A PROTEASE INHIBITOR OF THE SERPIN FAMILY IS A MAJOR PROTEIN IN CARP PERIMENINGEAL FLUID .2. CDNA CLONING, SEQUENCE-ANALYSIS, AND ESCHERICHIA-COLI EXPRESSION

A cDNA done, pCP9, has been isolated from a common carp liver cDNA lib rary by immunoscreening with polyclonal antiserum raised against purif ied 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 re sidues. The deduced amino acid sequence shows moderate homology to hum an 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 express ed in Escherichia coil. 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-anti trypsin, was capable of formation of serpin-enzyme complexes with tryp sin, chymotrypsin, and elastase. Therefore, we conclude that the prote in encoded by the pCP9 clone is indeed carp alpha 1-antitrypsin. Expre ssion of alpha 1-antitrypsin in brain was confirmed by reverse transcr iption and polymerase chain reaction performed on mRNA derived from bo th common carp and bighead carp brain.