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A Protease Inhibitor of the Serpin Family Is a Major Protein in Carp Perimeningeal Fluid: II. cDNA Cloning, Sequence Analysis, and Escherichia coli Expression
Author(s) -
Huang ChangJen,
Lee MingShyue,
Huang ForeLien,
Chang GeenDong
Publication year - 1995
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1995.64041721.x
Subject(s) - biology , microbiology and biotechnology , complementary dna , serpin , peptide sequence , immunoscreening , serine protease , masp1 , cdna library , molecular cloning , nucleic acid sequence , biochemistry , protease , gene , enzyme
A cDNA clone, pCP9 , has been isolated from a common carp liver cDNA library by immunoscreening with polyclonal antiserum raised against purified bighead carp α 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 α 1 ‐antitrypsin (38%), guinea pig contrapsin (35%), human α 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 α 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 α 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 α 1 ‐antitrypsin. Expression of α 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.