The immunostaining results were independently assessed by two pathologists

250 RPM for 48 hours in the presence of kanamycin and chloramphenicol. Protein Extraction and Purification Cell pellets were resuspended at 4uC in sample buffer supplemented with DNase, EDTA-free protease inhibitor cocktail and phenylmethylsulfonyl fluoride, and lysed by two passes through a cell disruptor at 30 Kpsi. Lysed cells were centrifuged at 18,000 RPM and 4uC for 45 minutes to remove cellular debris. The supernatant was applied twice to a pre-equilibrated nickel-nitrilotriacetic acid agarose resin to purify the hexahistidine -tagged target proteins. After the resin was washed with 40 column volumes of wash buffer to remove non-specific proteins, target proteins were eluted with 2 CV of elution buffer. Following the elution, the target protein was desalted into a low imidazole buffer to prevent protein precipitation during proteolytic cleavage of the fusion partner by TEV protease. To remove the fusion tag, the protein sample was filtered through a 0.45 mm filter and re-applied to reequilibrated nickel resin. The flow-through containing the target protein was concentrated to a volume of 7.5 ml and applied to an equilibrated gel filtration column. Protein fractions were collected and protein purity was determined on pre-cast SDS-PAGE. Fractions of highest purity were combined and concentrated to 7 mg/ml. Materials and Methods Plasmids and DNA Cloning DNA fragments encoding the N-terminal regions of different lengths from the LASV L protein were amplified by PCR with PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19645691 respective pairs of primers and cloned into the pMALC2X derivative plasmid pLou3 and the pEHISTEV plasmid, which includes a hexahistidine tag and a tobacco etch virus protease cleavage site at the N-terminus of the cloned genes. Based on the structure, a new plasmid construct was Protein Crystallization Protein crystallization was performed using the sitting-drop vapour diffusion technique. The purified N-terminal 200 residues of LASV L did not form crystals. However, crystallization was initiated after proteolysis of LASV L190 with subtilisin A in a Lassa LY3039478 chemical information Endonuclease Structure and Function 700:1 ratio for 6090 minutes on ice. Upon proteolytic treatment, the protein crystallized rapidly in various conditions with best crystals found in 100 mM Tris-HCl pH 89.5, 250 mM MgSO4 and PEG 10,000. Larger crystals grew after three days in 2 ml of protein and 2 ml of precipitant drops with 100 ml reservoir solution. Results Purification and Crystallization of the LASV L Endonuclease Domain DNAs encoding the first 200, 250, 300 and 500 amino acids of the LASV L polymerase were first cloned into the pLou3 plasmids and expressed in E. coli Rosetta cells as described in “Materials & Methods”. After multiple attempts using different induction conditions and media, the L200 containing the first 200 amino acid residues of the polymerase was the only construct that expressed well and was easy to purify. However, attempts to crystallize this protein failed. Similarly, L190 containing the first 190 amino acid residues of L was expressed and purified at a high level but again did not yield any crystals. A limited proteolysis screen revealed that, after subtilisin A treatment to cleave off some amino acids at the C-terminus of the L190 protein, crystals appeared in 100 mM Tris-HCl pH 9.0, 250 mM MgSO4 and 20% PEG 10,000 after 1 day of incubation. The final protein product for crystallization consists of residues 1173 based on the structure determined as described below. Data Collection P