Recombinant leech-derived tryptase inhibitor: construction, production, protein chemical characterization and inhibition of HIV-1 replication
| Autor: | Gerd P. Piechottka, Robert Morenweiser, Hans Fritz, Christoph Eckerskorn, Lutz G. Gürtler, Christian P. Sommerhoff, Ennes A. Auerswald |
|---|---|
| Rok vydání: | 1994 |
| Předmět: |
Electrospray ionization
Molecular Sequence Data Tryptase Saccharomyces cerevisiae medicine.disease_cause Virus Replication Biochemistry Mass Spectrometry law.invention law Leeches medicine Escherichia coli Animals Humans Amino Acid Sequence Cloning Molecular Cells Cultured chemistry.chemical_classification biology Molecular mass Base Sequence Oligonucleotide Circular Dichroism Proteins Virology Cassette mutagenesis Recombinant Proteins Amino acid chemistry Protein Biosynthesis biology.protein Recombinant DNA HIV-1 Electrophoresis Polyacrylamide Gel Spectrophotometry Ultraviolet Isoelectric Focusing Trypsin Inhibitors |
| Zdroj: | Biological chemistry Hoppe-Seyler. 375(10) |
| ISSN: | 0177-3593 |
| Popis: | A synthetic gene coding for leech-derived tryptase inhibitor, form C (LDTI-C), was designed, cloned and expressed. The gene assembled via 6 oligonucleotides contains linker sequences, stop codons and internal restriction recognition sites for cloning, expression and cassette mutagenesis. Periplasmatic expression products could not be detected in Escherichia coli (E. coli), but strong expression was found using Saccharomyces cerevisiae (S. cerevisiae) ( > 10 mg/l culture broth) if a variant of pVT102U/alpha was used as vector. The secreted material was isolated after cross-flow filtration and purified by cation exchange chromatography. The recombinant material proved to be pure and homogeneous by electrophoretic and chromatographic analyses. Amino acid sequencing and molecular mass determination (4737.6 +/- 0.77 Da) by electrospray ionization mass spectrometry confirmed that rLDTI-C was processed correctly and that it is indistinguishable from LDTI-C. The far UV-CD (circular dichroism) spectrum of the recombinant inhibitor is typical for a small folded protein. rLDTI-C is inhibitorily fully active, its complexes with bovine trypsin and human mast cell tryptase display equilibrium dissociation constants which are nearly identical to those with the natural inhibitor. Remarkably, the inhibitor blocked replication of HIV-1 in HUT-78 cells at a concentration of 20 microM. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|