ABSTRACT
The C-propeptide domains of the fibrillar procollagens, which are present throughout the Metazoa in the form of â¼90â kDa trimers, play crucial roles in both intracellular molecular assembly and extracellular formation of collagen fibrils. The first crystallization of a C-propeptide domain, that from human procollagen III, is described. Following transient expression in mammalian 293T cells of both the native protein and a selenomethionine derivative, two crystal forms of the homotrimer were obtained: an orthorhombic form (P2(1)2(1)2(1)) that diffracted to 1.7â Å resolution and a trigonal form (P321) that diffracted to 3.5â Å resolution. Characterization by MALDI-TOF mass spectrometry allowed the efficiency of selenomethionine incorporation to be determined.
Subject(s)
Procollagen/chemistry , Amino Acid Sequence , Crystallization , HEK293 Cells , Humans , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Procollagen/metabolism , Protein MultimerizationABSTRACT
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
This paper reviews the developments in high-throughput and nanolitre-scale protein crystallography technologies within the remit of workpackage 4 of the Structural Proteomics In Europe (SPINE) project since the project's inception in October 2002. By surveying the uptake, use and experience of new technologies by SPINE partners across Europe, a picture emerges of highly successful adoption of novel working methods revolutionizing this area of structural biology. Finally, a forward view is taken of how crystallization methodologies may develop in the future.
Subject(s)
Crystallography/methods , Proteins/chemistry , Crystallography/instrumentation , Crystallography/trends , Image Processing, Computer-Assisted , Nanotechnology , Plastics , Proteomics , Quality Control , RoboticsABSTRACT
Nine well-accepted cases of endometrial carcinoma associated with pregnancy have been reported. A 10th patient is added and the cases are briefly summarized. At the time of her definitive surgery 6 weeks postpartum, our patient was also found to have a Stage I endometrioid ovarian carcinoma. Nine of the 10 cases have been well-differentiated adenocarcinoma or adenoacanthoma and the lesions have tended to be focal with minimal or no invasion. Given the small number of patients, conclusions regarding prognosis and treatment are difficult to make. However, in younger patients with noninvasive adenocarcinoma of the endometrium, the adnexa may be spared. Endometrioid adenocarcinoma of the ovary, discovered incidentally in our patient, is associated with a concomitant adenocarcinoma of the endometrium in 5-29% of cases, but the present case in which the lesions coexisted with pregnancy appears to be the first such report in the literature.
