A protease inhibitor of the serpin family is a major protein in carp perimeningeal fluid: II. cDNA cloning, sequence analysis, and Escherichia coli expression.
A cDNA clone, pCP9, has been isolated from a common carp liver cDNA library by immunoscreening with polyclonal antiserum raised against purified bighead carp alpha 1-antitrypsin. This clone is 1,396 bp in length and has an open reading frame encoding a protein of 410 amino acid residues. The deduced amino acid sequence shows moderate homology to human alpha 1-antitrypsin (38%), guinea pig contrapsin (35%), human alpha 1-antichymotrypsin (34%), and human proteinase C inhibitor (31%), all members of the serine protease inhibitor (serpin) family. To confirm further that the cDNA clone was derived from the authentic carp alpha 1-antitrypsin gene, the presumptive mature protein of pCP9 was expressed in Escherichia coli. The molecular mass of the recombinant protein matched that predicted from the nucleotide sequence. This recombinant protein, which was recognized by antiserum against native alpha 1-antitrypsin, was capable of formation of serpin-enzyme complexes with trypsin, chymotrypsin, and elastase. Therefore, we conclude that the protein encoded by the pCP9 clone is indeed carp alpha 1-antitrypsin. Expression of alpha 1-antitrypsin in brain was confirmed by reverse transcription and polymerase chain reaction performed on mRNA derived from both common carp and bighead carp brain.
Huang
C J
CJ
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
Lee
M S
MS
Huang
F L
FL
Chang
G D
GD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001826:Body Fluids / Q000378:metabolism*; D002347:Carps / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D008578:Meninges / Q000378:metabolism*; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins; D017385:Sequence Homology; D015843:Serpins / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D015335:Molecular Probes; D009419:Nerve Tissue Proteins; D011480:Protease Inhibitors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015843:Serpins