This Mouse Kininogen 1 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Kininogen 1 protein (Cat: 50474-M08H) from the overexpression lysate was verified.
A DNA sequence encoding the mouse KNG1 isoform 3 (NP_001095882.1) (Glu 21-Ser 480) was expressed, with a polyhistidine tag at the C-terminus and a signal peptide at the N-terminus.
The secreted recombinant mouse KNG1 consists of 471 amino acids and has a predicted molecular mass of 52.5 kDa. In SDS-PAGE under reducing conditions, the apparent molecular mass of rm KNG1 is approximately 70-80 kDa due to glycosylation.
Cell lysate was prepared by homogenization of the over-expressed cells in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.
Kininogen-1, also known as high molecular weight kininogen, Williams-Fitzgerald-Flaujeac factor, Alpha-2-thiol proteinase inhibitor, Fitzgerald factor, KNG1, and BDK, is a secreted protein that contains three cystatin domains. Kininogen-1 / KNG1 is a protein from the blood coagulation system as well as the kinin-kallikrein system. It is a protein that adsorbs to the surface of biomaterials that come in contact with blood. Kininogen-1 / KNG1 circulates throughout the blood and quickly adsorbs to the material surfaces. Kininogen-1 / KNG1 is one of the early participants of the intrinsic pathway of coagulation, together with Factor XII (Hageman factor) and prekallikrein. Kininogen-1 / KNG1 is one of the kininogens, a class of proteins. As with many other coagulation proteins, the protein was initially named after the patients in whom deficiency was first observed. When the clinical data were combined, it turned out that all patients had a deficiency of the same protein. Defects in KNG1 are the cause of high molecular weight kininogen deficiency (HMWK deficiency) which is an autosomal recessive coagulation defect. Patients with HWMK deficiency do not have a hemorrhagic tendency, but they exhibit abnormal surface-mediated activation of fibrinolysis.
Ohkubo I., et al.,(1984), Isolation of a human cDNA for alpha 2-thiol proteinase inhibitor and its identity with low molecular weight kininogen. Biochemistry 23:5691-5697.
Takagaki Y., et al., (1985), Cloning and sequence analysis of cDNAs for human high molecular weight and low molecular weight prekininogens. Primary structures of two human prekininogens.J. Biol. Chem. 260:8601-8609.
Ota T., et al.,(2004), Complete sequencing and characterization of 21,243 full-length human cDNAs.Nat. Genet. 36:40-45.