Self-cleaving purification tags re-engineered for rapid Topo® cloning.

In this work, our previously reported ΔI-CM and ΔI(G)-CM mutant inteins were rationally re-engineered to be compatible with Invitrogen's Topo® cloning system. The resulting new inteins include the vaccinia virus topoisomerase I DNA recognition sequence TCCTT at their 3' ends, making them compatible with the highly convenient one-step Topo® cloning method. Addition of the Topo® recognition sequence resulted in an altered amino acid sequence at the C-termini of the inteins, changing their final five residues from VVVHN to VLVHN. Despite this change, these modified inteins retained their self-cleaving function, and continue to exhibit pH and temperature-sensitive cleaving characteristics as required for their use in generating self-cleaving affinity tags. Although the C-terminal modification decreased the intein cleavage rate under optimal conditions, cleavage can typically be completed within several hours at pH 6.5 and 37°C. In particular, the modified ΔI(GT)-CM intein is compatible with both the Topo® and Gateway® methods simultaneously, allowing fast parallel construction of multiple expression vectors with varying combinations of target proteins, self-cleaving affinity tags and promoters. These newly engineered inteins increase the functionality of intein-mediated technology, making it possible to explore a large number of combinations between target genes, self-cleaving affinity tags and expression hosts in a fast and efficient manner.

The use of recombinant activated factor VII in neurosurgery.

BACKGROUND: Bleeding complications in neurosurgery often take alarming proportions without major hemodynamic effect or impairment of coagulation physiology because severe neurologic deficits are to be expected. Any measures used to stabilize or normalize coagulation are therefore of great interest. Administration of packed red cells, fresh frozen plasma, and platelet concentrates is associated with volume loading, which is suspected to multiply the secondary brain damage, for example, by the development of an edema. In this respect, the administration of rFVIIa may develop into a new option associated with low-volume administration. CASE DESCRIPTIONS: We report on 5 neurosurgical patients to whom rFVIIa was given at doses of 51 to 202 microg/kg of body weight for the treatment of severe intraoperative bleeding (n = 3) or as prophylaxis of bleeding (n = 2). The operation was completed successfully in all patients after administration of rFVIIa, with stabilization of the coagulation status. CONCLUSION: Therefore, reported cases constitute an approach in treatment and prophylaxis of bleeding complications in neurosurgery. There are reports of thromboembolic events in use of rFVIIa, particularly in unlabeled use. But according to our findings and current literature, there is no evidence of higher risk of thromboembolic adverse events in treatment with rFVIIa. However, the number of patients presented does not allow any final assessment to be made as to whether the properties of rFVIIa are of particular benefit for neurosurgical patients. Further studies with appropriate study design are required to verify effects observed in this investigation.