novel, biologically interesting and crystallization-feasible targets that were then designed into 96 different constructs. Processing of the 96 constructs was performed in parallel using simple automated applications of ligation-independent cloning, small-scale bacterial expression and purification, and solubility assessment. After processing the 96 constructs of 12 targets, I found that 16 constructs of three targets (25%) yielded soluble protein. From the three soluble targets, I have spent most time on two of these protein.The first protein is a CARD domain containing protein that interacts with Bcl10. The primary function of Bcl10 is to interact with CARD proteins through CARD-CARD interactions to regulate its activity [2]. The crystal structure of this CARD containing protein solved at 1.5 Å resolution revealed six anti-parallel α-helices, showing that this protein is indeed similar to other CARD proteins with known structures. Approaches to determine the interaction between these two CARD domain containing proteins are currently being applied.The second protein I worked on is a DUF59 domain containing protein with no function characterized yet. However, it has been reported that a family member is part of the MMXD protein complex involved in chromosome segregation [3]. I solved two crystal structures of this DUF59 domain protein to 1.8 Å resolution revealing, unusually, two different types of domain swapped-dimer. Functional characterization of this DUF59 domain containing protein, and of its domain swapping, is currently being investigated.
A new mucopolysaccharide(FGAG) has been isolated from the cell wall of Stichopus japonicua. The molecular weight of FGAG is about 50,000. The component sugar of the FGAG are identified as galactosamine, glucuronic acid, fucose and sulfate with the molar ratioof 1:0.94:0.84:3.60,respectively. The anticoagulant effects of FGAG were studied. At low concentration(lμg/ml), FGAG completely inhibited the rabbit plateletaggregation induced by thrombin and prolonged thrombin time of not only human plasma but purified fibrinogen solution to a similar extent, suggesting that action of FGAG is not depend on plasma component(antithrombin III and/or Heparin c.ofactor II).After intravenous injection into rabbits(lmg/kg),significant prolongation of activated partial thromboplastin time(APTT) was observed. Although the in vivo antithrombin activity of the FGAG was weaker than that of heparin.it lasted longer than that of heparin and was not inhibited by platelet factor 4.These results suggested that FGAG is a new and unique mucopolysaccharide with antithrombin activity and may come into use for the treatment ofDiseminated Intravascular Coagulation.
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