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INTRODUCTION In inflammatory respiratory diseases, the imbalance between proteases and endogenous protease inhibitors leads to an exacerbated activity of human neutrophil elastase (a protease that destroys the extracellular matrix and stimulates proinflammatory cytokine release). Elastase is considered a target in the search for therapeutic treatments for inflammatory respiratory diseases. Pulmonary surfactant is a promising product for this purpose, because in addition to its biophysical function, it has anti-inflammatory properties. OBJECTIVE Evaluate effect of the Cuban porcine pulmonary surfactant (Surfacen), the rCmPI-II elastase inhibitor, and the Surfacen/rCmPI-II combination on activated neutrophil elastase activity in vitro, and determine if Surfacen's interface property changes in the presence of the inhibitor. METHODS The anti-elastase effect of Surfacen, rCmPI-II and the Surfacen/rCmPI-II combination was evaluated in an in vitro model of activated neutrophils, previously purified from the blood of healthy subjects. The cells were stimulated with LPS/fMLP and were incubated with diff erent concentrations of Surfacen, rCmPI-II and the Surfacen/rCmPI-II combination. Elastase activity was measured. The interface property was determined on a Langmuir surface balance. RESULTS Surfacen at 10 mg/mL inhibited 71% of stimulated neutrophil elastase activity. rCmPI-II at 0.1 µM reduced 20% of elastase activity; at 200 µM--the maximum concentration evaluated--inhibition was 68%. Both products had a dosedependent effect. The Surfacen/inhibitor combination (0.5 mg/ mL/80 µM) did not aff ect the surfactant interface property or the inhibitory activity of rCmPI-II against human neutrophil elastase. CONCLUSIONS Surfacen and the rCmPI-II inhibitor have an anti-elastase effect on an activated neutrophil model. rCmPI-II does not aff ect Surfacen's interface property and, therefore, both can be evaluated for combined use in treating inflammatory lung diseases. [ABSTRACT FROM AUTHOR]

P ichia pastoris is a highly successful system for the large-scale expression of heterologous proteins, with the added capability of performing most eukaryotic post-translational modifications. However, this system has one significant disadvantage - frequent proteolytic degradation by P . pastoris proteases of heterologously expressed proteins. Several methods have been proposed to address this problem, but none has proven fully effective. We tested the effectiveness of a broad specificity protease inhibitor to control proteolysis. A recombinant variant of the BPTI- Kunitz protease inhibitor Sh PI-1 isolated from the sea anemone S tichodactyla helianthus, was expressed in P . pastoris. The recombinant inhibitor (r Sh PI-1 A), containing four additional amino acids ( EAEA) at the N-terminus, was folded similarly to the natural inhibitor, as assessed by circular dichroism. r Sh PI-1 A had broad protease specificity, inhibiting serine, aspartic, and cysteine proteases similarly to the natural inhibitor. r Sh PI-1 A protected a model protein, recombinant human miniproinsulin (rh MPI), from proteolytic degradation during expression in P . pastoris. The addition of purified r Sh PI-1 A at the beginning of the induction phase significantly protected rh MPI from proteolysis in culture broth. The results suggest that a broad specificity protease inhibitor such as r Sh PI-1 A can be used to improve the yield of recombinant proteins secreted from P . pastoris. [ABSTRACT FROM AUTHOR]

Two new protease inhibitors (PIs), CmPI-II and AdKI were purified and characterized from mollusks Cenchritis muricatus and Aplysia dactylomela, respectively. They showed different specificities, CmPI-II for human neutrophil elastase (HNE) and AdKI for human plasma kallikrein (HPK). Purification procedures were established, rendering good yields and high purification degree. CmPI-II (UNIPROT: P84755) is a 5480 Da polypeptide of three disulphide bridges belong to the "non-classical" Kazal-type inhibitors. A new group was proposed according to the location of the CysI-CysV disulfide bridge. The presence of a basic residue at the inhibitor active site changed the pre-established requirement of a hydrophobic residue for elastase inhibition. The three-dimensional CmPI-II/HNE complex model contributes to explain the CmPI-II specificity for the enzyme. This is the first PI molecule isolated from C. muricatus. On the other hand, AdKI (2.9 kDa polypeptide) is an exception among invertebrate inhibitors in terms of inhibitory strength and selectivity against HPK. A new serine protease, AdSP, was also purified and characterized from the same extract, which could be the target for AdKI. CmPI-II and AdKI are the first inhibitors isolated from the phyllum Mollusca against ENH and HPK, respectively. [ABSTRACT FROM AUTHOR]