质。现在，我们正试图分析它们的动力学参数，并确定半胱氨酸蛋白酶的激肽原的真实的反应位点。

High and low molecular weight kininogens strongly inactivate cysteine proteases such as cathepsin B, H and L, calpains I and II, papain, and ficin in noncovalent and noncompetitive manners. It is thought that both kininogens play important physiological roles in the regulation of cysteine proteases. There are two inhibitory domains on the common heavy chain of both kininogens. It is speculated that domains 2 and 3 contain two regions as potential candidates for the reactive site of cysteine proteases : Gln-Val-Val-Ala-Gly (QVVAG) located at approximately two-thirds of the distance from the N-terminus of the domains and Gly located at 44 amino acid residues upstream from the QVVAG region.In this project, using a cDNA coding human low molecular weight kininogen, we have attemed to express the wild type protein and proteins with deletions of domains 2 and 3, and with deletions or substitutions of the QVVAG and Gly regions, in eukaryotic cells such as BHK, COS and CHO.Although the amounts of the wild type protein and two proteins with deletions of domains 2 or 3 (<50ng/ml) expressed by the induction with dexamethasone in BHK cells were very low, we collected these mediums from the cells (totally about 5 L each), partially purified these three proteins by using Red-Sepharose and Mono Q column chromatographies, and finally obtained about 3 ug of each protein. Now, we are attempting to analyze their kinetic parameters, and determine the real reactive site (s) of kininogens for cysteine proteases.