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The ability to precisely design large proteins with diverse shapes would enable applications ranging from the design of protein binders that wrap around their target to the positioning of multiple functional sites in specified orientations. We describe a protein backbone design method for generating a wide range of rigid fusions between helix-containing proteins and use it to design 75,000 structurally unique junctions between monomeric and homo-oligomeric de novo designed and ankyrin repeat proteins (RPs). Of the junction designs that were experimentally characterized, 82% have circular dichroism and solution small-angle X-ray scattering profiles consistent with the design models and are stable at 95 °C. Crystal structures of four designed junctions were in close agreement with the design models with rmsds ranging from 0.9 to 1.6 Å. Electron microscopic images of extended tetrameric structures and ~10-nm-diameter "L" and "V" shapes generated using the junctions are close to the design models, demonstrating the control the rigid junctions provide for protein shape sculpting over multiple nanometer length scales. [ABSTRACT FROM AUTHOR]

In order to design plasminogen activators with improved thrombolytic properties we sought to construct the bifunctional protein HLS-2 which combines both a plasminogen-activating and an anticoagulative activity. The chimeric protein comprises four elements: a derivative of thrombin inhibitor hirudin, a 6-amino acid spacer, the sequence of plasminogen-activator staphylokinase (Sak), and a 13-amino acid expression tag at the C-terminus. The gene of the fusion protein was obtained by SOE-PCR, cloned into pCANTAB5E, and expressed in E. coli BL21. HLS-2 was purified from periplasmatic extracts and characterized by Western blotting. Plasminogen-activation of HLS-2 and of Sak in equimolar mixtures with plasminogen showed near equivalence as measured by plasmin-mediated cleavage of chromogenic substrate S-2403. For catalytic amounts of plasminogen-activator, however, HLS-2 was less effective by a factor of 1.7. HLS-2 also inhibited both the amidolytic and the fibrinolytic activities of thrombin. Similar concentrations of either commercial HV1 (42 pmol/L) or HLS-2 (250 pmol/L) were required to halve the initial rate of thrombin reaction with fluorogenic substrate Tos-Gly-Pro-Arg-AMC, suggesting the retention of high-affinity inhibition of thrombin by the fusion protein sufficiently strong to substitute anticoagulative comedication during fibrinolytic treatment. The results provide a rationale for further testing the efficacy of HLS-2 for the lysis of platelet-rich arterial blood clots and for the prevention of reocclusion after thrombolysis. [Copyright &y& Elsevier]