Analysis of the funcional domains of the heavy chain of kininogens by using the 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灭活半胱氨酸蛋白酶，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包含两个区域作为半胱氨酸蛋白酶反应位点的潜在候选者：Gln-Val-Val-Ala-Gly（QVVAG）位于结构域N-末端的三分之二处，Glycine（Gly）位于GVVAG区上游44个氨基酸残基处。我们已经尝试在真核细胞如BHK和CHO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，尽管具有结构域2或3缺失的野生型低分子量激肽原的量在Bacureo病毒系统细胞和原核细胞如大肠杆菌中是显著的。BHK<less than or equal>细胞经地塞米松诱导后表达量为50 ng/ml。下一步，我们利用低分子量激肽原的定点cDNA，尝试在杆状病毒系统和大肠杆菌中表达野生型和其他缺失和/或取代蛋白

High and low molecular weight kininogens strongly inactivate cysteine proteases such as cathepsin B,H and L,calpain 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 region as potential candidates for the reactive site toward cysteine proteases : Gln-Val-Val-Ala-Gly (QVVAG) located at two-thirds of the distance from the N-terminus of the domains and Glycine (Gly) located at 44 amino acid residues upstream from the GVVAG region.In this project, by using a cDNA coding for human low molecular weight kininogen, we had attempted to express the wild type protein and proteins with deletions of domain 2 and 3, and with deletions or substitutions of the QVVAG and Gly regions, in the eukaryotic cells such as BHK and CHO,in Bacureo virus system cells and in the prokaryotic cells such as E.coli.Although the amounts of the wild type of low molecular weight kininogen with deletion of domains 2 or 3 (<less than or equal>50ng/ml) expressed by the induction with dexamethsone in BHK cells were very low. As the next steps, using site-directed cDNA for low molecular weight kininogen, we have been attemptting to express the wild type and other proteins with deletion and/or substitution in Bacuro virus system and in E