An inhibitor of collagen-stimulated platelet activation from the salivary glands of the Haementeria officinalis leech. II. Cloning of the cDNA and expression
| Autor: | P M, Keller, L D, Schultz, C, Condra, J, Karczewski, T M, Connolly |
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| Rok vydání: | 1992 |
| Předmět: |
Base Sequence
Genetic Vectors Molecular Sequence Data Gene Expression DNA Saccharomyces cerevisiae Blotting Northern Platelet Activation Polymerase Chain Reaction Recombinant Proteins Salivary Glands Leeches Cell Adhesion Animals Amino Acid Sequence Collagen RNA Messenger Cloning Molecular Salivary Proteins and Peptides Plasmids |
| Zdroj: | The Journal of biological chemistry. 267(10) |
| ISSN: | 0021-9258 |
| Popis: | Salivary glands of the leech Haementeria officinalis contain a protein, leech antiplatelet protein (LAPP), that specifically blocks collagen-mediated platelet aggregation (Connolly, T. M., Jacobs, J. W., and Condra, C. (1992) J. Biol. Chem. 267, 6893-6898). Degenerate oligonucleotides whose sequences were derived from two short peptides from V8 digests of the native LAPP were used as primers to generate a polymerase chain reaction (PCR) product which contains the cDNA region coding for the sequence between these two peptides. Using this PCR product as a hybridization probe, phage containing cDNA clones were isolated containing the entire deduced amino acid sequence for LAPP. Computer analysis of the amino acid sequence predicts a peptidase cleavage site between a 21-residue pre-peptide and a mature protein of 126 amino acids. A DNA insert to express the predicted mature LAPP protein was generated by PCR amplification using phage-derived cDNA clones as a substrate. This insert encoded a fusion protein with the leader sequence of the yeast alpha mating factor and the mature LAPP cDNA. These PCR products were cloned into the yeast expression vector pKH4 alpha 2. A KEX 2 Lys-Arg endopeptidase cleavage site was placed NH2-terminal to the predicted mature protein. This vector transfected into the yeast Saccharomyces cerevisiae directs expression of a secreted mature protein at levels up to 200 mg of LAPP/liter of culture medium. The recombinant protein was comparable to native LAPP in its electrophoretic mobility, its reactivity with anti-LAPP antisera, and its biological activity including inhibition of collagen-stimulated platelet aggregation and the adhesion of platelets to collagen. Availability of significant quantities of recombinant LAPP opens the way to further biochemical structure/function studies and to studies on the effects of an inhibitor of collagen-stimulated platelet aggregation in vivo. |
| Databáze: | OpenAIRE |
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