Application of Ni(II)-Assisted Peptide Bond Hydrolysis to Non-Enzymatic Affinity Tag Removal

In this study, we demonstrate a non-enzymatic method for hydrolytic peptide bond cleavage, applied to the removal of an affinity tag from a recombinant fusion protein, SPI2-SRHWAP-His6. This method is based on a highly specific Ni(II) reaction with (S/T)XHZ peptide sequences. It can be applied for the protein attached to an affinity column or to the unbound protein in solution. We studied the effect of pH, temperature and Ni(II) concentration on the efficacy of cleavage and developed an analytical protocol, which provides active protein with a 90% yield and ∼100% purity. The method works well in the presence of non-ionic detergents, DTT and GuHCl, therefore providing a viable alternative for currently used techniques

Dual delivery of hydrophilic and hydrophobic drugs from chitosan/diatomaceous earth composite membranes

Oral administration of drugs presents important limitations, which are frequently not granted the importance that they really have. For instance, hepatic metabolism means an important drug loss, while some patients have their ability to swell highly compromised (i.e. unconsciousness, cancer...). Sublingual placement of an accurate Pharmaceutical Dosage Form is an attractive alternative. This work explores the use of the beta-chitosan membranes, from marine industry residues, composed with marine sediments for dual sublingual drug delivery. As proof of concept, the membranes were loaded with a hydrophilic (gentamicin) and a hydrophobic (dexamethasone) drug. The physico-chemical and morphological characterization indicated the successful incorporated of diatomaceous earth within the chitosan membranes. Drug delivery studies showed the potential of all formulations for the immediate release of hydrophilic drugs, while diatomaceous earth improved the loading and release of the hydrophobic drug. These results highlight the interest of the herein developed membranes for dual drug delivery.The research leading to these results has received funding from Erasmus Mundus Joint Programmes, ERDF / POCTEP 2007-2013 under project 0687_NOVOMAR_1_P, from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number REGPOT-CT2012-316331-POLARIS, and from the North Portugal Regional Operational Programme (ON.2 - O Novo Norte), within the National Strategic Reference Framework (QREN 2007-2013) under the project NORTE-01-0124-FEDER-000018. Portuguese Foundation for Science and Technology is also acknowledged for the post-doctoral fellowship SFRH/BPD/112140/2015, for the doctoral fellowship SFRH/BD/112139/2015 and for the funds provided under the program Investigador FCT 2012 (IF/00423/2012). Dr. Helder Santos (University of Helsinki) is also acknowledged for valuable discussions on the concept.The research leading to these results has received funding from Erasmus Mundus Joint Programmes, ERDF / POCTEP 2007–2013 under project 0687_NOVOMAR_1_P, from the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement number REGPOT-CT2012-316331-POLARIS, and from the North Portugal Regional Operational Programme (ON.2 – O Novo Norte), within the National Strategic Reference Framework (QREN 2007-2013) under the project NORTE-01-0124-FEDER-000018. Portuguese Foundation for Science and Technology is also acknowledged for the post-doctoral fellowship SFRH/BPD/112140/2015, for the doctoral fellowship SFRH/BD/112139/2015 and for the funds provided under the program Investigador FCT 2012 (IF/00423/2012). Dr. Hélder Santos (University of Helsinki) is also acknowledged for valuable discussions on the concept.info:eu-repo/semantics/publishedVersio