Der p 1 is a major allergen from the house dust mite that belongs to the papain-like cysteine protease family. and its homolog Der f 1 LDN-57444 from genus feed on dander and small particles of shed skin which is commonly present in households. Some of their digestive enzymes are potent proteases that are abundant in the feces of dust mites and are highly allergenic. Der p 1 is usually a cysteine protease and a major allergen (3). Chronic exposure to Der p 1 occurs by inhalation and may lead to the production of IgE antibodies in susceptible atopic individuals. Der p 1 catalyzes the cleavage of the amide linkages in substrates like α1-antitrypsin the CD23 receptor on human B cells the IL-2 receptor (CD25) on human T cells and the Der p 1 pro-polypeptide sequence (4). Strong evidence suggests that Der p 1-related cleavage of these receptors contributes to its allergenicity (5 6 Structures of recombinant Der p 1 in both the proenzyme and mature forms were previously decided (7-9). The structure of natural Der f 1 which shares 81% sequence identity to Der p 1 was also decided (9). In addition structures of natural Der f 1 and natural Der p 1 in complex with the Fab fragment of the cross-reactive monoclonal antibody (mAb) 4C1 were also elucidated (10). Here we present the crystal structures of Der p 1 isolated from its natural source complexed with the Fab fragment of 5H8 (Der p 1-5H8) Der p 1 complexed with the Fab fragment of 10B9 (Der p 1-10B9) and the Fab fragment of mAb 10B9 alone. Both 10B9 and 5H8 are species specific whereas the 4C1 antibody is usually cross-reactive between Der p 1 from and Der f 1 from This enabled the Der p 1 epitopes for mAbs 10B9 5 and 4C1 to be compared with the corresponding surface on Der f 1 (9 10 It was discovered that the Der p 1 epitopes which bind 4C1 and 10B9 antibodies overlap and these two antibodies compete for the same binding site (11). The 5H8 antibody however binds to the epitope located on a different side of Der p 1 and does not compete with 4C1 or 10B9 for binding (11). The binding interfaces of Der p 1 with mAbs 4C1 5 and 10B9 with the binding interfaces of all currently known structures of complexes of proteins or peptides with monoclonal antibodies were also compared. Materials and Methods Production and Purification of Proteins Der p 1 was purified from mite culture as explained previously for Der f 1 (9 10 Briefly Der p 1 was purified from spent mite culture extract [100 g per 1 L of phosphate-buffered saline (PBS)] using affinity chromatography through a 4C1 mAb column. The mAb 5H8 and 10B9 were fragmented commercially by GenicBio Limited Shanghai (China) and Strategic BioSolutions (Newark DE) respectively. The fragmentation was performed using papain and the producing Fab were purified by Protein A affinity chromatography. The Fab from mAb 5H8 was further purified by gel filtration (Sephadex G-75). Both Der p 1-5H8 and Der p 1-10B9 complexes were prepared using the same protocol. In each case the allergen was mixed with the Fab fragment of antibody in a 1:1 molar ratio and incubated at 4 °C for 16 h for Der p 1-10B9 and 30 minutes for LDN-57444 Der p 1-5H8. After incubation the solution was concentrated using an Amicon Ultra concentrator (Millipore) with a LDN-57444 10 0 Da molecular mass cutoff and purified on a Superdex 200 column attached to an ?KTA FPLC system (GE Healthcare). LDN-57444 A solution composed of 10 mM Tris-HCl and 150 mM NaCl at pH 7.5 was utilized for gel filtration of both complexes. After gel filtration fractions made up of Der p 1-5H8 and Der p 1-10B9 were concentrated to about 5 mg/mL. The 10B9 Fab fragment utilized for Mouse monoclonal to Survivin crystallization of the antibody fragment alone was also purified LDN-57444 on a Superdex 200 using 10 mM Tris-HCl 50 mM NaCl LDN-57444 pH 7.5. Prior to crystallization the 10B9 Fab fragment was concentrated to 8 mg/mL. Crystallization Crystallization of Der p 1-10B9 Der p 1-5H8 and 10B9 was performed at 293 K. Crystals were produced using the hanging drop vapor diffusion method. The crystallization drops were a 1:1 mixture of the protein answer and the precipitant answer from your wells (100 mM MES 10 w/v PEG 6000 5 MPD at pH 7.0 for the Der p 1-5H8 complex 100 mM Na acetate 8 w/v PEG 4000 15 MPD at pH 4.5 for the Der p 1-10B9 complex and in the case of 10B9 100 mM sodium citrate 15 w/v PEG 6000 at pH 5.5 was used). Prior to data collection Der p 1-5H8 Der p 1-10B9 and 10B9 crystals were cryo-protected in LV CryoOil answer (MiTeGen Ithaca NY) a 1:1 mixture of Paratone-N and mineral oil or well answer respectively then immediately cooled in liquid.