Isolation, characterization, and recombinant expression of multiple serpins from the cat flea,Ctenocephalides felis
| Autor: | Nancy Wisnewski, Patrick J. Gaines, Kevin S. Brandt, Joely D. Maddux, A.M. Becher, Gary M. Silver, E.E. Jarvis |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
DNA
Complementary Physiology Sequence analysis medicine.medical_treatment Cat flea Molecular Sequence Data Gene Expression Genes Insect Sequence alignment Serpin Biochemistry Exon Immunoscreening Complementary DNA medicine Animals Cloning Molecular Serpins Genetics Protease Base Sequence biology Gene Amplification General Medicine biology.organism_classification Molecular biology Recombinant Proteins Larva Insect Science Cats Siphonaptera Digestive System Sequence Alignment Sequence Analysis |
| Zdroj: | Archives of Insect Biochemistry and Physiology. 55:200-214 |
| ISSN: | 1520-6327 0739-4462 |
| DOI: | 10.1002/arch.10139 |
| Popis: | Several clones encoding serine protease inhibitors were isolated from larval and adult flea cDNA expression libraries by immunoscreening and PCR amplification. Each cDNA contained an open reading frame encoding a protein of approximately 45 kDa, which had significant sequence similarity with the serpin family of serine protease inhibitors. The thirteen cDNA clones isolated to date encode serpin proteins, which share a primary structure that includes a nearly identical constant region of about 360 amino acids, followed by a C-terminal variable region of about 40-60 amino acids. The variable C-terminal sequences encode most of the reactive site loop (RSL) and are generated by mutually exclusive alternative exon splicing, which may confer unique protease selectivity to each serpin. Utilization of an alternative exon splicing mechanism has been verified by sequence analysis of a flea serpin genomic clone and adjacent genomic sequences. RNA expression patterns of the cloned genes have been examined by Northern blot analysis using variable region-specific probes. Several putative serpins have been overexpressed using the cDNA clones in Escherichia coli and baculovirus expression systems. Two purified baculovirus-expressed recombinant proteins have N-terminal amino acid sequences identical to the respective purified native mature flea serpins indicating that appropriate N-terminal processing occurred in the virus-infected insect cells. |
| Databáze: | OpenAIRE |
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