Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.

Means to prevent thrombus extension and local recurrence remain suboptimal, in part because of the limited effectiveness of existing thrombolytics. In theory, plasminogen activators could be used for this purpose if they could be anchored to the vascular lumen by targeting stably expressed, noninternalized determinants such as platelet-endothelial-cell adhesion molecule 1 (PECAM-1). We designed a recombinant molecule fusing lowmolecular-weight single-chain prourokinase plasminogen activator (lmw-scuPA) with a single-chain variable fragment (scFv) of a PECAM-1 antibody to generate the prodrug scFv/lmw-scuPA. Cleavage by plasmin generated fibrinolytically active 2-chain lmw-uPA. This fusion protein (1) bound specifically to PECAM-1-expressing cells; (2) was rapidly cleared from blood after intravenous injection; (3) accumulated in the lungs of wild-type C57BL6/J, but not PECAM-1 null mice; and (4) lysed pulmonary emboli formed subsequently more effectively than lmw-scuPA, thereby providing support for the concept of thromboprophylaxis using recombinant scFv-fibrinolytic fusion proteins that target endothelium. ( IntroductionPlasminogen activators (PAs; eg, uPA, urokinase plasminogen activator) help to restore perfusion after thrombotic vascular occlusion, the leading cause of human morbidity and mortality. [1][2][3] However, the clinical utility of PAs is limited by (1) inadequate delivery because of rapid elimination and inactivation en route and ineffective penetration into formed clots; (2) side effects, including extravasation leading to collateral damage in the central nervous system and other tissues; (3) lysis of "physiologic" (hemostatic) clots leading to hemorrhage; and, (4) reperfusion injury following a delay in restoring perfusion, where morbidity correlates with the duration of ischemia. [4][5][6] Clinical settings characterized by a high propensity for thrombosis have been identified, and means to diagnose early clot formation have been developed. 1,2 Although the indications for prophylaxis are known, PAs are not used prophylactically because of their unfavorable pharmacokinetics and side effects. Gene therapy approaches, effective in cell-culture and animal experiments, 7,8 are not practical when the need to enhance fibrinolysis is acute and of short duration. 9 Conceivably, prophylactic delivery of a PA derivative that rapidly restricts and sustains its activity in the vascular lumen can help to lyse nascent clots expeditiously, inhibit propagation of mural thrombi, and reduce the duration of ischemia.For example, PAs can be used for thromboprophylaxis by coupling to carrier red blood cells (RBCs), prolonging circulation and limiting extravasation. 10 This approach may have utility in settings in which RBC transfusion is part of current management. Drug targeting to suitable endothelial-cell-surface determinants 11-13 may provide an alternative approach and, in theory, localize PA activity in the affected intravascular compartment.For example, drugs coupled with antibodies to plateletendothelial ...

Conjugating tissue-type plasminogen activator (tPA) to red blood cells (RBCs) endows it with features useful for thromboprophylaxis. However, the optimal intensity and duration of thromboprophylaxis vary among clinical settings. To assess how the intrinsic properties of a plasminogen activator (PA) affect functions of the corresponding RBC/PA conjugate, we coupled equal amounts of tPA or Retavase (rPA; a variant with an extended circulation time, lower fibrin affinity, and greater susceptibility to PA inhibitors). Conjugation to RBC markedly prolonged the circulation of each PA in rats and mice, without detrimental effects on carrier RBC. The initial blood clearance of RBC/tPA was faster than RBC/rPA, yet it exerted greater fibrinolytic activity, in part due to greater resistance of tPA and RBC/tPA to plasma inhibitors versus rPA and RBC/rPA observed in vitro. Soluble and RBC-coupled tPA and rPA exerted the same amidolytic activity, yet RBC/tPA lysed fibrin clots more effectively than RBC/rPA, notwithstanding comparable fibrinolytic activity of their soluble counterparts. Conjugation to RBC suppressed rPA's ability to be activated by fibrin, whereas the fibrin activation of RBC-coupled tPA was not hindered. Therefore, the functional profile of RBC/PA is influenced by: pharmacokinetic features provided by carrier RBC (e.g., prolonged circulation), intrinsic PA features (e.g., clearance rate, resistance to inhibitors), and changes imposed by conjugation to RBC (e.g., loss of cofactor stimulation). These factors, different from those guiding the design of soluble PA for lysis of existing clots, can be exploited in the rational design of RBC/PA tailored for specific prophylactic indications.

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