Isolation, characterization, and recombinant expression of multiple serpins from the cat flea, Ctenocephalides felis.
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.
Brandt
K S
KS
Heska Corporation, Fort Collins, Colorado 80525, USA.
Silver
G M
GM
Becher
A M
AM
Gaines
P J
PJ
Maddux
J D
JD
Jarvis
E E
EE
Wisnewski
N
N
D000818:Animals; D001483:Base Sequence; D002415:Cats; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D004064:Digestive System / Q000378:metabolism; D005784:Gene Amplification; D015870:Gene Expression; D017344:Genes, Insect; D007814:Larva / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017421:Sequence Analysis; D015843:Serpins / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D005423:Siphonaptera / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D015843:Serpins