Purification, characterization and immobilization of proteinase inhibitors from Stichodactyla helianthus.

Isolation of proteinase inhibitors from the sea anemone Stichodactyla helianthus was achieved by trichloroacetic acid treatment of the aqueous extract followed by affinity chromatography on trypsin-Sepharose and ion-exchange chromatography on CM-cellulose. The average molecular mass of the major inhibitor (ShPI-I) obtained by fast atom bombardment mass spectrometry (FAB-MS) was 6110.6 Da. The amino acid sequence was determined by FAB-MS combined with manual Edman degradation, digestions with endopeptidases and exopeptidases and automatic sequencing. The sequence of ShPI-I (55 amino acids) was compared with those reported in the SwissProt database for several proteinase inhibitors and significant similarity to inhibitors belonging to the Kunitz family was observed. ShPI-I exhibits a broad specificity for serine, cysteine and aspartic proteinases. The dissociation constants of the complexes formed with different enzymes were determined. The affinity-purified fraction (PI) was immobilized on Sepharose and used in the purification of different classes of proteinases.

NMR solution structure of the recombinant tick anticoagulant protein (rTAP), a factor Xa inhibitor from the tick Ornithodoros moubata.

The solution structure of the recombinant tick anticoagulant protein (rTAP) was determined by 1H nuclear magnetic resonance (NMR) spectroscopy in aqueous solution at pH 3.6 and 36 degrees C. rTAP is a 60-residue protein functioning as a highly specific inhibitor of the coagulation protease factor Xa, which was originally isolated from the tick Ornithodoros moubata. Its regular secondary structure consists of a two-stranded antiparallel beta-sheet with residues 22-28 and 32-38, and an alpha-helix with residues 51-60. The relative orientation of these regular secondary structure elements has nearly identical counterparts in the bovine pancreatic trypsin inhibitor (BPTI). In contrast, the loop between the beta-sheet and the C-terminal alpha-helix as well as the N-terminal 20-residue segment preceding the beta-sheet adopt different three-dimensional folds in the two proteins. These observations are discussed with regard to the implication of different mechanisms of protease inhibition by rTAP and by Kunitz-type protein proteinase inhibitors.

Transient hydrogen bonds identified on the surface of the NMR solution structure of Hirudin.

Recombinant desulfatohirudin retains largely the thrombin-inhibitory activity of natural hirudin from Hirudo medicinalis and causes at most minimal immune response in humans. With regard to potential pharmaceutical applications it is of interest to further investigate the structural basis of hirudin functions. In this paper transient hydrogen bonds between backbone amide protons and side-chain carboxylates on the protein surface of desulfatohirudin (variant 1) have been identified using two-dimensional 1H NMR experiments and site-directed mutagenesis. The analysis of pH titration curves measured with NMR enabled the determination of the pK values of all 13 carboxylates, and downfield shifts larger than 0.2 ppm arising from weak bonding interactions with carboxylates were observed for the amide protons of Gly 25, Ser 32, Glu 35, and Cys 39. For these backbone amide protons virtually identical titration parameters were observed in intact desulfatohirudin and the mutant, truncated hirudin(1-51), demonstrating that the hydrogen bond acceptors are located in the N-terminal polypeptide segment 1-51. The hydrogen bonds Gly 25 NH-Glu 43 delta COO-, Ser 32 NH-Glu 35 delta COO-, Glu 35 NH-Asp 33 gamma COO-, Glu 35 NH-Glu 35 delta COO-, and Cys 39 NH-Glu 17 delta COO- were identified by considering spatial proximity in the NMR solution structure of hirudin(1-51), and comparing the pK values for the amide protons and the carboxylates in desulfatohirudin and the mutants hirudin(E43Q), hirudin(E35Q), hirudin(D33N) and hirudin(E17A).(ABSTRACT TRUNCATED AT 250 WORDS)

The NMR solution structure of a Kunitz-type proteinase inhibitor from the sea anemone Stichodactyla helianthus.

The solution structure of a 55-amino-acid Kunitz-type proteinase inhibitor, ShPI, purified from the Caribbean sea anemone Stichodactyla helianthus, was determined by NMR spectroscopy. Nearly complete sequence-specific 1H-NMR assignments were obtained at pH 4.6 and 36 degrees C, and stereo-specific assignments were determined for 23 pairs of diastereotopic substituents. A data set of 666 upper distance limit constraints and 122 dihedral angle constraints collected on this basis was used as input for a structure calculation with the program DIANA. Following energy minimization with the program OPAL, the average root-mean-square diviation (RMSD) of the 20 DIANA conformers used to represent the solution structure relative to the mean structure is 61 pm for all backbone atoms N, C alpha and C', and 106 pm for all heavy atoms of residues 2-53. This high-quality solution structure of ShPI has a nearly identical molecular architecture as the bovine pancreatic trypsin inhibitor (BPTI), despite a mere 35% of sequence similarity between the two proteins. Exchange rates measured for 48 out of the 51 backbone amide protons showed that the positions of 20 slowly exchanging amide protons correlate well with hydrogen bonds involving these protons in the energy-minimized solution structure. The solution structure of ShPI is compared to the four homologous proteins for which the three-dimensional structure is also available.

High level expression of streptokinase in Escherichia coli.

Streptokinase (SK), which activates human plasminogen by promoting its conversion to plasmin, is normally obtained from beta-hemolytic streptococci. Treatment with SK is an effective therapy for improving survival and preserving left ventricular function after coronary thrombosis. We report the cloning, expression in E. coli to levels of 25% of the total cell protein, and characterization of a novel SK (SKC-2) gene, the product of which is functionally equivalent to the naturally-derived protein. The availability of a recombinant streptokinase (rSK) in high yield and purity offers a potentially attractive alternative source of this important therapeutic agent.