ABSTRACT
A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target-selection strategy combining ;low-hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation-independent cloning (LIC) and expression systems, employing custom LIC-PCR, Gateway and In-Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X-ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target-selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs.
Subject(s)
Bacillus anthracis/genetics , Proteomics/methods , Bacillus cereus/genetics , Bacterial Proteins , Cloning, Molecular , Computational Biology , Crystallization , Crystallography, X-Ray , DNA, Bacterial/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Vectors , Magnetic Resonance Spectroscopy , RNA, Transfer/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Robotics , Spores, Bacterial/genetics , SulfurtransferasesABSTRACT
Protein characterization plays a role in two key aspects of structural proteomics. The first is the quality assessment of the produced protein preparations. Obtaining well diffracting crystals is one of the major bottlenecks in the structure-determination pipeline. Often, this is caused by the poor quality of the protein preparation used for crystallization trials. Hence, it is essential to perform an extensive quality assessment of the protein preparations prior to crystallization and to use the results in the evaluation of the process. Here, a protein-production and crystallization strategy is proposed with threshold values for protein purity (95%) and monodispersity (85%) below which a further optimization of the protein-production process is strongly recommended. The second aspect is the determination of protein characteristics such as domains, oligomeric state, post-translational modifications and protein-protein and protein-ligand interactions. In this paper, applications and new developments of protein-characterization methods using MS, fluorescence spectroscopy, static light scattering, analytical ultracentrifugation and small-angle X-ray scattering within the EC Structural Proteomics in Europe contract are described. Examples of the application of the various methods are given.
Subject(s)
Proteins/metabolism , Proteomics/methods , Crystallization , Hydrolysis , Light , Mass Spectrometry , Microscopy, Fluorescence , Models, Molecular , Protein Conformation , Reverse Transcriptase Polymerase Chain Reaction , Scattering, Radiation , Trypsin , Ultracentrifugation , X-RaysSubject(s)
Bismuth/poisoning , Organometallic Compounds/poisoning , Salicylates/poisoning , Aged , Aged, 80 and over , Female , HumansABSTRACT
Against Medical Advice (AMA) patients present troubling legal liabilities for emergency medicine physicians. One subset of AMA patients, those who present for care but leave the emergency department prior to evaluation by a physician, represent a potential source of liability. A total of 291 consecutive "Left Without Being Seen" (LWBS) patients were retrospectively studied to see how many died, required admission, or returned for outpatient medical care within 7 days. Nearly half (44%) were found to seek repeat care at clinics or the Emergency Department. Only 5 of 291 (1.7%) required admission. There were no documented deaths. Because of the difficulty in reliably predicting which patients would choose to leave without being seen, an attempt was made to identify specific LWBS patients who would seek a return visit or require admission. With the exception of one specific age group (infants less than 12 months of age), or a chief complaint referable to the cardiovascular system, no presenting parameter of the LWBS patient, including sex, chief complaint, duration of chief complaint, wait in the emergency department, and vital signs, was significantly helpful in differentiating those patients who would later return or require admission.
