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Isolation, characterization, and recombinant expression of multiple serpins from the cat flea, Ctenocephalides felis
Author(s) -
Brandt K.S.,
Silver G.M.,
Becher A.M.,
Gaines P.J.,
Maddux J.D.,
Jarvis E.E.,
Wisnewski N.
Publication year - 2004
Publication title -
archives of insect biochemistry and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.576
H-Index - 66
eISSN - 1520-6327
pISSN - 0739-4462
DOI - 10.1002/arch.10139
Subject(s) - biology , serpin , complementary dna , microbiology and biotechnology , peptide sequence , rapid amplification of cdna ends , serine protease , genetics , gene , protease , molecular cloning , biochemistry , enzyme
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. Arch. Insect Biochem. Physiol. 55:200–214, 2004. © 2004 Wiley‐Liss, Inc.